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IDENTIFICATION OF ROM10 AND PHRI-INTERACTING PROTEINS IN THE MAMMALIAN PHOTORECEPTOR
Rahim Akbarali Ladak
A thesis submitted in confornity with the requirements for the Degree of Master of Science, Gnduate Department of Molecular and Meâicai Genetics, in the University
of Toronto
@ Copyright by Rahim Akbadi Ladak 1999
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IDENTIFlCATION OF ROMl- AND PEKR1-INTERACTING PROTEINS IN TEE MAMMALIAN PHOTORECF2TOR Degree of Master OC Science, 1999, Rahim Akbarali Ladak, Graduate Department of Molecular and Medical Genetics, University of Toronto.
ABSTRACT OF THESIS
The photoreceptor is a unique neuron in the retina, and its highly specialized organelle, the
outer segment, is the site of phototransduction. Elucidation of the molecular basis of
outer segment structure and fùnction are two of the main goals of retinal research. Our
group has described one major outer segment protein, ROMl, and more recently
discovered a second outer segment polypeptide, PHRI, which contains a pleckstnn
homology (PH) domain. ROMl is an essential structural protein of the membranous disks
which fil1 the outer segment. The function of PHRl is unknown, but the presence of a PH
domain suggests that it may be a component of the cytoskeleton or participate in a
signalling pathway such as phototransduction. Using the two-hybrid system, 1 have used
the ROMl C-terminai tail and full-length PHRl in two different screens to identiQ
ROMl - and PHR1 -interacting proteins. The ROM1 C-terminal tail screen has identified
five novel putative ROMl-interacting proteins. Four novel cDNA clones encoding
putative PHR1-interacting proteins were identified, one of which contains a RING finger
domain, a well-known protein-protein binding motif. In situ hybridization revealed that
PHRI is expressed in photoreceptor inner segments, the outer plexiform layer, and the
ganglion ce11 layer of the retina. Immunohistochemical analysis indicates that PHRl is
specifically localized to the photoreceptor outer segments and to the plasma membrane of
ganglion cells. Retinal PHRl also migrates more slowly (-37kDa) than predicted
(-ZSkDa), suggesting that PHRl is post-translationally modified. The proteins which
associate with ROMl and PHRl in vivo are likely to encode important components of
photoreceptor outer segment structure and fûnctioa.
ACKNOWLEDGMENTS
1 would like to thank my supe~so r Dr. Roderick R Mclnnes for his support and
patience. 1 would also I l e to thank the members of my cornmittee, Dr. Sean Egan and
Dr. Barbara Fumeil, for their guidance and help. Special thanks to Danka Vidgen and
David Ng for their help during the writing of my thesis. And 1 would like to thank the
mernben of the McInnes lab for their assistance and scientific support.
To my family, thank-you so much for your patience, support and love. 1 could not
get through these last two years without your unconditional help.
TABLE OF C O m N T S
Abstract of' thesis
Acknowledgments
Table of Contents
List of Tables
List of' Figures
CHAPTER 1: INTRODUCTION
- Introduction
- An introduction to the vertebrate retina
- The vertebrate photoreceptor
- Visual transduction in the photoreceptor cell
- Photoreceptor outer kgment structure
- Outer segment disk rim proteins
- ROMl ami RDS/penpherin
- Associations between ROMl and RDS
- ROM1 and RDS are members of the transmembrane 4
superfamily (TM4SF)
- ~onrl" mouse, implication on photoreceptor structure
and fbnction
- Initial charactenzation of PHR1
- Plec kstnn homology domain
- PH domain structure
- PH domain ftnction
- Identifjing ROMl- and PHR1-interacting proteins using
the yeast two-hybrid system
CHAPTER 2: IDENTIFICATION OF ROM1-INTERACTING PROT'EINS
USING THE YEAST TWO-HYBRID SYSTEM
1 . INTRODUCTION
2. EXPERiMENTAL PROCEDURES
3. RESULTS
- Construction of the ROMl C-terminal tail bait
- Analysis of ROMl C-terminal tail bait expression in the
two-hybrid system
- Two-hybrid screen
- Characterization of the ROM 1 -interacting partners
4. DISCUSSION
CHAPTER 3: MOLECULAR CHARACTERIZATION OF PHRl AND
IDENTIFICATION OF PEIRI-INTERACTING PROTEINS
USING THE TWO-HYBRID SYSTEM
1. iNTRODUCTION 58
2. EXPERMENTAL PROCEDURES 63
3. RESULTS 68
- PHRI alternative splicing 68
- Expression of endogenous PHRI in retinal and brain lysates 72
- PHRl expression and localization in the neuroretina 74
- Presence of a putative C-terminal transrnembrane domain in PHRl 77
- Analysis of PHRl bait expression in the two-hybrid system 77
- The PHRl two-hybnd screen 80
4. DISCUSSION 94
CEIAPTER 4: CONCLUDING REMARKS
1. FUTURE DIRECTIONS
- Localizing expression of PHRl and ROMl putative
interactors in the retina
- Using afEnity chrornatography to confirm PHRl and
ROMl interactors
- Idenemg PHR 1 - and ROM 1 -interacting proteins
using afnnity chrornatography
- Future perspectives for PHRl
- Future perspectives for ROM1
REFERENCES
LIST OF TABLES
Table 1 - 1 Transmernbrane four superfamüy members (TM4SF) 15-16
Tables 2- 1 a e ROM1 C-terminai tail two-hybrid t&ets 47-5 1
Table 3 -A PH domain containhg proteins used or identified using
the yeast two-hybrid system 6 1-62
Table 3- l a-i PHRl two-hybrid targets 82-90
Table 3-2 The weak targets specific in their interaction with
the PHRl bait 93
Figure 1-1
Figure 1-2
Figure 1-3
Figure 1-4
Figure 1-5
Figure 1-6
Figure 1-7
Figure 2- 1
Figure 2-2
Figure 2-3
Figure 3- 1
Figure 3-2
Figure 3-3
Figure 3-4
Figure 3-5
Figure 3-6
Figure 3-7
Figure 3-8
Structure of the vertebrate retina
The vertebrate r d photoreceptor cell
Structural topology of ROM1
Light micrographs of retinal sections fiom 1 month old
~oml"' and ~ o m l " mice
Genomic structure of PHRI
The predicted PH21 protein sequence
Cornparison of PH domains
A. The mouse ROM 1 C-tenninal tail two-hybrid
construct, referred to as MRC
B. Western blot of yeast lysates expressing the MRC bait
Quantitative liquid P-galactosidase assay
Hypothetical mode1 of the rod disk rim
Alternative splicing of PHRI in the retina, brain, kidney,
and liver
Alternative splicing of exon 7 of PHRl
PHRl expression in HEK293 cells, mouse retinal and
brain lysates
In situ hybridization of mouse PHRl in adult mouse retina
PHRl is localized to the photoreceptor outer segment and
to the ganglion cells of mouse adult retina
PHRl protein sequence anaiysis
PHRl two-hybrid bait
Strong PHRl putative interactors identified in the
two-hybrid screen
CHAPTER 1
Introduction
INTRODUCTION
The retina is a highly organized, well-defined, and conserved neurosensory
structure. As the visual transducer of vertebrates, it captures photons and converts the
energy into an electrophysiological signal which is carrieci to the higher visual centers of
the brain (Jindrova, 1998). A thin layer of tissue, the retina, is situated at the posterior of
the vertebrate eye (Wechsler-Reya and Barres, 1997). The retina is composed of two
components, the neuroretina and the pigment epithelium. The neuroretina consists of
three parallel cellular layen: the ganglion ce11 layer (GCL), the i ~ e r nuclear layer (INL),
and the outer nuclear layer (ONL) (Wechsler-Reya and Barres, 1997). The outer nuclear
layer is comprised entirely of photoreceptor cells. The most notable structural feature of
the photoreceptor is a specialized ciliary denvative called the outer segment. The outer
segment is a finger-like structure, composed of a plasma membrane surrounding a stack of
-1000 membranous disks (Morrow et al., 1998). Three of the most interesting features of
the outer segment are that i) the disks are the site of photon absorption and light
transduction (Jindrova, 1998), ii) the disks are constantly renewed at a rate of -10% per
day, with new disks being formed at the base of the outer segment by disk morphogenesis,
and older disks being phagocytosed at the tip of the outer segment by the adjacent retinal
pigment epithelial (RPE) cells (Young, 1976; Anderson et al., 1978), and iii) in addition to
the symbiosis between the RPE and the photorecepton that is manifest by outer segment
phagocytosis, the RPE also provides important metabolites and nutrients to the
photoreceptor (Robinson, 1 99 1). Identification of the proteins involved in
phototransduction, outer segment structure, disk morphogenesis and turnover are three of
the main goals of retinal research.
The cloning and characterization of retinal gpecinc, gbundant, and onserved
(SAC) cDNAs has been an effective strategy for the identification of cDNAs encoding
proteins important for retina structure, finction, and development (Bascorn et al., 1992).
My research is concemed with two photoreceptor proteins, ROM1 and PHRI, identified
by the SAC cDNA cloning strategy. These two proteins are both abundantly expressed in
the outer segment of marnmalian photoreceptor, but their precise fwictions are unknown.
The overd goal of my research has been to begin to determine how these protehs
contribute to photoreceptor structure or hction by identdjing photoreceptor proteins
with which they interact.
ROMl is an essential integral membrane protein of the outer segment;
photoreceptors lacking the ROMl protein die (Bascom et al., 1992; G. Clarke
unpublished). PHRl is a novel retinal, abundantly expressed gene which is also present at
reduced levels in other tissues including the brain, kidney, and liver. The presence of a
plekstnn hornology (PH) domain in PHRl (gleckstrin hornology in the -na) suggests
that PHRl may be a component of the outer segment cytoskeieton, or of photoreceptor
signal transduction, including possibly phototransduction. In addition to identifjmg retinal
proteins with which PHRl may associate, 1 also evaluated the quality of PHRl antibodies
that were used to determine the cellular and subcellular localization of the protein.
The identification of ROMI- or PHR1-interacting proteins will provide critical
information about the fiinction of ROMl and PHRI, in the photoreceptor. The
identification of ROMl- and PHR1-interacting proteins using the yeast two-hybnd system
is the major subject of this thesis.
Chapter 1 presents an o v e ~ e w of retinal anatomy and fùnction, with particular
attention to the photoreceptor cell. In addition, information on ROMl and PHRl is
reviewed in this chapter.
Chapters 2 describes the results obtained from the yeast two-hybrid screen using
the ROM1 C-terminal tail as the bait. Chapter 3 contains further information on the
expression, localization and characterization of PHRI, as well as the results of a PHRl
two-hybrid screen. Concluding remarks are given in Chapter 4, with emphasis on the
further charactehtion of the putative ROM1 - and PHR1 -interacting proteins.
AN INTRODUCTION TO TEE VERTEBRATE RETINA
The retina consists of two major components, the pigment epithelium (PE) and the
neuroretina (NR) (Robinson, 1991). The vertebrate neuroretina is several hundred
microns in thickness. As mention4 in the introduction, the neuroretina is composed of
three parallel nuclear layers (outer nuclear layer (ONL), inner nuclear layer (INL) and
ganglion ce11 layer (GCL)). In addition, the three parallel nuclear layers are separated by
two piexifon layers (imer and outer plexifonn layen) (Fig. 1 - 1). The ONL is adjacent to
the retinal pigment epithilium (RPE), and is closest to the back of the eye with respect to
the other retinal ce11 layen (Fig. 1-1).
Light transverses al1 the layen of the retina until it is captured by the outer nuclear
layer. The ONL contains the ce11 body and nuclei of the photoreptor (PR) cells (Fig. 1-1).
The outer plexifonn layer (OPL) is the region in which synaptic co~ections are made
between cells of the ONL and imer nuclear layer (N) (Fig. 1-1). The ZNL consists of
interneuronal nuclei for al1 horizontal, bipolar, and interplexifom cells, the majority of
amacrine cell nuclei and a few ganglion cell nuclei (Famiglietti, 1990). Adjacent to the
IM. is the imer plexiform layer (PL) where synaptic processes between intemeurons and
ganglion cells connect. The ganglion ce11 layer (GCL) is composeci primarily of ganglion
ceil nuclei and a few amacrine cell nuclei (Robinson, 1991). The axons fiom the ganglion
cells bundle to collectively form the optic nerve which carries the light transduction signal
to the visual centers of the brain. The non-neuronal glial Müiler cells span al1 three ce11
layers of the retina, although their nuclei are located in the iNL (Robinson, 1991).
The neuroretina consists of six major neuronal ce11 types (photoreceptor cells,
bipolar cells, ganglion ceils, horizontal cells, amacnne cells, and interplexifom cells) and
two types of glial cells (Muller cells and in some species astrocyte cells). There are also
different neuronal ce11 classes. For example, ganglion cells can be subdivided into several
subtypes, including a, p. y, small-soma, and large-soma, based on morphology and time of
birth (Robinson, 199 1).
The second component of the retina is the RPE. The RPE consists of a
monolayer of pigrnented cells. These cells contribute to the enhancement and resolution
of the visual image by absorbiig scattered light via melanin granules (Bok, 1990). In
addition, the RPE plays an important role Ui photorezeptor fwictior The presence of
mannose receptors on RPE celis facilitates phagocytosis of rod outer segments (ROS)
(Lutz et al., 1995) whereas the activation of protein kinase C (PKC) in ROS inhibits
phagocytosis of ROS by the RPE (Hali et al., 1991). Rezently, it has been shown that
RPE (Retinal Pigment Epitheiium)
OS (Outer Segments)
IS (Imer Segments)
ONL (Outer Nuclear Layer)
OPL (Outer Plexiform Layer)
INL (Inner nuclear Layer)
1 GCL (Ganglion
8orm Layer)
Cell Layer)
A I 1 I
Light
Figure 1-1. Structure of the vertebrate retina. The vertebrate retina is laminar in structure and consists of five layers as shown. Light tranverses al1 the layers to reach the back of the retina, where the rhodopsin bound chromophore 1 1 4 s retinal, in the outer segments of the photoreceptors, captures photons and initiates the iight transduction cascade. The signai then proceeds to the ganglion cells, whose axons comprise the optic nerve, which relays the signai to the visual centen of the brain. Photograph fkom G. Clarke.
proteins secreted by RPE cells (RPE-CM) are important for photoreceptor cell s u ~ v a l
and development as suggested by retinal explant cultures and by rats injecteci intravitreaily
with RPE-CM (Sheedlo et al., 1998).
The Vertebrate Photoreceptor
The vertebrate photoreceptor (PR) is a specialized post-rnitotic neuron which
primarily functions to capture and convert light into an electrical signal. There are two
types of PR cells in the retina of vertebrates, rods and cones. The average human retina
contains approximately 92 million rods and 4.6 million cones (Curcio, 1990). Each PR
ce11 is specialized for vision under low or high light intensity, with rods fùnctioning
opitmally at low intensities of light and cones under high light intensity. Cones also
fascilitate colour vision.
The distribution of rod and cone photoreceptors is not random. Generally, at the
periphery of the eye there are few cones but many rods groups of which make a synaptic
connection with a single bipolar neuron, making this region of the eye highly sensitive to
light with low acuity (Bumside and Dearry, 1986). In contrast, the fovea of the eye
consists oniy of cones each making a single synaptic connection with its own bipolar
neuron.
Rods and cones cm be divided into an OS, IS, ceIl body and a synaptic terminal
(Fig. 1-2). The OS is the site of light absorption and transduction. The IS, which
connects to the OS by the ciliary process, contains the major metabolic machinery of the
ceIl and cm be subdivided into the myoid and ellipsoid regions (Fig. 1-2). The ellipsoid is
proximal to the OS and is highiy packed with mitochondria which generates ATP
providing energy to metabolic functions of the PR such as ion pumping and disk
morphogenesis (Farber and Shuster, 1986). The myoid region of the IS contains the
major metabolic machinery including organelles such as the endoplasmic reticulum and
Golgi apparafus (Farber and Shuster, 1986). The nuclei of the PR cells are located within
the cell body. Signal transfer to higher order neurons occurs at the synaptic terminal of
the PR (Fig. 1-2).
pigment epithelium
R d outer segment
Connecting cilium .
Rod inner segment
Nucleus
plasma membrane - Disks
Open disk (evagination)
EUipsojd (mitochondria)
Myoid (golgi complex and endoplasmic recticulum)
Celi body
Axon
Synaptic terminal
Figure 1-2. The vertebrate rod photoreceptor cell. The photoreceptor ceii is divided structurally into four regions, the outer segment (OS), the imer segment (ES), the ce11 body which houses the nucleus, and the synaptic terminal. Functionaiiy, the outer segment is involved in light absorption and transduction whereas the inner segment contains the major metabolic machinery of the ceil. The retinal pigment epithelium ce11 is important for photoreceptor survival and disk turnover.
Visual Transduction in the Pbotoreceptor CeIl
The visual transduction pathway is compriseci of several proteins, many of which
have been characterized extensively. Biochemical and structurai studies have led to a very
detailed description of the events in phototransduction.
The highly sensitive üght transduction pathway is the process by which energy
from photons is converted to an electrophysiological signal. The initial step in the
phototransduction pathway is the absorption of photons by the rhodopsin bound
chromophore 1 1-cis retinai causing it to isomerize to the 1 l-ull trm-retinal form
(Hargrave, 1986). Photoisomerization induces a conformational change in rhodopsin, a
member of the G-protein coupled receptor family (Farber and Shuster, 1986). Activated
rhodopsin then binds inactive transducin-GDP (TaPy-GDP) causing the activation of Ta
by catalyzing the exchange of bound GDP for GTP. Consequently, Ta-GTP dissociates
fiom TBy.
Released Ta-GTP proceeds to activate the heterotrimenc rod outer segment
(ROS) cGMP phosphodiesterase (PDE). PDE is inhibited from functioning by its intemal
inhibitor PD& (Granovsky et al., 1997; Artemyev, 1997; Artemyev et al., 1998). The
displacement of PD& by Ta-GTP bindhg activates PDE.
Enzymatic hydrolysis of cGMP by activated PDE decreases the cytoplasmic
concentration of cGMP which lads to the closure of plamsa membrane cGMP-gated
cation channels (Farber and Shuster, 1986). In the dark, cGMP binds to cation channels
of the plasma membrane thereby maintai~ng them open (Hargrave, 1986). Closure of the
channels results in the transient hyperpolarization of the photoreceptor plasma membrane.
The cation channels and ~ a ' - ~ a ~ ' exchanger are the principal ion transporters in the
photoreceptor (Hargrave, 1986). Hyperpolarization at the synaptic terminal of the
photoreceptor leads to a reduction in ca2' influx and subsequently decreases the rate of
neurotransxnitter release. As a result, the neurotransmitter is no longer capable of
inhibiting post-synaptic neurons dowing them to become excited and continuing the
signal through the retina.
In order to terminate the visual cascade activated rhodopsin is deactivated by
phosphory lation. Phosphorylation of rhodopsin is performed by rhodo psin kinase and
occurs at multiple serine/threonine residues at the C-temiinal end of rhodopsin (Hargrave,
1 986).
Phosphorylated rhodopsin, however, still retains some activity. To elirninate
residuai activation of the phototransduction pathway by rhodopsin, the phosphorylated
form is bound by arrestin. The binding of arrestin leads to the effective blockage of
transducin access to its binding sites on phosphorylated rhodopsin (Palczewski et al.,
1992; GureMch and Benovic, 1993). Arrestin dissociates fiom rhodopsin once the
potential binding capacity for transducin has been removed and the I 1-aff ~r<us-retinal has
been released. Binding of new 1 1-cis retinal restores rhodopsin and the process of arrestin
dissociation is dependent upon the regeneration of rhodopsin.
In addition, restoration of the dark state requires inactivation of transducin, an
increase in calcium concentration, and a retum to dark levels of cGMP concentration
(Farber and Shuster, 1986). Inactivation of Ta is accomplished by the hydrolysis of GTP
to GDP. Reessociation of Ta with TPy pennits the release of PDEy which retums to
PDEaP to restore its inhibitory state (Artemyev et al., 1998). Reopening of calcium
charnels through the binding of cGMP, which is synthesized fiom GTP by guanylate
cyclase, increases ca2' concentration (Hargrave, 1 986).
The concentration of calcium in the outer segment is thought to rnediate the
capacity of the visual transduction system to adapt to varying light intensities. ca2' ions
enter the outer segment by cGhdP-gated channels and are removed by ~ a ' l ~ a " , K'
exchanging pumps. The closure of cGMP-gated cation channels causes a cytoplasmic
decrease in ca2' which is detected by recoverin, a calcium binding protein. Recoverh is
thought to participate in visual transduction by preventbg the phosphorylation and thus
deactivation of rhodopsin (Enkson et al., 1998).
An interesting feature of the light transduction cascade is its abiiity to ampli@ the
signalling process. One activated, rhodopsin protein is capable of activating 500 G-
protein molecules. Furthemore, activation of PDE lads to the hydrolysis of 2000 cGMP
molecules per second. In effect, the absorption of a simgle photon causes the amplification
of its detection 106 fold (Bumside and Dearry, 1986).
Photoreceptor Outer Segment Structure
The photoreceptor outer segment is highly specialized for photon absorption and
phototransduction. It comprises of flattened membranous disks which are stacked on top
of one another. The most abundant protein in the disk membrane is rhodopsin which
represents 95% of the protein in bovine disk membranes (McDowell, 1993).
Morphological differences between rods and cones exist in their OS organüration. In
cones, disks are continuous with the plasma membrane (PM) while in rods the disks are
pinched off from the PM to fonn closed intracellular vesicles, except for those at the base
of the OS (Fig. 1-2) (Cohen, 1970). The continuity of disks and the PM in cones is
thought to reduce the siie of cone disks as they are displaced apically, resulting in the
typical tapered shape of the cone OS (Corless and Fetter, 1987).
Although the mature photoreceptor is not mitotic, it still undergoes the dynamic
process of disk renewal in its OS (Usukura and Obata, 1995). Primate rods contain
approximately 1000 disks making up 95% of the total md outer segment membranes with
the average rate of disk renewal calculated to be 10% per day (Young, 1976; Anderson et
ai., 1978).
The process of disk renewal is considered essential for PR cells in order to reduce
the accumulation of light darnaged components during time. As old disks are removed by
phagocytosis fkom the apical tip of PR cells by RPE cells, new disks are fonned at the base
of the OS. Thus, a balance is maintained so that the net length of the OS remains constant
(Besharse, 1986). The mechanism of disk morphogenesis is mediated by an actin-myosin
motor (Williams et al., 1992). Actin filaments are localized to within the ciliary axoneme
at the base of the rod OS by immunoelectron microscopy (Chaitin et al., 1984; Chaitin et
al., 1989; WiUiarns, 1991). The involvement of actin filaments in disk morphogenesis is
thought to be important because addition of cytochalasin D causes their depolymenzation
and perturbs disk formation (Vau& and Fisher, 1989). Recently, the product of the
Usher 1B syndrome gene was identified to be myosin VIIa and was determined to be
concentrated at the connecting cilia of PR cells (Kubota et al., 1997; Liu et al., 1997).
The role of myosin W a in photoreceptor biology is currently unknown, but it is thought
to maintain a barrier against difision of proteins between the imer and outer segments of
the PR (Liu et al., 1997).
Outer Segment Disk Rim Proteins
The vertebrate outer segment disk can be divided into two domains: 1) the central
lamellae, and 2) the highly curved domain refend to as the disk rim (Corless and Fetter,
1987). One of the key differences between the disk rim and lameliar regions is the
distribution of proteins in the two domains. For example, the integral membrane protein
rhodopsin is only present in the lamellar region, and is excluded fiom the disk rims
(Corless and Fetter, 1 987). Despite advances in understanding the molecular events
involved in the light transduction pathway, the components which provide the structural
integrity of the photoreceptor outer segment are less understood. The cloning and
characterization of RDS, ROMl and recently ABCR has highlighted the importance of
proteins located only at the disk rims in the photoreceptor outer segment. The fùnction of
ABCR is unknown, however, it is a member of the ATP transport superfarnily based on
protein sequence analysis (Sun and Nathans, 1996). In the cases of ROMl and RDS, the
roles of these two disk rim proteins has been greatly advanced by biochernical and genetic
analyses.
ROMl and RDS/Peripherin
ROMl and RDS are both integral membrane proteins which localize to the nms of
the rhodopsin containhg disks which comprise the outer segment (Bascom et al., 1992).
The two proteins are 35% identical at the amho acid Ievel. Despite the low degree of
identity between ROMl and RDS, the two proteins share similar structural characteristics.
Hydropathy plots for both ROMl and RDS reveal that both proteins contain four
transmembrane (TM) domains and a large hydrophilic stretch of amho acids (-140 aa)
between the third and fourth TM regions (Bascom et al., 1992). Furthemore, membrane
topology analysis on ROMl in microsomal membranes in vitro verined that ROMl is a
transmembrane protein with cytoplasmic N and C-te& and that the large central
hydrophilic loop lies within the lumen of microsornes (Bascorn et al., 1992). Interestingly,
the membrane topology of ROMl is consistent and comparable to that of RDS (Co~el l
and Molday, 1990). In addition, both proteins are comparable in site (37 kDa and 39.1
kDa, respectively ).
Polypeptide sequence analysis on ROMl and RDS hiflght fiirther similarities
between the two proteins. The most conserved region between ROMl and RDS occurs in
their hydrophilic loops (49% identity) with seven of seven cysteines present in this region
being conserved (Bascom et al., 1992). In addition, there is a stretch of arnino acids
(15/16) in the hydrophilic loop which is highly conserved and cornposed mainly of
cysteines and prolines (Fig. 1-3). There are also two other regions present in ROMl and
RDS which show consenration. The predicted junctions between the N-terminus and
TM1 as well as between TM4 and the C-terminus are identical between ROMl and RDS
(Fig. 1-3). These two regions of identity, each seven arnino acids long, are of unknown
fùnction, but could be required for important protein-protein interactions.
In contrast to the similarities between ROM1 and RDS, there are signifiant
differences to note which may have functional implications for the two proteins. For
instance, there are differences between the charges, and consequently the isoelectric points
of the two proteins, at the N and C-termini. The N-terminus of ROMl is absent of
negatively charged residues unlike RDS which does not appear to show an
underrepresentation of such amino acids (Bascom et al., 1992). With respect to the C-
terminus, RDS has been noted to be highly charged. ROMl, however, is only modestly
negatively charged in this region. Overall the theoretid pI of RDS, not accounting for
glycosylation, is 5.3 which is considerably lower than that of ROMl (5.98) (Boesze-
Battaglia et al., 1997). Finally, there are no observed Klinked glycosylation sites in
ROMl whereas one such site is present in RDS (Comell and Molday, 1990).
Associations behveen ROMl and RDS
Various independent experiments have been perforrned to show that ROMl and
R D S are capable of fonning disulphide-lied homodimers and non-covalent multimers
(Bascom et al., 1992; Goldberg and Molday, 1996; Goldberg et al., 1998). Bascom et al.
(1992), for instance, provided evidence of ROMl diaulphide linked homodimers with non-
Cytoplasm
Disk Membrane
Disk Lumen
PXXC
Figure 1-3. Structurai topology of ROM1. Amino acids conserved between RDS and ROM 1 are shown as open circles while substitutions are indicated by closed circles. The four domains that are identical or near identical between ROM1 and RDS are shaded in grey. Also shown are the motifs, referred to as the CCG, PXSC, PXXC, and ECG, in the hydrophiiic Ioop which are present in members of the TM4SF (Tomlinson and Wright, 1994). Each letter of the motifs stands for an arnino acid (C = cysteine, G = glycine, E = glutamate, P = proline, and X = any amino acid). Modified from R. Bascom.
reducing and reducing SDS-PAGE. ROMl and RDS non-covalently associated
heterotetrarners were identified through an anti-RDS monoclonal Ab 2B6-Sepharose
affinity column passed with detergent solubilized bovine ROS membranes (Bascorn et al.,
1992).
Recently, Goldberg and Molday (1998) have evaluated the ability of the 13
cysteine residues present in bovine RDS to form disulphide-linked homodimers, associate
with ROMl, and assemble into tetramers by replacing each cysteine with a senne residue.
Mutations involving the six non-conserved cysteines had no effect on dimer formation,
folding or subunit assembly . However, re placing any of the seven conserved cy steine
residues, al1 of which are present in the hydrophilic loop, dismpted these properties. Six
of the seven cysteine residues (including a C214S mutant linked to autosoma1 retinitis
pigmentosa) when altered a k t e d the normal folding of RDS, interactions with ROMl
and self-assembly into homotetramers. The seventh cysteine residue when rnutated
(C 1 SOS) did not affect the association of RDS with ROM 1 . However, it did incapacitate
the ability of RDS to fonn intennolecular disulfide bonds (Goldberg and Molday, 1998).
ROM1 and RDS are memben of the transmembrane 4 superflmily (TM4SF)
The structural similatities observed between ROMl and RDS have been observed
recently in a number of proteins, many of which are leukocyte cell-surface molecules.
Interestingly, each protein has the same membrane topology as that observed for ROMl
and RDS (Table 1-1). Currently, there are 19 TM4SF members which are found in
organisms fiom schistosomes to humans (Table 1-1).
The basic architecture of a TM4SF member is the presence of four hydrophobic
TM domains, cytoplasmic N and C-tennini and a large extracellular domain between TM3
and TM4. The membership of ROM1 and RDS into the TM4SF has been somewhat
controvenial due to the fact that they both contain long cytoplasmic C-terminal tails (65-
70 amino acids) which is uncharacteristic of other TM4SF members (5 -14 amino acids)
(Wright and Tomlinson, 1994). In addition, the hydrophilic loops of ROMl and RDS are
not extracellular but in fact protrude into the lumen of OS disks. It should be noted,
however, that the lumen of OS disks is equivalent to the extracellular environment
Table 1-1. Transmembrane four superfamily membus (TMMF).
Bascom et al., 1992
Protein
ROM1
Expression Pattern Rod photoreceptors, Ou ter Segment disk rims
Peri p heri n i RDS
XRDS3S distant relative of RDS
XRDS36 distant relative of RDS
Size (kb/ae and/or B a ) 37 kDa
Rod and Cone photoreccptor outer segment disk rims
Rods only
Rods only
Function
rnaintaining outcr segment stiucnirc
39 kl)a
maintainhg outer segment structure
345 aa
crds2 (chick)
Uropfakinla
Othtr Points
interacts with RDS non- covalently
345 aa
low expression in photoreccptors
bladder
364 aa
rnaintainiag outcr segment stnicturt and function
forms higher- order complexes with XRDS3S and
27 kDa
developing B cells, platelets, neuroblastoma cell lines, activated T cells, neurons, ocular ciliary epithelial cells
rnaintaining outer segment structure
Kedzierski et al., 1996
not known
Àsyrnmetrical Unit Membrane (AUM) prevents bladder
samc as Uroplaskin Ia
interacts with ROM1 non- covalently
avart of
signal transduction in platelets, cell adhesion, ce11 motility
Travis et al., 1991
forms higher- order complexes with XRDS38 and
expression scen in the retina at
Uroplaskin 11 (15 D a ) oligomerizes
1 interacts with Uroplaskin III (47 kDa)
Kedzierski et al., 1996
Weng et al., 199 8
embryonic &y 18 interacts with
Wu et al:, 1995; Finch ci al., 1997
associates with alpha 3, 4, 5, 6 and Bcta 1 integrins, CD19
Yu et ai.. 1994;
Yu et al., 1994; Wu et al., 1995; Finch et al.,
ubiquitious: heart, spleen, kidney, lung, brain, liver, testis, skeletal muscle
1997 Loffler et al., 1997; Bcrditchevski et al., 1996; Shaw et al., 1995; Banejee et al.. 1997; Martin- Alonso et al., 1992; Horvath et
26 kDa Imai et al., 1993;
, Mannion et al., 1996; Seldin et
1 al., 1995;
signal transduction in B cells, ceIl adhesion in B, T and non- lyrnphoid cells
component of B ceIl signalling cornplex CD19KD211CDS l ,neu-13 associates with CD63, P14K and
:O-O29
Tomlinson et al., 1996
2D37
rectal & colorec- tal carcinomas mature B cells low expression in T celis lymphoid tissues
32 kDa
281 aa 1.2 kb
not known
downrcguiated on B ce11 activation role in B ceIl poliferation
see CD82
associates with CD53, CD81, CD82, MHC class II, CD19, CD21 in B ccils
Sala et al.. 1990
Table 1-1 cont. Transmernbrane four superfamily members (TM4SF).
Protein
SAS
CD63ME49 1
AIS-
SFA- 1PETA-3
TI- 1
low expression iri
1 heart
spleen, thymus, heart, lung, kidney lower expression B and T cells, thymocytes number of tumour cell lines platelets, mclanosomes, lysosomes high in kidncy B, T and non- lymphoid cells ocular mclanoma tissues Schistosoma mansoni
Antigen
hem, lung, pancreas & prostate tissue epithelial cclls
human lung, breast, colon, ovarian carcinomas & heaithy epithelial tissue
1 Fonction
signal transduction in B cells
signal transduction in B cells, monocytes, granuloctyes, rat macrophages, NK and T mlIs growth remlation? growth regdation cell motility may link P14K ta alpha3, Betal integrin
not known
not known
metastasis signal transduction? involved in growth arrest
not known
Otber Points
associates with CD37, CD53, CD8 1, MHC ciass II, CD19 & CD21 in B celis & CD4 CD8 & CD81 in T cells expresscd on early CD4" CD&' cells immunoprecipitai es with phosphatase 12q13-14 hurnan
associates wi th alpha3 and Betal and alpha6 and Bctal intcgrins
most homologous to
llp15.S human
negati vcly ngulated by TGF- Beta
most distant relative of 4TM superfamily 3q21-25 human
Reterences
Adachi et al., 1996; Nagira et al., 1994; Imai et al., 1993
Tomlinson et al., 1993; Wright et al., 1993; Carmo et al., 1995; Tomlinson et al., 1995 Jankowski et al., 1995 Wang et al., 1992; Gwynn et al., 1996; Radford et al., 1997; Radford et al., 1996
Wright et al., 1990
Emi et al., 1993; Vinaneva et al., 1994 Hasegawa et al., 1997; Fitter et al,, 1995 Kallin et al., 199 1
Maruyama et al., 1996; Kurihara et al., 1997
(Hargrave, 1986). ROMI, RDS and their homologs may, in fact, represent distant
relatives of the characteristic TM4SF members (Wright and Tomlinson, 1994).
Furthermore, in addition to their structural topology, three other reasons strengthen their
incorporation into the TM4SF. First, both molecules contain three cysteine motifs in their
hydrophilic loops, a characteristic shared in aimost al1 TM4SF members (Fig. 1-3).
Second, uroplakins ta and lb, which are not leukocyte proteins, are restricted in location
and function to a terminally differentiated structure callea the asymmetric unit membrane
in the mamrnalian bladder epithelium. In the case of ROMl and RDS their functions and
locaiization are restricted to the photoreceptor OS in the adult retina. Third, many
TM4SF members participate in interactions with one another, as is the case for ROMl and
RDS (Table 1-1, see the column Other Points).
oni il'" mouse, implications on photomeptor structure and function
ROMl and RDS are related proteins that are physically associated at the disk nm.
The colocalization of ROMl with RDS at the disk rims of the outer segment, where they
are thought to be pan of the disk rim protein complex, suggests that both these proteins
may play a key role in outer segment function a d o r structure. Complete loss of RDS
function has dramatic consequences - (a) no outer segments form, and @) there is ce11
death (Travis et al., 1991a). In addition, RDS heterozygotes have - (a) disorganized outer
segments, @) larger-than-normal disks, and (c) ce11 death (Travis et al., 199ia). To
examine the effect of ROM1 on photoreceptor structure and function a mutant mouse
which is homozygous for the Roml allele was constmcted in our lab (G. Clarke,
unpublished).
Interestingly, a complete loss of ROMl function is associated with a less severe
phenotype than the complete l o s of RDS. Homozygous Roml mutants resemble Rds
heterozygotes, having - (a) disorganized outer segments (Fig. 1-4), (b) larger-than-normai
disks, and (c) ce11 death (G. Clarke, unpublished). The formation of large disks in the
R m l homozygous mutant animais demonstrates that ROMl is critical for the termination
of disk biogenesis, a property it shares with RDS (Travis et al., 1991a). Roml mice
heterozygous for a loss of fùnction dele, on the other hand, have Wtually nomal
Light micrographs of retinal sections from 1 month old ~oml+l+ and ~oml- l ' mice. Outer segments arc f m e d in the ~ornl-'- mouse, but are disorganized. All other layen appear normal. RPE - retinai pigment epithelium, OS - outer segment, IS - inner segment and ONL - outer nuclear layer. A lOOx magnification of the neuroretina is shown. Courtesy of G. Clarke.
photoreceptors (G. Clarke, unpublished). Altogether, these findings suggest that the
ROM1 protein plays a less criticai role than RDS in the biogenesis of the outer segment
and in disk morphogenesis, although it is essential to the mamrnaiian photoreceptor. The
different roles ROM1 and RDS have may reflect diaerences in the protein-protein
interactions in whidi these two proteins participate.
Initial characterization of PERl
The identification of retinal specific, abundantly expressed, and conserved (SAC)
cDNAs for genes has been an important step in understanding the structure, function, and
development of the mammalian eye. Two SAC cDNAs which have been cloned in the
McInnes lab are ROM1 (Bascorn et al., 1992) and CHXl O (Li et al., 1994). PHRl was
also identified in the McImes lab as an abundantly expressed and conserved retinal gene.
However, PHRl is also expressed at lower levels in the brain, kidney, liver and lung
(McInnes and Valle, unpublished). The PHRl gene has been mapped to 1 1 q 13.5- 14.1 by
FISH, oncor mapping panel and radiation mapping panel (Taylor and Mches,
unpublished).
The PHRl gene has nine exons, with exons 3-6 coding for a pleckstrin homology
(PH) domain (Fig. 1-5) (hicimes and Valle, unpublished). All PHRl transcripts encode
proteins that contain a PH domain at the N-terminus and a transmembrane (TM) domain
at the C-terminus. In the retina, two PHRl tninscnpts have been identified. The larger
transcript contains al1 nine exons (-729 bp) whereas the smaller transcript originates fiom
the sarne promoter as the full-length transcript, but is altematively spliced resulting in the
removal of exon seven. Two brain specific transcripts which are produced from an
alternative promoter located in intron have also been identified by RT-PCR (personal
communication, S. Xu) (Fig. 1-5).
The full-length PHRl predicted protein consists of 243 arnino acids (Fig. 1-6).
The PH domain is encoded by amino acids 22 to 127. Exon 7 encodes 35 amino acids
(13 1-165) which are immediately C-terminal to the PH domain (Fig. 1-6). A
transmembrane domain (amino acids 124 to 243) is present at the C-terminal end of PHRl
(Fig. 1-6).
Exons coding for the PH domain
Figure 1-5 . Genomic structure of PHRI. The gene has 9 exons, represented as boxes, one of which, exon 7 (shown in purple). is alternatively spliced. The PH domain is encoded by exons 3 to 6, as shown in red gradient boxes. The brain specific transcripts of PHRI are indicated by the pink lines. Brain specific franscripts are transcribed from a brain specitic promoter located in intron 2, as indicated by the B 1 box.
PHRl contains a region of similarity to seven characterized PH domain-containing
proteins: Akt (30% identity), cytohesin-1 (2g0/0 identity), GRPl (29% identity), ARNO
(29% identity), Cdc25 (23% identity), dynamin (24% identity), and oxysterol binding
protein (OSBP) (24% identity). The region of similarity between Akt (Fig. 1-7),
cytohesin- l , GRP 1, ARNO, and Cdc25 and PHRl is present only at the N-terminal part of
the PH domains of these proteins and ranges in length fiom 59 - 74 arnino acids. The
region of similarity between dynamin (Fig. 1-7) and OSBP and PHRI, however, occurs
over the entire PH domain.
Pleckstria homology domain
In 1993 two groups identified a family of sequences, of approximately 100-120
amino acids, which were originally discovered in pleckstrin, a major protein kinase C
(PKC) substrate in activated blood platelets (Tyers et al., 1988; Mayer et al., 1993;
Haslam et al., 1993). To date, more than 100 proteins involved in a variety of cellular
signalhg and cytoskeletal functions have been observed to contain PH domains. Many of
these proteins cm be grouped into seven distinct finctional categones: 1) senne/threonine
kinases, such as Akt/RAC, and PARK, 2) tyrosine kinases, like Btk, 3) regdators of small
G proteins, such as Ras-GAP, SOS1 and 2,4) endocytotic GTPases, like dynamin-1 and - 2, 5) adaptors, including IRS- 1, and 3BP2, 6) cytoskeletal associated molecules,
particularly spectrin, and pleckstrin and 7) lipid associated enzymes, such as PLC isoforms
(Shaw, 1998). In the majonty of cases, proteins containing PH domains have one copy of
this motif, however two copies of the PH domain have been identified in severai proteins
including pleckstnn, GRF and syntrophin.
Arnino acid alignments of the PH domains frMn diierent proteins reveals that low
identity exists in the PH domains of these molecules, fiom 10-20% (Lemmon et al., 1997).
The only invariant residue of the PH domain is a tryptophan residue in the C-terminal a-
helix. This residue is beiieved to be important for the structural stability of the domain
(Rebecchi and Scarlata, 1998) (Fig. 1-7).
The PH domain is an evolutionarily conserved motif that is present in a large
number of molecules ranging fiom yeast to human, suggesting an important biological role
Met Ser P r o A l a Ala Pro Val Pro Pro Asp Ser Ala Leu Glu Ser Pro
Thr P r o A l a Pro A l a Gly Ala T h r Val Pro Pro Arg Ser A r g Arg Val
Cys S e r Lys V a l Arg Cys Val Thr Arg Ser Trp Ser Pro Cys Lys Val
Glu Arg Arg X l e Trp Val Arg Val Tyr Ses Pro Tyr Gln Asp Tyr Tyr
Glu Val Val Pro P r 0 Asn Ala H i s Glu Ala Thr Tyr Val Arg Ses Tyr
Tyr Gly Pro Pro Tyr Ala Gly Pro Gly Val Thr H i s Val Ile Val Arg
Glu Asp Pro Cys Tyr Ser Ala Gly Ala Pro Leu A l a MetFly Met Leu
Aïa Gly Ala Ala Thr Gly Ala Ala Leu Gly Ses Leu Met Trp Ses Pro
Figure 1-6. The predicted PHRl protein sequence. Deduced primary amino acid sequence of PHRI. The PH domain is highlighted in blue while the amino acids encoded by the al tematively spliced exon 7 are shaded in yellow. Possible sites of protein-protein interaction are underlined. Predicted C-terminal transmembrane domain is bracketed.
Figure 1-7. Cornparison of PH domains. A. A cornparison between the PH domains of the dynamin family (DYN 1 and DYN2) from human, rat and mouse for DYNl and hunian for DYN2 aligned with the entire PH domain fro~n PHR 1. B. The PH domains of the AKT orthologs frorn mouse, bovine rat and human are aligned with the N-terminal pan of the PH domain from PHR 1. Amino acids shown in red and listed as the consensus sequence are identical between PHRl and AKT or dynamin. Amino acids shown in blue are conserved whereas amino acids shown in grey are not conserved. The first 63 amino acids of the PH doniain from PHRl are 30% identical to those of AKT whereas the entire PH domain of PHR 1 is 24% identical to the dynamin n~olecules (Altschul et al., 1997). The only invariant residue (W - tryptophan) between PH domains from differeiit inolecules is iiidicated by the arrow.
for this motif. In addition, many enzymes, such as Akt, Dbl, Vav, Bcr and Rac kinase
(Ingley et al., 1995) possess PH domains and have critical regdatory îùnctions.
Mutations in such proteins are implicated in oncogenesis and developmental disorders
(Ingley et al., 1995). In one of the most well-studied examples, mutations have been
found to cluster in the PH domain of Btk, a protein required for B-ce11 development and
poliferation. These mutations are responsible for some types of human X-linked
agarnmaglobulinemia ( K A ) and m u ~ e X-Linked irnmunodeficiency (Xid) (Rebecchi and
Scarlata, 1998). Interestingly, single amino acid substitutions in the PH domain of Btk,
corresponding to mutations found in XLA, have been associated with distinct fùnctional
defects. Two such mutations, R28C and E41K have been implicated to reduce or
enhance, respectively, the binding of Btk to phosphatidylinositol phosphate (PtdIns)
groups (Fukuda et al., 1996; Kojima et al., 1997).
PH domain structure
Currently, the structures of the PH domains of pleckstnn (N-terminal), p-spectrin,
dynarnin-1, PLC 6,, Son of sevenless (SOS1 - both human and murine), Btk and PARK
have been solved to high resolution (Rebecchi and Scarlata, 1998). In two cases, PLC 6 ,
and P-spectrin, the PH domains were determined in the presence of phosphatidylinositol
4,5-biphosphate (NP2) (Lemmon et al., 1997).
Although the primary sequence of PH domains are loosely conserved, the structure
shows a remarkable conservation of three dimensionai organization. The structure
consists of a pair of nearly orthogonal beta sheets comprised of four and three antiparallel
strands, forming a j3-sandwich. The P-sandwich is closed onand stabilkd at one corner
by a C-terminal amphipathic a-heh (Cohen et al., 1995; Lemmon et al., 1997; Rebecchi
and Scarlata, 1998). Moreover, each PH domain is electrostaticdy polarized. Typically
the positively charged face of the domain coincides with the hypervariable loops (Lemmon
et al., 1997). Loops separating the P-strands are variable in both length and sequence.
Furthemore, they can tolerate large insertions. For instance, the PH domain of PLCy is
split at the P3434 loop of its PH domain by three src homology domains (Rebecchi and
Scarlata, 1 998).
Structural studies pedomed on PARK and SOS 1 have reveaied severaî interesting
features of PH domains. In the case of PARK, a particularly unique aspect of its PH
domain is the presence of an extended C-teminal a-helix that behaves as a molten helix
required for protein-protein interactions (Fushman et al., 1998). The SOS 1 protein
contains a Dbl homology P H ) domain involved in GTP binding. Al1 proteins containing a
DH domain also contain a PH domain. In fact in al1 cases the DH domain is foflowed
imrnediately C-terminal by the PH domain. Recently, NMR studies on the DH-PH
domains in SOS1 indicated that critical interactions between the two motifs occur at the
most poorly conserved regions (Soisson et al., 1998). In addition, the PH domain retains
its characteristic three dimensional fold despite the nearby presence of another modular
domain, indicating that the PH domain is structurally stable (Soisson et al., 1998).
PH domain runction
Identifying PH domain ligands has largely involved in vitro studies exarnining
possible protein-protein or protein-lipid interactions. Based on structural similarities
between PH domains and retinol binding protein, it was speculated that PH domains may
be capable of binding to lipophilic molecules (Harlan et ai., 1994). To examine the site of
interaction between the PH domain of pleckstnn and vesicles containing PtdIns(4,5)P2, 'Il, 13 C, and 1 5 ~ chernical shifis were followed as a finction of added iipid ("Harlan et al.,
1994). Residues K13, K14, S16, V17, N19, T20, W21, K22 and G46 were al1 detennined
to be involved in PtdIns(4,5)Pz binding. interestingly, Harlan et al. (1 994) noted that al1
residues involved in lipid binding were located in the N-terminal region of the PH domain.
Furthemore, substitution of each lysine residue for asparagine caused mutant pleckstnn
molecules to exhibit a -1 0 fold loss in binding afanity for PtdIns(4,5)Pz indicating that the
positively charged lysines are required for interacting with the negatively charged
phosphates (Harlan et al., 1995). Further evidence has also been collecteci fiom the X-ray
crystai structure of the PH domain fiom PLCG1 complexed with PtdIns(l,4,5)Pi. The
solution of this complex revealed that the positively charged face of the PH domain is
required for interacting with phosphatidylinositol phosphates (Ferguson et al., 1995).
The specificity of interaction between PH domains and phosphoinositides has also
been shown through comparative binding analysis of PH domains from different proteins.
Such studies utilize 3~-labelled phosphoinositides or examine quenching of intrinsic
tryptophan fluorescence afler binding. For instance, Akt has been observed to interact
with PtdIns(3,4)P2 and PtdIns(3,4,5)P3 specificaily, whereas dynamin is capable of
associating only with PtdIns(4,5)P2 (Frech et al., 1997; Rameh et al., 1997; Hirata et al.,
1998; Barylko et al., 1998).
The involvement of the PH domain with phosphatidylinositol phosphates suggests
that a role of this motif is to recruit proteins to the membrme, thereby targetting the PH
domain-containing molecules to the appropriate cellular cornpartment. Evidence for the
plasma membrane association of PLCG1 and PLCy has been obtained by
immunofluorescence studies using recombinant proteins in Cos and MR33 cells,
respectively (Paterson et al., 1995; Falasca et ai., 1998). Positively charged N-terminai
residues in the PH domain of PLCGi have been altered to show that they have a critical
role in phosphatidylinositol phosphate binding (Yagisawa et al., 1998). The plasma
membrane recruitment of pleckstrin and spearin has also been assessed by
immunofluorescence (Wang et al., 1995; Wang et al., 1996; Ma et al., 1997). In the case
of pleckstrin, Ma et al. (1997) have shown that pleckstrin is required for the formation of
membrane projections in transfected Cos-1 cells, indicating that the recruitment of
pleckstrin to the membrane is of structural and functional significance.
The specific binding of phosphatidylinositol phosphates to the PH domain of
proteins such as Akt and GRPl has been associated with the function of these molecules.
Binding of PtdIns(3,4,5)Pi, but not PtdIns(4,5)P2, makedly enhanced the guanine
nucleotide exhange activity of GRPl (Klarlund et ai., 1998). Activation of the kinase
domain of Akt requires the association of its PH domain with PtdIns(3,4)Pz7 and not
PtdIns(3 ,4,5)P3 (Klippel et al., 1997; Franke et al., 1997).
As with protein-lipid binding, protein-protein interactions are another major source
of signal transmission in various intraceliular signalling cascades. Associations between
PH domains and proteins such as PKC and G protein Py-subunits have been observed.
The ability of the PH domains of Btk and RAC-protein kinase to bind to PKC has been
demonstrated both in vivo and in vitro (Yao et al., 1994; Yao et al., 1997; Konishi et al.,
1996). The functional consequence to Btk when PKCP binds to it is that Btk becomes
phophorylated (Yao et al., 1994). As a result of phosphorylation by PKCB, the activity of
Btk is dom-regulated. In addition, Yao et al. (1994) have exarnined the effm of PKCP
binding with altered fonns of Btk. The GST-Btk(xid) protein with the Ala28Cys mutation
showed lower PKCP binding, suggesting an essential need for the arginine residue in Btk-
PKCP association. Reduced Btk-PKCP binding may in fact have a major impact on the
disease manifestations of Xid rnice by reducing B-cell poliferation and, thus the immune
system. In the case of RAC-PK, binding has been observed in vitro between its PH
domain and the PKCG isofonn resulting in the phosphorylation of PKCG (Konishi et al.,
1996). The association of the PH domain of RAC-PK and PKCG was found only aîter
cells were heat-treated, suggesting that RAC-PK may participate in the cellular response
to stress through its PH domain (Konishi et al., 1996). Interestingly, the region to which
PKC isofonns bind to the PH domain has been shown in vitro to map to the amino-
terminal portion of the PH domain of Btk (Yao et al., 1997). In other proteins this region
of the PH domain also binds phosphatidylinositol phosphate groups. Yao et al. (1997),
suggest that molecules such as PtdIns groups, which bind to the N-terminal portion of the
PH domain, may act to regulate the binding of the PH domain to PKC isoforms.
The ligand-induced activation of many receptors leads to the dissociation of the
py-subunits from the a-subunit of heterotrimeric G proteins, both of which regulate many
different effector molecules involved in signal transduction. In many cases, proteins
involved in intracellular signal cascades contain PH domains. In vitro studies involving
GST fusion proteins containing PH domains fiom several signalling molecules such as
Btk, Akt, oxysterol binding protein, PARK, IRS-1, and PLCy have been observed to
interact with GPy subunits (Touhara et al., 1994; Konishi et al., 1995). Studies involving
truncated PH domains have indicated that the critical region for interaction with GPy
subunits includes only the C-terminai portion of the PH domain and sequences just distal
to it (Touhara et al., 1994). Furthemore, GPy subunits are known to contain W 4 0
repeats, a 40 amino acid motif with multiple trp-asp dipeptides. Interactions between the
PH domains and WD40 repeats have been noted in vitro, suggesting that the WD40 motifs
are important for protein-protein associations with PH domains (Wang et al., 1995).
Recently, the IRS-1 and IRS-2 PH domains have been show to bind to acidic
motifs in proteins (Burks et al., 1998). Using the yeast two-hybrid system and the PH
domains of IRS-1 and iRS-2 as baits, Burks et ai. (1998) identified three proteins Lon
protease, myeloblast protein, and nucleolin. These results are the first exarnple of the
identification of PH domain interacting proteins using the yeast two-hybnd system.
Although the roles of these molecules in insulin action are not known, each protein
contained an acidic motif of aspartate and glutamate residues capable of interacting
specifically with the PH domain of IRS-2. Only the acidic motif present in nucleolin
bound to IRS- 1, suggesting that the PH domains of IRS- I and IRS-2 are not identical. By
using peptides containing the acidic motifs identified in the two-hybrid system, Burks et al.
(1998) were able to show that binding of the acidic peptides dismpted IRS-1 and IRS-2
coupling to the activated insulin receptor.
Understanding the function of the PH domain has relied mostly on in vitro studies.
Analysis in vitro has dissected the PH domain into two functional components. The N-
terminal region of the PH domain is required for phosphatidylinositol phosphate and PKC
binding, whereas the C-terminal region is essential for G protein association. In vivo
experiments showing interaction between the PH domain of the Btk and G proteins, or
PKCP or c, have been obtained using co-irnrnunoprecipitation (Yao et ai., 1997; Jiang et
al., 1998). To further elucidate the significance of various protein-protein or protein-lipid
associations observed, more detailed in vivo investigations are required for this
biologically important domain. Subsequent in vivo analysis can help determine the
physiological relevance of observed interactions involving PH domains in terms of
functional effects on signalling pathways, cellular morphological changes or regdatory
events.
Identifying ROM1 and PHRI interacting proteins using the yeast two-hybrid
system
The two-hybrid system relies on the structure of particular transcription factors
that have two physically separable domains: a DNA binding domain and a transcriptional
activating domain. The DNA binding dornain targets transcription to specific promoter
sequences (UAS, upstream activating sequence), whereas the activation domain serves to
initiate transcription by facilitating the assembly of the transcription complex. The fact
that a fùnctional transcription factor can be reconstituted through non-covalent
interactions of two hybrid proteins, with one containing the DNA-binding domain and the
other containing the activation domain, is the basis of the two-hybrid system (Bai and
Elledge, 1996). The hybrid proteins are usually transcnptionally inactive alone or in the
presence of a non-interacting hybrid protein. However, if two interacting hybrid proteins
are co-expressed, a reconstituted transcription complex can be assembled at an upstream
activating sequence, thereby activating expression of a testable reporter gene (Bai and
Elledge, 1 996).
The two-hybrid system has three major advantages over biochemical methods such
as co-immunoprecipitation and chromatography in detecting protein-protein
interactions. First, the yeast two-hybrid system was designed to identify cDNAs encoding
proteins that physically associate with a given protein in vivo. Second, with the
development of cloning vecton such as pACTXI, large representative cDNA libraries from
a vanety of tissues can be constructed and screened efficiently. Third, this method of
identifjmg protein-protein interactions can be used to define or test the domain necessary
for the interaction of two proteins. For these reasons, 1 chose the two-hybrid system as
the method to identify putative ROM1 and PHRt interacting proteins.
CHAPTER 2
Identification of ROM1-interacting proteins using the yeast two-hybrid system
The vertebrate photoreceptor cells are unique neurons, both stmcturaily and
functionally (Besharse, 1986; Bumside and Dearry, 1986). In particular, the
photoreceptor outer segment is a highly specialized component of the photoreceptor. In
primates, the outer segment is composed of flattened disks stacked one on top of the
other. Each disk is independent and separated from the plasma membrane in rod
photoreceptors, whereas in cones the disks are exposed to the extracellular environment
(Besharse, 1986). Each disk is composed of a rim and a centrai region called the lamella,
the latter containing the majonty of the proteins involved in the phototransduction
pathway (Farber and Shuster, 1986). The nms have generaily been thought to have only a
structural role, seMng as points of attachent for proteios to the plasma membrane and to
other neighboring disks.
Presently, only three disk rim proteins have been identified: ROMl, RDS and
ABCR. The ABCR protein is a member of the ATP transport farnily (Sun and Nathans,
1996), which generally mediate ion transport. Although, the ligand transported by ABCR
is not known, mice homozygous for mutant abcr show no outer segment abnormality at
least to two months of age (personal communication G. Trais) suggesting that ABCR
may have no critical structural role in maintainhg the integnty of the outer segments.
Outer segment structural roles for both ROMl and RDS, however, have been identified.
Mice homozygous for mutant R h fail to develop outer segments, even though an aborted
attempt at disk biogenesis is made, indicating that RDS is essential for disk formation
(Chaitin, 1991). Mice homozygous for mutant Roml, in contrast, do form outer
segments, but they are disorganized and contain enlarged disks (personal communication
G. Clarke), demonstrating that ROMl plays a criticai role in disk rnorphogenesis.
ROMl and RDS are related protehs, each capable of forming disulde linked
homodimers (Bascom et al., 1992). Moreover, interaction between ROMl and RDS
homodimers lads to the formation of non-covalent multimers (Bascom et al., 1992).
Both ROMl and RDS share the same structurai topology (Bascom et al., 1992). The
presence of four transmembrane dornains, two cytoplasmic t d s and a large loop between
TM3 and TM4 places ROMl and RDS in the four transmembrane superfamily (TM4SF)
(Wright and Tomlinson, 1 994).
There are currently 19 known TM4SF members, seven of which are expressed by
leukocytes. Generally, more than one TM4SF protein is present in the sarne cell, as is the
case for ROMl and RDS (Wu et al., 1995). In addition, almost al1 TM4SF members are
capable of interacting with another member of the superfamily (Table 1-1). Furthemore,
TM4SF members are also capable of interacting with other proteins. For example, CD9
and CD63 associate with alpha and beta integrins (Rubinstein et al., 1994; Nakamura el
al., 1995; Radford et al., 1996), uroplakin la and uroplakin 1 b interact with uroplakin II
and uroplakin III, respectively (Yu et al., 1994; Wu et al., 1995). Identifjmg protein
associations involving TM4SF members has generally relied on co-immunoprecipitation
expenments, however, characterization of the TM4SF proteins regions mediating the
obsewed interactions studied have yet to be determined.
ROM1 and RDS appear to be part of a multi-protein cornplex at the disk rim. In
order to elucidate the function of ROMl through the identification of proteins present at
the disk rim capable of interacting with ROMl besides RDS, a two-hybrid screen was
initiated. Identifjmg ROMl interacting partners is a first step to begin to understand the
biochemical role of ROMl in the outer segment of the photoreceptor. In addition, the
ROMl interacting proteins may in fact be essential disk rim components. Thus, mutations
in such important disk rim protehs may be the moleculai defect behind uncharacterized
retinal degeneration diseases.
Since ROMl is a transmembrane protein which traverses the disk membranes four
times, it cannot be used in its entirety as a bait in the two-hybrid system. For this reason,
only hydrophilic regions were chosen as bait to test for protein-protein interactions in the
two-hybnd system. Hydrophilic regions of transmembrane proteins have been used to
identiQ protein hteractors successfbily using the two-hybnd system (Bauch et al., 1998;
Shiratsuchi et al., 1998).
1 chose to do the first ROMl two-hybnd screen with the C-terminal tail for two
reasons. First, its placement in the cytoplasm offers a putative site for protein-protein
interactions with cytoplasrnic proteins present near or at the disk rims. Secondly, the C-
terminal tail is the lest conserved region between ROMl and RDS, suggesting that
unique ROM1-interacting proteins might be identifieci by using this part of ROMl as the
two-hybrid bait. In addition, there is a perfkctly conserved seven peptide sequence in
ROMl and RDS, present at the boundary between the Gterminal tail and the fourth
transmembrane domain (Fig. 1-3). This region of conservation between the two proteins
may be a site of protein-protein interactions.
The C-terminal tail of ROMl was fused to the G U binding domain and used as
a bait to screen an adult bovine oligo-dT retinal library. From the screen 1 have isolated
five different cDNAs which encode polypeptides capable of interacting specifically with
the ROMl C-terminal tail. Whether the interaction between ROMl and these proteins is
physiologically relevant will require confinning the interactions with direct in vitro
approaches such as afijnity chromatography and detennining their expression in the retina
by itt siiu hybridization.
EXPERIMENTAL PROCEDURES
PCR
The DNA encoding the mouse ROMl C-terminal tail (amino acids 285 - 35 1)
encoding DNA (Bascom et al., 1993) was amplified Rom plasmid DNA containing full-
length Rom1 cDNA cloned into pBIuescript KS (Stratagene). The primer set used in the
PCR reaction consisted of the 3 1 -mer 5'
GGAATTCCATATGGGTTTGCGGTATTTGCAG 3', which contained a NdeI linker and
the 26-mer 5' CGGGATCCCTAGGCCTCAGCTAGAAC 3', which contained a Pst1
linker. The components used in the amplification were: 10 mM Tris, pH 8.0, 50 mM
KCI, 1.5 mM MgCli, 0.01% gelatin, 10 pM of each (MTP, 50 ng of each primer, and 2 ng
of plasmid DNA. Folowing an initial denaturation at 98OC for 5 min, 2.5 U of PFU
polymerase (Stratagene) were added and the tube was sealed with mineral oil. Each PCR
cycle (total 30) was carried out as follows: a denaturation step at 9S°C for 30 sec, an
a~eal ing step at 59OC for 1 min, and an extension phase at 72°C for 2 min.
PCR products were purified using the Qiagen PCR purification kit according to
the manufacturer's specifications (Qiagen, CA). The ampliecl product was then digested
with NdeI and Pstl (BRL) at 37OC for 2 hrs and purified by a phenol:chlorofotm
extraction method (Sambrook et al., 1989).
Su bcloning
The two-hybrid vector used in the subcloning procedure was pASl
(CLONTECH). It was digested with NdeI and Pst1 (BRL) at 37OC for 2 hrs and
subsequently isolated and purified fiom a 1% TBE agarose gel in 1X TBE buffer using a
QIAEX II gel extraction kit (Qiagen, CA). Purifieci mouse ROM1 C-terminal tail cDNA
fragments were ligated into 100 ng of vector for a 1 :2 molar ratio of vector to insert with
1 unit of T4 ligase (BRL) in 1X ligase buffer. The ligation reactions were incubated
overnight at 14OC. DHSa competent cells (BRL) were transformed with haif of the
liçation reaction and plated ont0 LB agar plates containing 100 pg/ml ampicillin. Plates
were incubated at 37°C overnight.
Plasmid DNA from cultured transformants were prepared using the Qiagen
Plasmid Miniprep kit (Qiagen, CA). Restriction enzyme digestions were perfonned to
identiQ positive transformants.
Sequencing
Sequencing reactions were perfonned with the T7 dideoxy DNA sequencing kit
(Pharmacia) and [ 3 ' ~ ] d ~ ~ ~ (NEN) according to the protocol designed by the
manufacturer. Reaction products were separateci on 6% polyacrylamide gels
(acrylamide/N'N/ -bis-methy lene-acrylamide ratio 3 6.51 1, 0.053% APS, 0.052% Temed) at
80 W for 2-4 hrs. Gels were fixed with 10% glacial acetic acid and 10% methanol
solution, dried for Ihr at 80°C and exposed to XAR film (Kodak). The oligonucleotide
primer used to sequence the two-hybnd bait construct was 5'
TCATCGGAAGAGAGTAG 3'.
Transformation o f Y 190 with bait construct
The yeast strain Y 190 (CLONTECH) was transforrned with the ROMl C-terminal
tail bait construct using a slightly modified version of the CLONTECH procedure. An
ovemight culture of Y 190 was grown at 30°C in synthetic dropout (SD) media lacking
uracil until an ODm of 0.5-0.8 was achieved. The cells were pelleted at 3000 rpm for 5
min, resuspended in 1 ml of sterile double deionized H20 (ddHZO) and transferred into
eppendorf tubes. The resuspended pellet was then centriiùged at 14000 rpm for 1 min.
The supernatant was cleared and the remaining pellet was resuspended in 5 volumes of
1 M lithium acetate (LIAOC) to make the cells competent. In separate eppendorf tubes,
the following components were added: 300 pl of PEG rnix (40% PEG/ 1 X TE/ 1 X
LiOAC), 10 pl of 5 mglml denatured, single-stranded salmon spenn (Pharmacia), 3-5 pl of
plasmid DNA, and 100 pl of competent Y 190 cells. The transformation mix was vortexed
for 30 sec, shaken at 300 rpm for 30 min at 30°C, and then heat-shocked at 42OC for 20
min. Ceils were centrifùged at 14,000 rpm for 1 min, resuspended in 100 pl of ddH20 and
plated ont0 appropriate selective plates. Transformations were incubated for two days at
30°C.
Preparation of yeast protein Iysates
Overnight 10 ml cultures of the ROMl bait and pVA3 positive control (p53/GAL4
DNA-binding domain hybnd) in Y190 were grown in SD selection medium lacking
tryptophan to an ODW of 0.5-0.8 units. The bait vector, pAS1, contains the TRPl gene
for selection in trp- auxotropic yeast strains. Cells were pelleted and resuspended in 1 ml
of sterile dd&û containing 10 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma). The
resuspension was transferred to an eppendorf tube and centrifùged at 14000 rpm for 1
min. The supernatant was cleared and the pellet was resuspended in 3 vol of 3X laemmli
sample buffer (Sambrook et al., 1989) with 10 mM PMSF and 1 vol of acid washed glass
beads (Sigma). The cells were lysed by vortexhg for 30 sec and cooling on ice for 1 min
(six times), boiling for 5 min, placing sarnples on ice for 5 min and then centrifughg for 5
min at room temperature. The supernatant was transferred to a fresh eppendorf tube and
stored at -20°C.
SDS PAGE
Yeast protein lysates were resolved using 5% stacking and 10% separating
acrylamide gels, 0.75 mm thick, in the Bio-Rad Minigel system. The amount of yeast
lysate loaded per lane was 5-10 pg. Gels were stained with either Coomasie blue
(Sambrook et al., 1989) or the proteins were transferred to nitrocellulose for Western blot
analysis.
Western blots
Proteins were electrophoretically transferred to Hybond C nitrocellulose. The
transfer was performed at 4OC for 1 hr at 100 V in transfer buffer which consisted of 25
mM Tris, 192 mM glycine and 20% rnethanol. M e r transfer, the nitrocellulose blot was
washed with Tris-buffered saline (20 rnM Tris-HCI, 0.5 M NaCl, pH 7.2) including 0.05%
Tween-20 (TBST). Non-specific background was elirninated by blocking the blot with
5% milk solution dissolved in TBST for 1 hr at room temperature, followed by one 5
minute wash at room temperature with TBST. The blot was then incubated ovemight at
room temperature with anti-GALA anti-mouse IgGh monoclonal antibody (Santa Cruz)
diluted in 1% milk in TBST (1500 dilution). Unbound pnmary antibody was removed by
three 30 min washes with TBST, at room temperature. The nitrocellulose blot was
subsequently incubated for 1 hr at room temperature with the secondary antibody anti-
mouse IgG (whole molecule) peroxidase conjugate (Sigma) diluted 15000 in TBST with
1% milk. Excess secondary antibody was removed by three 30 min washes with TBST
solution at room temperature. The electrochemical luminesence @CL) detection system
(Dupont) was employed to visualize the protein bands after exposure to film.
Beta-galactosidase assays
a) Filter assay for P-galactosidase
Whatman filter papers were layered over agar plates containing appropriate
selection medium. Colonies to be assayed were streaked in small patches directly ont0 the
filters. The plates were incubated at 30°C for one day to allow the cells to grow. The
filters were then lifted out and placed ceIl side up in liquid nitrogen in order to penneablize
the yeast cells. M e r 30 seconds of exposure to liquid nitrogen, the filters were placed on
Whatman 3 MM chromotography paper in unused petri dishes, soaked with a Z buffer/X-
gal solution. The Z buffer (pH 7.0) contained 16.lgL NaZHPOJH20 , 5.5 g/L
NaH$04*H20, 0.75 g/L KCI, and 0.246 g/L MgSO1m20. From a stock X-gal solution
of 50 mg/ml, 10 pL was added to 2.5 ml Z buffer. The filters were then placed at 30°C
between 30 min to overnight.
b) Liquid culture assay using ONPG (O-nitro P-D-galactopyranoside) as substrate
Ovemight cultures of 5 ml were prepared in liquid SD selection medium and
grown to saturation. The next day 2 ml of the overnight cultures were transferred to 8 ml
of YPD and incubated at 30°C for 3-5 hrs with shakuig (250 rpm) until the cells reached
an OD600 of 0.5-0.8 units. On the day of the experiment, ONPG (Sigma) was dissolved in
Z buffer to a concentration of 4 mg/ml by shaking for 1-2 hrs.
M e r reaching mid-log phase, the cells were hawested by placing 1.5 ml of each
culture into eppendorf tubes, centrifûged at 14,000 rpm for 1 min, decanted and then
resuspended in 1 .S ml of Z buffer. The cells were vortexed and centrifbged again. The
supernatant was removed and the pellets were resuspended in 300 pl of Z buffer. To a
fresh microcentrifùge tube, 0.1 ml of the ce11 suspensions were added. The tubes were
placed first in liquid nitrogen for 1 min and then in a 37OC water bath for 1 min to thaw
the cells. This process was repeated three times to ensure ceii fiactionation. A blank tube
was also set up containing only 0.1 ml of Z buffer. To 0.7 ml of Z buffer 1.9 pl of P- mercaptoethanol was added. The Z buffer/P-mercaptoethanol solution was dispersai into
each reaction tube, and 160 pl of ONPG in Z buffer was then added to each tube. The
time it took for the yellow colour to develop at 30°C was measured. Once the yellow
colour appeared 0.4 ml of 1 M Na2C03 was added to the tube and the timer was stopped
for each reaction and blank tube. The tubes were centrifbged for 10 min at 14,000 rpm.
The supernatants were transferred to clan cuvettes. Samples were then read in a
spectrophotometer (Beckman DU-65) at an OD of 420 nrn relative to the blank. The
following equation was used to detennine the amount of activation present (Miller, 1972):
P-galactosidase units = 1000 x ODud(t x 0.5 x ODa)
where: t is the elapsed time in min
Two-Hybrid sçreen
The bovine adult retinal oligo-dT primed two-hybnd library was a gift from Ching-
Hwa Sung of Corne11 University. The two-hybrid protocol followed was designed by D.
Gietz (Gietz and Schietl, 1995). A 10 ml ovemight culture of Y 190 cells with the bait
plasmid was begun in SD lacking tryptophan (trp) and uracil (ura). The following
morning the culture was diluted into 300 ml SD lacking trp and ura and grown until an
OD, reading of 0.6 was achieved the next day. Two flasks containing 250 ml YPD and
50 ml of 300 mg& adenine were warmed at 30°C. The yeast culture was diluted into the
YPD media (total volume 500 ml) to yield a starting O D a of 0.15 and then grown for
four hours or until the ODsW reached 0.6.
The cells were centrifùged at 5000 rpm for 5 min. To the pellet, 25 mls of sterile
water was added and the pellet was disrupted. Resuspended pellets were combined into
one GSA bottle. A lithium acetate mix (40 ml) was prepared containing 100 mM LiOAC
and iX TE, and added to the resuspended ceils. The cells were centrifùged at 1500 rpm
for 5 min. Finally, the pelîeted cells were resuspended in 5 mls LiOAC mix to achieve
cornpetence. As a control for the two-hybrid screen, 100 pl of the LiOAC treated cells
were removed and to each aliquot was added one of the following: i) no DNA plated
ont0 SD -leu, -trp, -ura; ii) pGAD plated ont0 SD -leu, -trp, -ura; and iii) pGAD plated
ont0 SD -leu, -trp, -ura, -his with 30 mM amino-triazole (Sigma). The plasmid pGAD
contains the full-length GAIA protein cloned into pASl (CLONTECH). To the remaining
cells were added LOO pg of the retinal target library DNA and 5 mg of denatured salmon
sperm (Pharmacia). Retinal denved cDNAs were subcloned into the p ACT11 vector which
contains the LEU2 gene for selection in leu- auxotropic yeast strains. CeUs (0.5 ml
aliquots) were then transferred into 10 round bottom polypropylene tubes and to each
tube 3 rnls of PEG mix (40% PEG/lX LiOAC/lX TE) was added; tubes were inverted to
rnix the contents. The transformation mix was incubated at 30°C for 30 min and then
heat-shocked at 42°C for 20 min. Cells were centrifùged at 1500 rpm for 3 min, decanted
and resuspended in 2 mis of sterile water.
In order to obtain 1x10' transformants per 150 mm plate, 0.65 mls of the library
transformation was added to each plate containing SD -leu, -trp, -his with 30 mM arnino-
triazole (Sigma), for a total of 30 plates. To determine the transformation efficiency a
I / 100 fold dilution of the bait plus library was made and plated as 20, 100 and 500 pl
aliquots ont0 3 separate SD - leu, -trp plates. Library plates were incubated for 5-7 days
at 30°C while the control plates were grown for 2 days at 30°C. Colonies which appeared
much larger than background colonies were selected and placed onto fiesh SD -leu, -trp,
-ura plates and then tested for lac2 expression by filter f&galactosidase assays.
isolation of two-hybrid targets
From the two-hybrid screen, yeast transformants that were capable of growth on
-his 7 3-AT plates and were Lac' were subject to additional analysis. For this purpose,
the targets from each independent positive were isolateci. A 3 ml ovemight of each
positive transformant was inoculated into SD liquid media lacking leucine. Cells were
centrifbged at 3000 rpm for 10 min. Each pellet was resuspended in 200 pl of sterile
water and transferred into 1.5 ml eppendorf tubes. To each tube, 0.2 ml of yeast lysis
solution (2% Triton X-100, 1% SDS, 100 mM NaCI, 10 mM Tns-HCI pH 8.0, and 1 .O
mM EDTA), 0.2 ml phenol/chloroform/isoamyl alcohol (2924: 1) and 0.3 g of acid-
washed glas beads were added. Each tube was vortexed for 3 min to lyse the cells and
then centrif'uged for 10 min at room temperature. Supematants were transferred into fresh
eppendorf tubes. DNA was precipitated by adding 1110 volume 3 M NaOAC and 2.5
volumes 100% ethanol. Tubes were centrifùged for 10 min at 4OC and then decanted.
DNA pellets were washed with 70% ethanol, centrifùged for 10 min at 4°C and then dried
under a vacuum for 10 min. Yeast minipreps were resuspended in 50 pi 10 mM Tns-HCI
pH 8.0.
Yeast minipreps were cleaned using the QIAEX II gel extraction kit. Each
purified miniprep (5 pl) was transformed into MH6 E. coli cells (gift from B. Andrews)
which are ampicillin sensitive and have a defect in the leuB gene that can be complemented
by the LEU2 encoded plasmid gene. Transformed cells were plated on LB agar plates
containing 100 pg/ml ampicillin. Colonies isolated the foUowing day after growth at 37OC
were patched ont0 M9 minimal plates (Sambrook et al., 1989) lacking leucine. Cells
capable of growth after incubation of plates ovemight at 37°C contained the target
plasmid only. Target DNA was then isolated by a standard alkaline lysis procedure
(Sarnbrook et al., 1989) and sequenced to identify the target capable of interacting with
the bait in yeast. Target DNA was sequenced with a primer upstream of the insert 5'
CTACAGGGATGTTTAATACC 3'.
RESULTS
Construction of the ROMl C-terminal tail bait
To investigate the role of ROMl in photoreceptor structure and function, a two-
hybrid screen was initiated. The ability of ROM1 to interact with RDS has been well
analyzed, however, the disk rim cornplex, to which they both belong to, is still poorly
characterized (Bascorn et al., 1992). Further insight into the fbnction of ROMl requires
identification of the proteins with which ROM1 associates. In order to identifL ROMI-
interacting partners a two-hybrid screen was initiated with the C-terminal tail of ROMl.
The DNA fragment encoding the C-temiinal tail of mouse ROMl was subcloned i n - h e
into the bah vector pAS1. Thus, the resulting bait fiision protein consisted of the ROMl
C-terminal tail and GALA bhding domain (Fig. 2- 1 a).
Analysis of ROMl Gtenninal bait expression in the two-hybrid system
Before proceeding with the two-hybrid screen, 1 optimized the screening
conditions by establishing expression of the bait in yeast celis and determining the ability
of the bait fusion aione to cause activation of the reporter genes. The bait consmict was
initially transformed into the yeast strain Y 190. This strain contains two reporter genes
GAL4 Binding Domain
HA epitope Mouse Rom1 C-ter tail - 22 arnino acids
Protein Markers ( k W
Figure 2-1. A. The mouse ROM1 C-terminal tail two-hybrid constmct, referred to as MRC. The constmct contains the mouse C-terminal tail (66 amino acids) fused in-frame to the GALA binding domain including the seven amino acid peptide present at the junction beween TM4 and the C-ter tail. An HA epitope separates the two polypeptides. B. Western blot of yeast lysates expressing the MRC bait Yeast lysates were separated on a 10% SDS-PAGE. Proteins were transferred ont0 Hybond C, probed with anti-GALA antibody and visualized using the ECL system. Lanes 1 and 2 show MRC fusion protein migrating at 34 kDa. Lane 3 shows the positive control, murine p53 (amino acids 72- 390) migrating at 54 kDa (see arrow) and lane 4 negative conml (Y 190 alone).
(HL93 and lad) used to analyze bait-target interactions identified in a two-hybrid screen.
The HIS3 gene is under the control of the native GALl UAS, while the locZ gene is under
the control of a synthetic UA& 17-mer consensus sequence (Bai and Elledge, 1996). The
upstream activation sequences are sites that the GALA binding domain can recognize. It is
important to note that the lac2 reporter gene was integrated into the Y190 yeast genome
along with a uracil marker (Bai and Elledge, 1996). Thus, maintenance of the lac2
reporter gene requires growing cells in the absence of uracil.
Western blot analysis of yeast lysates was performed in order to confirm that the
bait was appropriately expressed (Fig. 2-lb). Using an anti-GAL4 antibody, the mouse
ROMl C-terminal tail-GALA fusion protein (Fig. 2-1 b lanes 1 and 2), as well as a positive
control (GAL4 binding domain-p53) were readily detected in yeast lysates.
To determine whether the mouse ROMl C-terminai tail-GAZA fiision alone could
activate the two reporter genes, expression was assessed by examining the ability of the
bait strain to grow in media lacking histidine or by detecting P-galactosidase activity by
tilter or liquid assays. Conditions were optimized to identiQ the appropriate
concentration of 3-aminotriazole (3-AT) to be added to the agar plates lacking
tryptophan, histidine and uracil. The compound 3-aminotriazole inhibits the HIS3 gene
product, which is produced by low expression of the reporter gene in Y 190.
Various concentrations of 3-AT were added to plates, ranging from 15 m M to 50
rnM. Slight activation was observed with the ROMl C-terminal tail bait in the presence of
15, 20 and 25 m M 3-AT. However, this degree of activation was repressed by 30 mM 3-
AT. Thus, in order to perform the two-hybrid screen and reduce the number of false
positives identified, 30 rnM 3-AT was used in the medium of each library plate.
The extent to which the ROM 1 C-terminal bait activated the lacZ reporter gene
was also analyzed. Filter assays were used to test for the activation of P-galactosidase in a
qualitative assay. Functional B-galactosidase is capable of cleaving X-gal, causing the
yeast cells to produce a blue colour during filter incubation at 30°C. A qualitative P- galactosidase filter assay was perfomed with the ROMl C-terminal tail bait. Although
the bait was capable of activating of the IacZ reporter gene, this activation was very weak
compared to the positive control. To deterrnine the extent of the activation, a quantitative
B-galactosidase assay was perfonned (Fig. 2-2). The bait alone is capable of activating the
lm2 reporter gene (Fig. 2-2, MRC alone). in additon, the bait was tested in the presence
of a target protein which should not interact the ROM1 C-terminal tail. The ROMl C-
terminal bait with a negative control target plasmid pTDI, which encodes an SV40 large
T-antigenGAL4 activation domain fiision activated the lacZ reporter gene (Fig. 2-2,
MRC + pTD1). However, this degree of bait activation in cornparison to the positive
control, which contained an interacting bait and target, indicated that it is weak and
therefore managable in a two-hybnd screen (Fig. 2-2, pVA3 + pTD1).
Two-hy brid screen
Having determined that the ROMl C-terminal tail bait was expressed in Y190 and
having defined the appropriate concentration of 3-AT (30 mM) required to suppress
background activation of the HIS3 reporter gene, 1 performed a two-hybrid screen. The
oligo-dT primed adult bovine retinal library contains retina cDNAs cloned into the
p ACT11 target expression vector (EcoRI-XhoI). A total of 1 .5 million transformants were
screened using the methods designed by D. Getz (Gietz and Schietl, 1995).
The initial screen examined the expression of the HïS3 reporter gene. This step in
the two-hybrid system was accomplished by transforming the library DNA into Y190 cells
already containing the ROMl C-terminal tail bait and then plating the transformations
onto plates containing 30 mM AT and lacking leucine, tryptophan and histidine. Plates
were incubated at 30°C for 5-7 days until positive colonies were identified by their larger
sire in companson to background growth. From the total of 1.5 million clones screened,
233 HIS' transformants were isolated. Each yeast two-hybnd positive was picked and
streaked ont0 fresh plates which were incubated at 30°C for two days.
1 next exarnined the ability of the target protein to interact with the bait by
m e a s u ~ g activation of the lac2 reporter gene in P-galactosidase filter assays. The use of
a second reporter gene reduces the number of false positives by identifying those cDNA
encoded proteins which are capable of activating ody the HIS3 reporter gene. This
analysis led to the identification of 66 HIS' Lac' putative interactors.
Quantitative Liquid Beta-Gal Assay
Figure 2-2. Quantitative liquid beta-galactosidase assay. Samples were grown in appropnate yeast media and tested for expression of the lac2 marker. Quantitative vaiues were obtained using standard Müller uni6 shown on the y-axis. The negative control contained no plasmid DNA. The MRC bait was tested alone for autoactivation, with a non-specific bait (pTDl), and with each two-hybrid target isolated from the screen (l4a, 15q, 3 la, I 18, 233).
The target plasmid was isolated fkom the 66 HIS' Lac' yeast transfonnants.
Target isolation was accomplished by selecting for plasmids which express the LEU2
marker and can complement a defect in the leuf3 gene of bacterial cells (MH6).
After isolating each target cDNA, specificity tests were perfomed to identifL those
targets capable of interacting only with the ROMl C-terminal tail bait and not with other
baits. Three non-specific baits fused to the GALA binding domain were chosen. These
included the standard baits pVA3 and pLAM5' described above, and a pCHXlO bait
(provided by B. Muskat), which contains full-length CHXl O (Liu et al., 1994) excluding
the C-tenninal acidic domain. Each target cDNA was subsequently transfonned into
Y 190 cells expressing one of the three non-specific baits, which were previously show
not to cause activation of the reporter genes on their own, and, in parallel, into yeast cells
expressing the ROMl C-terminal tail bait. Bait specificity interaction was determined by
exarnining lac2 reporter gene activation using P-galactosidase filter assays. Of the 66
HIS' ~ a c ' putative interactors identified, only five were shown to interact specifically with
the ROMl C-terminal tail bait. The remaining 61 putative interactors activated l d gene
expression in the presence of each non-specific bait.
To quanti@ the interactions between the five specific targets and the ROMl C-
terminal tail bait, quantitative P-galactosidase liquid assays were performed. In the
presence of the ROMl C-terminal tail bait, al1 five specific targets were capable of
activating the lac2 reporter gene to a level at least 2-3 times greater than that of the
negative controls (Fig. 2-2, MRC + 14% 1 Sq, 3 la, 1 18, or 233).
Characterization of the ROMl interacting partners
To acquire information about the target proteins found to interact specifically with
the ROMl C-terminal tail bait, the cDNA insert of each of the five isolated cDNAs was
sequenced. Since the sequence coding for the GALA activation domain (AD) is known,
the frame of each target protein was determined by analyzing the GAL4 AD/target
sequence.
Once the sequence of the target proteins identified in the two-hybrid screen were
known, they were compared to each other and used to search the NCBI database
(Altschul et al., 1997). The cDNAs are refemd to by the number they were onginally
given when isolated fiom the library plates (14% 1 Sq, 3 la, 11 8, and 233) (Table 2-la-e).
The cDNA 159 encodes the smallest polypeptide, of 48 amino acids while the cDNA 233
encodes the largest polypeptide, of 126 amino acids (Table 2-lb and e). To assess the
possibility that the same cDNA was identified in the screen more than once, the five
isolated cDNAs were compared to each other using the Geneworks alignment program.
The result of the cDNA alignrnents revealed that each one was unique (results not show).
Thus, five different target proteins are putative interactors with the ROM1 C-terminal tail.
To gain further insight into the identity of the target proteins, proteins cornparisons were
performed using the database for each cDNA.
The results of the database protein comparisons are sumrnarized in Tables 2-1 a-e.
Al1 of the five cDNAs are novel based on protein and nucleotide searches. Database
searches performed with the nucleotide sequence of cDNA 15q revealed that it is
homologous to a human PAC clone 102G20 and is homologous to an uncharacterized
mRNA from a brain library. However, there are no retinal ESTs. An EST contains
partial nucleotide sequence of a randornly picked cDNA fiom a particular tissue library.
The protein sequence encoded by the cDNA 1Sq is novel, sharing 50% identity over 28
amino acids with ICB-1 (Table 2-1 b), a recently identified protein putatively involved in
cell matrix interactions (Treeck et al., 1998). The cDNA 1 18 encodes a 1 14 arnino acid
polypeptide that is 45% identical to a region of MCBP, a RNA-binding protein (Table 2-
Id) (Funke et al., 1996). The region showing homology between the predicted protein
encoded by cDNA 118 and MCBP, when used in a database search did not contain a
known motif currently present in the database. The cDNAs 14a and 233 encode two
different novel cDNAs each of which have some homology to different known proteins in
the database over a stretch of 37 arnino acids (Table 2-la and e). However, the regions of
homology do not contain any known domains, as determined either by using the database
or by analyzing published literature on the proteins identitied fiom the database. Finally,
cDNA 31a encodes a 68 amino acid novel polypeptide. Protein database andysis and
sequence examination revealed a 42 amino acid probe and histidine rich region (Table 2-
Protein Homology:
40% identity to retina derived POU-domain factor 1 (human) and 34% identity to secretin (porcine)
14a polypeptide sequence:
PACPLGDREI DGRDHPLTLP HPRCSWPGVW 30 ELVALGNLPL LLLDPKALRL GPWICRSWAE 60 SPCSLSWMPS RGPGSQLTVP LEGPGHLRVA 90 RHPLASLAVS PTVALMLPVQ GLX
Table 2-la. ROM1 C-terminal iail two-hybrid target 14a. Proteins homologous to the polypeptide encoded by 14a are listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.
Protein Homology:
50% identity to ICB-1 (human)
15q polypeptide sequence:
PVLASIEKTT CRAYLQVCLL PAIKTSSSTC PTLPASTPGF LLVVFWKLX
Table 24b, ROM1 C-terminal taii two-hybrid target 15q. Protein homologous to the polypeptide encoded by 15q is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.
Protein Homology :
3 1 a polypeptide sequence:
EKGRKKSEGK PCCLFKCTEV LAQGPPTHHT 30 TTHHPHPQLE HSPPAPCSH IPPRGRDVEG 60 RQGRRAGPX 68
Table 2-1c. ROM 1 C-terminai tail two-hybnd interac ting target 3 1 a.
Protein Homology:
45% identity to mCBP (mouse)
1 18 polypeptide sequence:
VSGVPDAIIL CVRQICAVIL ESPPKGATIP 30 YHPSLSLGTV LLSTNOGFSV GOYGTVTPA 60 EVTKLQQLSG HAVPFASPSM VPGLDPSTOT 90 SSQEFLVPND LIGCVIGRQG NKIKX 114
Tabte 2-Id. ROM1 C-terminal tail two-hybnd target 118. Protein homologous to the polypeptide encoded by 118 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are underlined.
Protein Homology:
48% identity to RRG4 (rat)
233 polypeptide sequence:
KRGRFPAAQH SDSGFELRSQ PWQSPRGPAQ 30 GKEPLGPRDA AVPARVPRRC RAVASSRPLP 60 GIPELVTEDH IQAPGSGHP TALSPEAGQA 90 ALSPSTIGAG HLKEAAFGPT DLVTLKEASF 120 PEVSHPX 126
Table 2-le. ROM1 C-terminal tail hvo-hybrid target 233. Protein homologous to the polypeptide encoded by 233 is listed, with the percent identity observed. The amho acids in the polypeptide showing homology are underlined.
1 c). Fifieen of the 42 amino acid present in the polypeptide encoded by 3 la is either a
proline or histidine nsidue.
DISCUSSION
ROMl was identified as an evolutionarily consented, abundant and retinal specific
cDNA in a diflerential hybridization screen (Bascom et al., 1992). Characterization of
ROM1 indicated that it shared overall structural similarity to RDS, the two proteins being
comparable in their cellular specificity, subcelluiar localization and membrane topology
(Bascom et al., 1992). Furthemore, at least 19 dierent proteins share the same topology
as ROMl and RDS, even though the primary sequences are not conserved (Wright and
Tomlinson, 1994).
Despite similanties between ROMl and RDS the two proteins are not functionally
redundant. For instance, lack of either murine RDS or ROMl causes photoreceptor ce11
death (Travis et al., 199 la; G. Clarke unpublished). The roles of ROMl and RDS in the
photoreceptor outer segment is currently unknown, but recent advances into the
biochemical roles for ROMl and RDS have been made. Boesze-Battaglia et al. (1998)
have shown that RDS is capable of mediating membrane fusion, at least in vitro,
suçgesting that RDS may be required for rod outer segment membrane fusion. The
authors have also determined that residues 3 13 to 327 in the RDS protein promote the
observed membrane fusion (Boesze-Battagiia et al., 1998). This fiisogenic region is
present in the C-terminal tail of RDS. Previous analysis performed on RDS have shown it
to be phosphorylated at the C-terminal tail, and have established, that the phosphorylated
form of RDS enhances membrane fusion (Boesze-Battaglia et al., t 997). Furthemore, the
phosphoqlation occured on a serine residue (BoeszeBattaglia et al., 1997). There are
two serine residues present in the C-terminal tail of RDS. The authors speculate that
ser321 located within the fùsogenic region is perhaps the serine that is phosphoiylated in
RDS thereby contnbuting to the fusogenic nature of the protein (Boesze-Battaglia et al.,
1 998).
The C-terminai tail of ROMl, used in the two hybrid screen, revealed that it
contains no serine residues. In fact, homology between ROMl and RDS is weakest at the
C-terminal tail. Although this difference in peptide sequence rnay suggest that ROMl is
not involved in disk morphogenesis, ~oml-' mice have longer disks in the outer segments
of photoreceptor cells (personal communication G. Clarke). Taken together t hese findings
indicate that ROMl does have a critical role in proper disk nm formation, however, the
differences highlighted above between ROMl and RDS rnay well in fact reflect dserent
protein associations the two partake in during disk morphogenesis.
To begin to identi@ ROMl interacting partners a two-hybrid screen was
performed using the C-terminal tail of ROMl. Findings fiom the analysis of the
photoreceptors fiom the ~ o m " mouse has revealed a role for ROMl in disk formation and
in maintaining outer segment structure. Thus, ROM1 interacting proteins identified from
the two-hybrid screen could have roles in disk biogenesis andor outer segment structure
and maintenance.
The two-hybrid screen performed using the ROMl C-terminal tail identified 66
HIS' Lac' transformants. Only 5 of these 66 HIS' Lac' transformants were found to be
capable of specificaily interacting with the ROMl C-terminal tail bait. Quantitative P- çalactosidase assays showed that the interaction between the predicted proteins encoded
by the cDNAs isolated and the ROMl C-terminal tail bait was weak (Fig. 2-2). These
weak interactions rnay be due to one of five possible situations. First, the weakness of the
interaction between the targets and the ROMl C-terminal tail is indicative of the in vivo
condition. Second, full-length ROMl is required to strengthen the interaction. Thus, the
other regions of ROMl rnay interact with the proteins isolated in the two-hybrid screen,
thereby stabilizing the binding. Third, the conformation of the C-temiinal tail rnay be
important for the interaction with the targets, and the only appropriate confonnation is
obtained wit h full-lengt h ROM 1. This qualification rnay also apply to the targets. Fourth,
other proteins rnay be needed to enhance the weak binding observed. Fah, none of these
interactions occun in vivo, and the weak nature of the two-hybnd result reflects the fact
that the interaction between the targets and the ROMl C-terminal tail is spunous.
Sequence analysis of the five cDNAs isolated indicated that none of the targets
were isolated more than once and that the predicted proteins encoded by the cDNAs were
al1 novel (Table 2-la-e). Considering that the only three known proteins at the disk rims
of photoreceptor ceils are ROMl, RDS and ABC& the identification of five novel cDNAs
from the two-hybrid screen is not surpnsing (Fig. 2-3). Assuming that the putative ROMl
interacting proteins do indeed associate with ROMl in vivo, possible insights into the
fùnctions of these proteins was investigated using protein and nucleotide sequence
cornparisons of each cDNA to known proteins. Although some of the predicted proteins
encoded by the cDNAs shared some homology with known proteins in the database
(Table 2-la-e), no recognizable motifs were identified in any of the protein sequences
encoded by the cDNA insert of the target plasmid.
Understanding the function of proteins present in the disk rim complex requires
their identification and examining their associations with known disk rim proteins such as
ROMI. Despite the lack of information available on the cDNAs isolated from the ROMl
C-terminal tail two-hybrid screen, it is conceivable that the proteins identified may be
involved in structural maintenance of the disk nms and photoreceptor outer segments.
Additiondly, as discussed earlier, a role for ROMl in disk morphogenesis has been
identified by the analysis of ~ o m l " mice. Perhaps some of the cDNAs isolated encode
proteins that are components of the myosin Wa-actin motor known to be responsible for
disk morphogenesis (Williams, 1992). Finally, proteins present at the disk rims do not
necessarily have to be required for the strutural integrity of the photoreceptor outer
segment but may in fact be necessary for outer segment function as is the case with
ABCR, a putative ATP transporter. It is possible that some of the proteins encoded by
cDNAs isolated in the ROMl C-terminal tail two-hybrid screen are involved in processes
such as light transduction which occurs largely in the outer segment.
Despite the interesting possible roles polypeptides encoded by the cDNAs isolated
may engage in, fùrther experiments are required in order to substantiate the interaction
obsexved between the targets and ROMl C-terminal tail bait. First, in situ hybndization
c ytoplasm
unknown disk - rim protein
ABCR
RDS RDS disuiphide l inked homodimer
@ R O M 1 linked ROM 1 homodimer disulphide heterotetramer RDS-ROM 1 non-covalent
Figure 2-3. Hypothetical mode1 of the rod disk rim. The only three known proteins at the disk rim are: ROM 1, RDS and ABCR. Rotein-protein interactions may be required to stabilize the disk rim curvature. This figure shows one possible interaction, between ROM1 and cable-like proteins extending between disks and from disks to the plasma membrane. Unidentificd disk rim proteins such as those identified in the MRC two-hybrid screen may associate with ROM 1.
studies are needed in order to dehe the spatial and temporal expression similarities
between the putative interactors and ROMI. Second, the interaction between the target
and bait must be shown using alternative methods such as a n i t y chromotography. The
advantage of affinity chromotography is that the protein-protein inteïactions are tested
directly without the possibility of another unknown protein mediating the detected
association first observed in yeast. Third, protein localkation studies using antibodies
raised to the ROM l interacting proteins can be perfonned. Addressing these points will
help to validate the interaction observed in the two-hybnd system between the isolatecl
targets and ROM 1.
CHAPTER 3
Molecular characterization of PHRl and identification of PHR1-interacting proteins using the yeast two-hybrid
system
The majority of the work presented in this chapter is mine. Jonathan Horsford and 1 performed the PHRI Ni situ hybridization on adult mouse retinal sections. The monoclonal antibodies were made by Greg Lee at the Centers of Excellence. The polyclonal antibodies were a gifi from S. Xu of Dr. Valle's group at Johns Hopkins University. The immunofluorescence was peformed by Danka Vidgen.
Abundant mRNAs of a differentiated ceIl often encode molecules that are required
for the differentiated cellular phenotype (Bascom et al., 1992). Evolutionarily consewed
genes that are expressed abundantly in the retina are likely to have an important role in its
structure, function, and development. Such genes, when mutated, are also likely to
represent candidate genes for diseases Secting the eye. The isolation of specific,
abundant, and conserved (SAC) or abundant and conserved (AC) cDNAs by differential
hybridization is an effective strategy for identifjmg genes required for retina structure,
function, and development. Two important rethal abundant and conserved genes isolated
by differential hybridization were ROM1 and CHXlO (Bascom et al., 1992). ROM1 is ah
important disk rim protein required for photoreceptor outer segment function and
structure (Bascom et al., 1992; G. Clarke unpublished) whereas CM10 is a homedomain
protein involved in the function and development of the neuroretina and inner nuclear
layer (Liu et al., 1994). The PHRl retinal AC cDNA was also identified by differential
hybridization, and may therefore to encode a protein important for retinai biology.
Mer the cloning of the PHRl cDNA, its expression was detennined to be
restricted to the retina, brain, kidney, liver, and lung by Northern blot analysis (Girami,
1994), with highest expression found in the retina. PHRl protein sequence analysis
revealed that it has regions of similarity to several proteins including dynamin, Akt,
oxysterol binding protein, and h o , dl of which contain a pleckstrin homology (PH)
domain. The region of hornology between these proteins and PHRl was restncted to
sequences in their respective PH domains, indicating that PHRl contains a PH domain.
PH domains have been identified in over 100 signalhg (Ma et al., 1997). The PH
domain is a loosely conserved 100-120 amino acid motif, with the only invariant amino
acid being a tryptophan residue present in the C-terminal region of the domain. Despite
the low degree of identity (generally 10-20%) beiween pleckstrin homology domains of
different proteins, it has been detennined, by NMR spectroscopy and X-ray
crystallography, that the six PH domains which have been solved to date ali share the same
basic fold. Each domain consists of a P-sandwich formed by two neariy orthogonal
antiparallel fbsheets of four and three strands, respectively. An amphipathic C-terminal a
helix closes off one end of the sandwich (Lemmon et al., 1997).
Based on structural similarities between the PH domain and retinol-binding
proteins, it was thought that the PH domain rnay be capable of binding to lipophilic
molecules (Jakoby et ai., 1993; Harlan et al., 1994). in vitro assays using radioactively
labelled phosphatidylinositol phosphates (PIPs) have show that the PH domain is capable
of interacting with phosphoinositides and that this association is required for recruitrnent
of PH domain-containing proteins to membranes (Harlan et al., 1994). The interaction
between PIPs and PH domains of different proteins is also specinc (Lernmon et al., 1997).
Thus, PLC& interacts most strongly to phosphatidylinositol-1,4,5-triphosphate, while
pleckstrin specifically associates with phosphatidylinositol-4,5-biphosphate (Harlan et al.,
1994; Ferguson et al., 1995).
The participation of many PH domain-containhg proteins in signalling cascades
has suggested that PH domains may act as effkctors of G proteins or PKC in signai
transduction pathways (Cohen et al., 1995). The PH domain-containing proteins such as
Btk, rasGAP, PARK, PLCy, Akt, P-spectrin, oxysterol binding protein, and IRS-1 have al1
been shown to interact with the Gpy subunits of G proteins (Touhara et al., 1994; Jiang et
al., 1998). Interactions between the PH domains of Btk and RAC-protein kinase and PKC
have also been demonstrated in vitro and in vivo (Yao et al., 1994; Yao et al., 1997;
Konishi et al., 1996). PKC is also important for regulating the cytoskeletal reorganization
functions of PH domain-containing proteins such as pleckstrin (Abrams et al., 1995) and
ARNO (Frank et al., 19%). Identification of protein-protein interactions involving the PH
domains of different proteins has been determined by anity chromatography and CO-
irnmunoprecipitation expenments. The results corn these experiments have indicated that
the C-terminal region of the PH domain and amino acids immediately C-terminal to the PH
domain (-30-35 amino acids) are required for interactions with G proteins whereas the N-
terminal betaZbeta3 strands of the PH domain are required for interactions with PKC.
Other proteins capable of interacting with the PH domain alone have been
identified for the PH domains of IRS-1 and IRS-2 using the yeast two-hybnd system
(Table 3-A). The PH domains of iRS-1 and IRS-2 have been shown to bind to the acidic
motifs present in Lon protease, myeloblast protein, and nucleolin (Table 3-A).
PH domain-containing proteins such as IRS-1, IRS-2, BO1 1, BOIZ, hGrb 1 Oy, and
Trio have also been used in their entirety as baits in the yeast two-hybrid system
successfully (Table 3-A). In al1 these examples, the proteins identified in the yeast two-
hybrid system interacted with other regions of the baits outside or including the PH
domain. PHRI, for example, contains other possible sites for protein-protein interaction
besides its PH domain, including a cluster of prolines at the N-terminus and a cluster of
tyrosines C-terminal to the PH domain (Fig. 16). SH3 domains, for exarnple, are known
to bind proline rich seqences (Cohen et al., 1995) while clusters of tyrosine residues are
potential sites of phosphorylation (Songyang et al., 1995). In addition, the yeast two-
hybnd system has also been successful in identifying PH domain-containing proteins
capable of interacting with baits such as Syk, HAPI, rho, racl, BCR, insulin receptor (IR),
MEK 1, phosphatidylinositol3-kinase, and SHP-2 (Table 3-A).
As an initial step in defining the role of PHRl in photoreceptor and neuronal
biology we have exarnined the location of PHRl in adult mouse retina and have
undertaken a two-hybnd screen. In collaboration with S. Xu, we have recently
detedned the ability of PHRl to bind to the py-subunits of the G protein transducin
(personal communication S. Xu). Furthermore, the localization of PHRl to the outer
segment of photoreceptors, the site of phototransduction, suggests that PHRl may
fùnction as a previously unrecognized modulator of the light transduction pathway.
The two-hybrid screen was performed using full-length PHRl as the bait. The
PHRl two-hybrid screen has generated 50 potential PHRl interactors, 36 of which
interacted strongly with PHRl while the remaining 14 interacted weakly. Two dBerent
PHRl specific interacting proteins were isolated twice in the two-hybrid screen. Both of
these potential PHR1-interacting proteins are novel, based on sequence analysis, with one
containing a protein-protein interaction motif cded a RING hger domain. Only two of
the fourteen weak PHR1-interacting proteins were specific in their interaction with PHRl.
Both of these potential PHR1-interacting proteins are daerent and novel.
Table 3-A. PH domain containing proteins used or identifiai using the yeast two-hybnd system. The only PH domain containhg protein interacton capable of interacting with oniy the PH domain were found for IRS- 1 and IRS-2.
PH domain containing proteins used in the two- hybrid system
, (sait) Insulin receptor substrate- 1 (ES- 1) IRS-1 PH domain
'Y contains SH2 and
only Insulin receptor substrate-2 (IRS-2) IRS-2 PH domain O ~ Y
BOX1 and BO12
PH domains
-
containing proteins identified in a two- hybrid screen
nucleotide exchange factor) contains a Dbl hornology @H and
PH domain -Duo which contains a GEF
PH domain containing protein interactors identified from the yeast two-hybrid system
-Cytoplasmic portion of the insulin receptôr (R) -Acidic motif of nucleolin
-Cytoplasmic portion of the insulin receptor (IR) -Acidic motifs of Lon protease, myeloblast protein, and nucleolin -Beml~ binds to the SH3 domai; of BO11 and BOi.2. -The Rho-type GTPase Cdc-42p binds to a segment of BO11 which contains its PH domain -hGrblOgarnma bound to a segment of hGrb 10 w hich included the insert between the PH and SH2 domains (IPS) and the SH2 domain -Interaction between the N- terminal region of hGrb logamma involves the IPS/SH2 region and the PH domain of a second hGrb 1 Ogamrna -GEF domain- 1 of Trio interacts with actin binding protein füamin
Syk tyrosine kinase
HAPl (Huntingtin- associated protein 1)
References
Sawka-Verhelle et al., 1996 Burks et al..
et al., 1996 Burks et al..
Bender et. al., 1996
Dong et al., 1998
Bellanger et al., 1998
Deckert et al., 1998
Colomer et ai., 1998
Table 3-A.
PH domain containing proteins used in the two- hybnd system (Baitl
PH domain containing proteins identified in a two- hybrid screen
-citron which contains a C6N2 zinc finger, PH domain, a long coiled-coil forming region including 4 leucine zippers, and the rho 1 rac binding site -APS adaptor molecule which contains PH and SH2 domains
-PSM which is pro- rich and contains PH, and SH2 domains -GrblO which contains a pro-nch region and a central PH domain and a C-terminal SH2 domain -Gab 1 an insulin receptor substrate with a PH domain
-ffirb 14 adaptor protein SH2 and PH domains
PH domain containing protein interactors identined from the yeast two-hybnd system
GTP-bound forrns of rho and rac 1 (GTPases)
Associates with BCR, and potentiaily with the immuuoreceptor tyrosine- based activation motif (ITAM), Lnlc signal transducer which links T- cell receptor to PLCgamma, Grb2, and phosphatidy . . linositol3- kinase. Cytoplasmic fragment of the insulin rece3or (IR)
The MEKI kinase interacts with the SH2 domain of Grb 10
Tyrosine phosphorylated Gabl interacts with phosphatidylinositol3- kinase and SHP-2 The SH2 domain and the region between the PH and SH2 domain mediate the interaction with insulin receDtor (IR)
References
Madade et al., 1995
Yokouchi et al., 1998
Riedel et al., 1997
Nantel et al., 1998
Rocchi et ai., 1998
Kasus-Jacobi et al., 1998
EXPERIMENTAL PROCEDURES
RT-PCR
RNA from mouse retina, brain, kidney and liver were isolated using the ~ ~ l z o l ~
Reagent (GIBCO BRL). Mouse retina, brain, kidney, and liver single-stranded cDNA was
made by adding 5 pg of total RNA to 0.5 vg of oligo d(T) (1 2- 18 T's - GIBCO BRL) and
9 pl of diethylpyrocarbonate (DEPC) treated water. The mix was incubated at 70°C for
10 min and then chilled on ice for at least one minute. During ice incubation, the PCR
machine was set for one cycle with the following conditions: 42°C for 1 hr, 70°C for IS
min and then to the 4°C soak temperature. To each reaction tube was added 4 pl 5x first
strand buffer (GIBCO BRL), 2 pL 0.1M DTT (GIBCO BRL) and 1 pl 1OmM dNTP
(Pharmacia). The components were mixed and incubated initially at 42OC for 5 min and
subsequently, 1 pl of Superscript U RT (GIBCO BRL) (200 U/pl) was added to each tube
and incubated for a further 50 min at 42T. The reaction was terminated by incubation at
70°C for 15 min and then treating each tube with 1 pl RNase H (3.8 U/pI) (GIBCO BRL)
for 20 min at 37OC.
The entire coding region of PHRl was amplified by PCR for each reverse
transcribed RNA sample. The PCR reaction perfomed consisted of the primer set 5'
GTTCCACCCGACTCCATC 3' and 5' CCAAGAGCAGCACCCGTG 3'. The
components used in the amplification were: 10 rnM Tris, pH 8.0, 50 mM KCI, 1.5 rnM
MgCl*, 0.01% gelatin, 10 pM of each dNTP, 50 ng of each primer, and 1 pl of reverse
transcribed RNA. The PCR machine was set with an initial denaturation temperature of
98OC for 5 min. Following denaturation, PFU polymerase (2.5 U/p1, Stratagene) was
added to each reaction and the PCR machine was set for 30 cycles of amplification. Each
PCR cycle was carried out at a denaturation temperature of 95OC for 1 min, an annealing
temperature of 59OC for 2 min and an extension setting of 72OC for 2 min. The same
conditions were applied to plasmid DNA containing full-length human PHRl except that
the primer set used consisted of 5' GGAATTCCATATGAGCCCTWAGCCCCG 3' and
5' ACGCGTCGACGGCTCAGAACCAGCAGGG 3'.
In situ Hybridization
A 624 bp PstI-BamHI fiagnient fiom the coding region of mouse PHRI
subcloned into pBluescript SK (Stratagene) was used as a template to generate [a-
3 3 ~ ] ~ ~ ~ (DuPont-N'EN) labelled riboprobes. Sense probe was made from the T7
promoter by in vitro transcription of the BanilII linearized template and antisense probe
was in vitro transcribed fiom the T3 promoter using EçoRI digested template.
Riboprobes were generated using the Stratagene in vitro transcription kit according to the
instructions of the manufacturer.
Mouse adult retina tissue slides purchased fiom Novagen were hybridized at 5S°C
oveniight with 150 pl of hybridization solution containing 3 3 ~ labelled probe at a
concentration of 1 x 105 cpm/pl. The washing protocol was as follows (Millen and Hui,
1 994):
a) 20 min at 6S°C in shaking water bath with a solution containing 50%
formamide, 2X SSC, and 0.1% P-mercaptoethanol @Me) (wash buffer 2).
b) Three washes of 10 min each with prewarmed (37'C) 0.5M NaCI, lOmM Tris-
HCl pH 8.0, and 5mM EDTA at 37OC (wash buffer 3).
c) Treat with 20 ~<g/ml RNAse in prewarmed wash buffer 3 at 37OC for 30 min.
d) Rinse in wash buffer 3 at 37OC for 15 min.
e) Repeat above wash with wash buffer 2 at 65 OC for 20 min.
f ) Rinse in 2X SSC, the 0.1X SSC, 15 min each at 37OC.
g) The sections were dehydrated by quickly processing through 30%, 60%, 80%,
95% ethanol made with 0.3M ammonium acetate, and hnce through 100%
ethanol.
The sections were allowed to dry and then dipped in Eastman Kodak NTB-2
Nuclear emulsion. Sections were exposed at 4OC for 3-5 days. Each section was
developed, counterstained with toluduine blue and mounted with permount (Milien and
Hui, 1994). Dark field and bright field pictures were taken with the Leica Leitz DMRB
microscope using Kodak Elite ï I Ektachrome 100 film.
Transient expression of PBRl in manmalian celb
To transfect mammalian ceUs with PHRI, tùll-length PHRl cDNA was subcloned
into the pcDNA 3.1 + A marnmalian expression vector (Invitrogen). FulClength PHRI
was arnplified Rom plasmid DNA by PCR with PFU polymerase. The primer set used in
the PCR reaction was as follows: 5' CCGGAATTCCGGATGAGCCCTGCAGCCCCG 3'
which includes an EcoRl site and 5' GCTCTAGAGCGAACCAGCAGGGCGACCA 3'
which contains a XbaI restriction site. The PCR reaction was performed as outlined in the
RT-PCR section above. The PCR product was purified by the Qiagen PCR purification
kit. The amplified product and vector were then digested with EcoRI and XbaI at 37OC
for 2 hrs, mn on a 1% agarose gel and purified by the Qiagen gel extraction kit following
manufacturer's instructions. The PHRl PCR product was subsequently subcloned into the
vector using the rapid ligation kit (BoeMnger-Mannheim). Ligation products were
confirmed to be correct by sequencing using the Tt primer site present in the vector. The
entire PHRl insert subcloned into the pcDNA 3.1 + A vector was sequenced to confimi
the absence of any PCR induced errors.
The following standard protocol was used for transient transfection of adherent
HEK293 cells (ATCC) in 60-mm dishes (supe&ectm, Qiagen). The day before
transfection cells were seeded to a concentration of 2-8 x 10' cells per 60-mm dish in 5 ml
of DMEM @ulbecco's Modified Eagle Media) growth medium containing 10% serum
(fetal calf senim) and incubated ovemight at 37OC with 5% CO2. Transfmion was
performed with no DNA, pcDNA vector only and the PHRl expression constmct. DNA
was prepared using the Qiagen Maxi prep. A total of 10 pg of DNA was diluted in 300 pl
of growth medium without senun. A reporter constnia containing the lacZ gene (5 pg)
was also included in the reactions to confimi efficient transfe*ion by Western blot analysis
of cell lysates with anti-P-galactosidase antibodies (Promega). To the DNA solutions was
added 30 pl of superfectThi transfection reagent (Qiagen). The reaction was mixed by
trituration. Sarnples were incubated for 10 min at room temperature to allow the
superfectTM transfection ragent to complex with the plasmid DNA. During complex
formation, growth media was removed from the petri dishes by gentle aspiration and cells
were washed with 4 ml of 1X PBS (phosphate buffered saline). M e r the 10 min
incubation period, 3 mls of growth medium with serum was added to each reaction tube
containing transfection complexes and mixed by trituration. The total volume was
inunediately transferred to the ceils in 60-mm dishes. Cells were incubated at 37°C for
two-three hours with the complexes. After the incubation period medium was removed by
gentle aspiration and cells were washed with 4 ml of 1X PBS. New cell growth medium +
serum was added. Cells were incubated for 24 hrs.
CeU lysate preparation
Once cells were incubated for 24 hrs at 37OC, they were removed h m the
incubator and placed on ice. Growth medium was removed by gentle aspiration and cells
were washed with cold 1X PBS ( 5 ml). Cells were lysed with 1X RIPA buffer (Sambrook
et al., 1989) which contains: 15 mM NaCl, 50 mM Tris-CI pH 8.0, 0.1% triton X-100,
0.5% deoxycholic acid (Sigma), 0.1% SDS, 1 m M PMSF, 1 m g h l STF (serum fetal calf
trypsinase inhibitor). Lysis was performed for 15 min with 1X RIPA buffer (2 d60-mm
petri dish). Lysates were collected and centnfbged at 4°C for 15 minutes. Supernatant
was transferred to a new tube and stored at -20°C.
Protein electrophoresis and immunoblotting
Tissue and ceIl protein lysates were resolved using 5% stacking and 10%
separating, 0.75 mm thick, acrylamide gels in the Bio-Rad Minigel system. The amount of
tissue and cell lysate loaded per lane was 1 pg. The amount of purified his-tagged PHRl
loaded was 0.2 pg. Gels were either stained with Coomasie blue (Sambrook et al., 1989)
or the proteins were transferred to Ntrocellulose for Western blot analysis.
Proteins were electrophoretically transferred to Hybond C nitrocellulose as
outiined in Chapter 2. Non-specific background was elhinated by blocking the filter with
5% milk solution dissolved in TBST for 1 hr at room temperature, foliowed by one 5 min
wash at n o m temperature with TBST. The filter was then incubated overnight at room
temperature with anti-PHR1 rabbit IgG polyclonal antibody diiuted in 1% miik in TBST
(1 : 5000 dilution). The following day fiesh primary antibody was added to the filter at a
dilution of 1: 1000 and incubated at room temperature for 2-3 hrs. Unbound prirnary
antibody was removed by washing 3X 10 min with TBST, at room temperature. The
nitrocellulose filter was subsequently incubated for 1 hr at room temperature with the
secondary antibody anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma),
diluted 1 : 5000 in TBST with 1 % milk. Excess secondary antibody was removed by 4X 1 O
min washes with TBST solution at room temperature. The enhanced cherniluminesence
detection system (Dupont) was employed to visualite the protein bands after exposure to
film.
Immunofluorescent Iabelling of PHRl in the adult mouse retina
Wild type mice were sacrificed by ceMcal dislocation and adult mouse eyes were
quickly enucleated and transferred into cold 4% paraformaldehyde in phosphate buEered
saline (PBS) for ovemight fixation at 4°C. M e r PBS washes eyes were stored in 30%
(w/v) sucrose PBS overnight. Tissue was embedded in OCT compound (Tissue-TEK)
and quickly frozen in isopentane over iiquid nitrogen. 7-14 Pm cryostat sections were
collected on Sigma, ~ i l a n e - ~ r e ~ ~ ~ slides and stored at -20°C until use.
Cryostat sections used for immunfiuorescent staining were rehydrated in PB S pH
7.3 for 15 min. Retinal sections were blocked in 10% goat serum (Sigma) in PBS for 30
min and then permeablizied with 10% goat serum with 1% triton X-LOO in PBS for 10
min. Primary antibodies were fieshly diluted and spun down at 3000 rpm for 3 min.
Monoclonal antibody (clone 6) was diluted to either 150 or 1:200 while polyclonal
antibodies (2605 and 2606, provideci by S. Xu) were diluted to 1500. Incubation with
primary antibodies took place overnîght at 4OC. After washing with 3X 10 min PBS,
sections were treated with fluorescein-conjugated F(ab')Z fiagrnent secondary antibody
fieshly diluted in PBS. For the monoclonal antibody (clone 6), goat anti-mouse FITC
1: 100 was used, whereas for the polyclonal antibodies goat anti-rabbit FITC 1:70 was
used. Sections were incubated for 1 hr at room temperature with secondary antibodies,
and then were washed with 3X 10 min PBS and rinsed with double deionized HzO
(ddH20). S lides were mounted with Imrnunofloor (ICN Biomedical). Sections were
Mewed with a confocal microscope Zeiss LSM 4 10 invert. The extemal laser used was set
at Ar 488m while the intemal laser used was set at HeNe 543 nm and 633 m.
Two hybrid screen
The procedure for the two hybrid screen has been outlined in Chapter 2 (see
experimental procedures). The bait constnicted for the human PHRl screen included full
length PHRl subcloned into the PAS 1 vector. The primer set used to ampli@ full length
PHRl fiom plasmid DNA was 5'
GGAATTCCATATGAGCCCAGCAGCCCCGGTCCCGCCT 3', which contained a NdeI
restriction site and 5' AACTGCAGAACCAATGCATTGGTCAGAACCAGCAGGGCGA
3', which contained a Pst1 restriction site.
An oligo dT prirned adult bovine retinal two hybnd library was used in the screen.
The yeast strain used in the two hybnd screen was a gift fiom D. Gietz and was referred to
as KGY 3 7.
RESULTS
PHRl alternative splicing
PHRl was originally identified in a difEerential hybridization screen and was
initially referred to as clone 198L. The insert isolated fiom clone 198L was used to screen
a human adult retinal cDNA library to isolate the full-length cDNA. Three overlapping
clones were identified. Sequence analysis indicated that the PHRI gene exhibits
alternative splicing due to the absence of a 105 bp coding segment in two of the three
isolated clones. Analysis of the PHRI genornic structure and the sequences of the three
overlapping clones revealed that exon 7 was identical to the 105 bp fiagrnent removed by
alternative splicing. This exon encodes 35 amino acids C-terminal to the pleckstnn
homology domain of PHRI.
To directly determine the involvement of alternative splicing in the expression of
PHRI, RT-PCR was pedonned with mouse retina, brain, kidney and liver RNA. Each
PCR reaction used a primer set which amplifieci the entire coding region of PHRI. The
results from the RT-PCR conf'irmed the existence of two transcripts for PHRI, one
containing nill-length coding sequence and the other lacking exon 7 (Fig. 3-1). As
expected fiom the sequence of PHRI, the full-length PHRI coding region PCR product is
approximately 730 bp while the alternatively spliced version of PHRl is approximately
620 bp (Fig. 3-1). To determine when the increase in PHRI PCR products was linear,
samples from each RT-PCR reaction were separated by gel electrophoresis &er 10, 15,
20, and 27 cycles of amplification (data not shown). The relative amount of the two
PHRl PCR products could be compared since both amplified products from each PCR
reaction were made with the same primer set and cornparisons were made while the
increase in amount of products was linear. With respect to the retina, full length PHRl is
expressed twice as abundantly as the altematively spliced product, whereas in the other
tissues tested the altematively spliced product is more than four times as abundant as full
length PHRI. In addition, dunng the linear amplification of the PHRI coding products by
RT-PCR, it was noted that the abundance of the coding PHRI message was highest in the
retina, then brain, kidney, and liver as expected based on the Northem blot for PHRI (data
not shown).
To confimi that the alternatively transcribed product from the RT-PCR was the
outcome of the removai of exon 7, the two fragments produced by the RT-PCR were
digested with an enzyme (Pvu II) whose restriction site is only present in exon 7. An RT-
PCR was performed with mouse retina RNA (Fig. 3-2 lane 4). When digested with Pvu
II, only the top band corresponding to fuli-length PHRl was digested to produce two
bands, and the sizes of the two bands together equded that of full-length PHRI. Plasrnid
DNA containing full-length human PHRl was also amplilied to produce a 750 bp band
(Fig. 3-2 lane 6). The full-length product amplified from plasrnid DNA when digested
with Pvu II produced the expected two bands (Fig. 3-2 lane 9, the sizes of which together
equalled the undigested product (lane 6). To c o n t h that the alternatively spliced product
did not contain exon 7, the 620 bp product fiom the RT-PCR with mouse retinal RNA
Figure 3-1. Alternative splicing of PHRl in the retina, brain, kidney, and liver. PHRl RT-PCR with RNA from retina, brain, kidney and liver. The PCR reactions were perfomed with the same set of pnmers and for 30 rounds of amplification. Lane 1 shows the positive control PCR with plasmid DNA containing full-length coding product of PHRl (-730 bp). Lane 2 shows the negative control PCR lacking a DNA template. Lanes 3,4,5 and 6 are RT-PCR results with RNA from rnouse retina, brain, kidney and liver, respectively. In the retina, the full-length coding message of PHRl is twice as abundant as the alternatively spliced coding product (-620 bp). In contrast, the altematively spliced coding product of PHRl is more than four times as abundant as the full-length coding product of PHRI. Marker sizes are shown to the left of the figure.
Figure 3-2. Alternative splicing of exon 7 of PHRI. PHRI RT-PCR with RNA from mouse retina was digested with Pvu II (lane 2), a restriction enzyme whose recognition site is only present in exon 7. Only the full-length PHRl DNA product was digested by Pvu II producing two bands whose sizes together equalled that of full-length PHRI. Lane 3, undigested PHRl RT-PCR products. Lane 5 contains the full-length PHRl PCR product amplified from plasmid DNA containing full-length coding human PHR I . The PHRI product from lane 5 was digested with Pvu II, producing two bands (lane 4) whose sizes together equalled the full-length coding PHRI product. Lane 6 contains only the altematively spliced product (- 620 bp) purified from a RT-PCR reaction with adult mouse retinal RNA. The PHRl coding altematively spliced product from lane 6 was not digested with Pvu II. Lane 1 shows the lambda marker with the size of the fragments on the left, in nucleotides (nt).
was purified and digested with Pvu 11. No digestion was observed with Pw II providing
further evidence for the lack of exon 7 in the PHRI aitematively spliced product (Fig. 3-2
compare lanes 7 and 8).
Expression of endogenous PHRl in retinal and bnio lysates
To establish the expression and apparent mass of the PHRl protein in retina and
brain lysates, Westem blot analysis was performed with polyclonal antibodies raised
against full-length PHR1. Transient transfections were pediormed in HEK293 cells with
either the pcDNA 3.1 mammalian expression vector only as the negative control or with
the full-length PHRl cDNA subcloned into pcDNA 3.1. Lysates were prepared from
independent duplicate transfection experiments with cells expressing PHRl and cells
containhg vector only. Expression of P-galactosidase was tested by Westem blot analysis
in order to confirm proper transfection efficiency. Lysates fiom both PHRl expressing
cells and from cells transfected with the empty vector expressed the P-galactosidase
marker at similar levels indicating that al1 cells were transfected correctly (data not
show).
Once PHRl expression was confirmed by Western blot analysis in HEK293 cells
transfected with the PHRI rnamrnaiian expression constmct, positive and negative control
cell lysates were electrophoresed with lysates from mouse retina and brain in a 12.5%
SDS-PAGE. Proteins were ~ansferred to a nitroce~lulose fiher and probed with the
polyclonal antibody 2605 raised against full-length PHRl. His-tagged PHRl purified from
bacteria served as a positive control for the Westem.
The size of the bacterially puriîïed his-tagged PHRl was -25 kDa (Fig. 3-3 lane 8),
as predicted from the open reading of the iÙU-length cDNA. Expression of PHRl in the
HEU93 ce11 lysates, however, produced two major bands not observed in the negative
controls, one migrating at -37 kDa and the other migrating at -33 kDa Fig. 3-3 lanes 2
and 4). The srnalier band is believed to be a major degradation product. In the retina
lysate a doublet migrated at -37 kDa and -35 kDa. The larger band of the doublet in the
retina migrated at the same rate as the PHRl protein transiently expressed in the HEK293
cells, aithough it was less abundant (Fig. 3-3b compare lanes 2 and 4 with lane 6). The
Figure 3-3. PHR 1 expression in HEK293 cells, and mouse retinal and brain lysates. Two independent transfections were perfonned in HEK293 cells with full-length PHRI subcloned into pcDNA. An immunoblot, exposed for 10 sec panel A or 1 min panel B, of an SDS-PAGE with lysates from HEK293 cells transfected with the PHRl expression construct (3 v g ) (lanes 2 and 4), HEK293 cells transfected with vector only (3 pg) (lanes 3 and 5), mouse retina (1 pg) (lane 6), and rnouse brain (3 pg) (lane 7), probed with polyclonal antibodies to PHR 1. Full-length PHR 1 migrates at 37 kDa in both HEK293 cells (lanes 2 arrow and 4) and retina (panel B, lane 6 arrowhead). A major degradation band migrates at 33 kDa in the HEK293 ceIl lysates with hill-length PHR 1 (lanes 2 white arrowhead and 4). The altematively spliced PHRl prduct may be migrating at 35 kDa (panel B, lane 6 arrow). The 40 kDa band in panel A lane 7 (arrow) may represent full-length PHR 1 in the brain. As a control, his-tagged bacterially purified full-length PHRl migrates at 25 kDa (lane 8). Lane 1 shows the marker bands, the sizes for which are on the left (in kDa) for each panel A and B.
73
lower product of the doublet in the retina migrateci at -35 kDa and may be the result of
the translation of the altematively spliced product, since the expecteù size of the protein
produced by translation of the altematively spliced transcript would be 2-3 kDa smaller
than that of full-length PHRI.
The expression of PHRl in the brain differs fiom its expression in other tissues in
that transcription cm begin either at exon one or in intron two. The use of an alternative
promoter in intron IWO results in the expression of two brain-specific transcripts, one
containing exons 3-9 and the other containing exons 3-9 but lacking exon 7 as a result of
alternative splicing (Fig. 1-5) (personal communication S. Xu). The imunoblot of the
brain lysate probed with PHRl polyclonal antibodies differed fiom the others in that four
prominent bands were detected at 4 0 kDa, -28 kDa, -26 kDa and -25 ma. These
bands do not correspond to bands detected in the PHRl expressing HEK293 ce11 lysates
or retina lysate. Thus, it is difficult to determine which bands in the brain lysate
correspond to full-length PHRl or PHRl isofonns. The fact that PHRl migrates at a
larger than expected size (-25 kDa) based on its amino acid composition in HEK.293 cells,
retina and perhaps brain (-40 kDa) suggests that some form of post-translational
modification is taking place.
PHRl expression and localization in the neuroretina
1'1 situ hybridization of mouse adult retinal sections with 3 3 ~ labelled probe was
performed to determine if the PHRl message is expressed throughout the neuroretina or if
it is cell type specific (Fig. 3-4a). PHRI expression was seen most abundantly in the imer
segments of the photoreceptor cells (Fig. 3-4b). It was also detected in the outer
plexiform layer, and in the ganglion cetl layer (Fig. 3-4b). No significant labelling was
found with the sense probe (Fig. 3 4 ) .
To determine the cellular and subcellular localization of PHR1, monoclonal and
polyclonal antibodies to full length PHRl were used on cryostat sections of mouse retina.
Immunofluorescent staining showed that PHRl abundantly localizes to the outer segments
of the photoreceptor ceUs in the outer nuclear layer (Fig. 305% and b). in addition, both
monoclonal and polyclonal antibodies detected PHRl in the ganglion cell layer (Fig. 3-Sa,
Figure 3-4. In situ hybridization of mouse PHRl in adult mouse retina. Sections were probed with 3 3 ~ labelled PHRl (nt 8-632). A. Bright field of the mouse retina stained with toluduine blue. B. Dark field picture of hybridization with the antisense probe. C. Dark field picture of hybridization with control sense probe. PHRI is expressed abundantly in the photoreceptor inner segment (1s) and at lower levels in the outer plexiform layer (OPL) and ganglion ce11 layer (GCL) as shown by the white signal (panel B). INL = inner nuclear layer, IPL = inner plexiform layer, ONL = outer nuclear layer and OS = outer segment.
Figure 3-5. PHRl is localized to the photoreceptor outer segment and to the ganglion cells of mouse adult retina. Panels A and B show micrographs of adult mouse retinal sections labelled by immunofluorescence with PHRl monoclonal or polyclonal antibodies, respectively. C. higher magnification of the ganglion cells stained with monoclonal antibodies to PHRl. PHRl staining is localized in ganglion cells to the plasma rnembrane(arrow).
and b). At higher magnifications on thinner sextions, PHRl is localized to the plasma
membrane of the ganglion ceUs (Fig. 30%). This finding is in agreement with in vitro
studies which have shown that PHRl is capable of associating with bovine rod outer
segment membrane fractions (personal commwication S. Xu).
Presence o f a putative Gterminal trrinsmembrane domain in PBRl
To determine the presence of transmembrane domains in PHRI, a hydropathy plot
was performed on full-length PHRl using the Kyte-Doolittle algorithm (1 982). The
hydropathy profile of full-length PHRl shows that PHRl contains a single putative
transmembrane domain at the C-temiinal end of the protein (Fig. 3-6). The putative
rnembrane-spanning domain consists of 22 residues and has an average hydrophobicity of
approximately +1.6 (Fig. 3-6). Thus, the only two known protein domains present in
PHRl are the C-terminal TM domain and the N-terminal PH domain.
Analysis of PHRl bait expression in the two-hybrid system
To identi@ proteins capable of interacting with PHRl in the retina, a two hybrid
screen was performed. The entire coding sequence for PHRl was subcloned into the
PAS l bait vector (Fig. 3-7a). As for the ROMl C-terminal tail screen, appropriate control
experiments were performed to establish that correct expression of the PHRI bait
occumed and to cietennine whether the bait was capable of autoactivating the reporter
genes.
The yeast strain used in the PHRl two hybrid screen was KGY37 (provided by D.
Gietz). Unlike the Y 190 strain used in the ROMl C-terminal tail screen, KGY37 does not
produce as many background colonies during the screening procedure and does not
spontaneously lose the IacZ reporter gene unless grown on SD media lacking uracil, the
marker used to integrate the lac2 gene into the yeast genorne (Gietz and Schiestl, 1995).
The P m 1 bait construct was transfonned into KGY37 afler it was demonstrated not to
contai. any PCR induced artefacts by sequencing analysis.
Before beglluiing the PHRl two-hybnd screen, expression of the PHRl bait in
KGY37 was evaluated by Western blot analysis. Fustt, yeast protein lysates were prepared
Figure 3-6. PHRl protein sequence analysis. Kyte-Doolittle (1982) hydropathy plot of PHR 1. The last 20 arnino acids code for the putative transmembrane domain. PHRl profde was plotted as a mean hydrophobie index against amino acid number, using a moving window of 19 aa with a one amino acid interval.
A GALA Binding Domain
+H3N COO'
- 22 amino acids
Protein Markers W a )
HA epitope PHRl
Figure 3-7. PHRl two-hybrid bait. A. Human PHRl two-hybrid constnict. The constnict contains full-length PHRl (243 arnino acids) fused in hune to the GALA binding domain. An HA epitope separates the two polypeptides. B. Western blot of yeast lysates expressing the PHRl bait. The PHRl construct was transformed into KGY37 cells. Lane 1 shows the PHRl fusion protein migrating at 50 Wla. Lane 2 shows the positive control, murine p53 (72-390) and lane 3 negative con001 (KGY37 alone).
from cells containing either a positive control plasmid (pVA3), the PHRl bait, or no
plasmid. The lysates were separated in a protein gel and subsequently transferred to a
nitrocellulose filter for Western blot analysis. An anti-GAIA binâing domain antibody was
used to detect PHRl bait expression (Fig. 3-7b). As predicted, a band rnigrating at -50
kDa was detected, indicating expression of the PHRl bait in KGY37 cells.
To evaluate the autoactivating potential of the PHRl bait, the expression of both
the HIS3 and lac2 reporter genes were analyzed. No P-galactosidase activity was
detected by filter assays, demonstrating that the PHRl bait did not autoactivate the lad
reporter gene. To determine if the PHRl bait autoactivated the HE3 reporter gene,
KGY37 cells expressing the PHRl bait were plated on SD media lacking histidine, but
containing various concentrations of 3-AT ranging fiom 15 mM - 50 rnM. Frorn this
analysis, it was determined that the addition of 15 rnM 3-AT was sufficient to minirnize
background growth to levels observed with the negative control.
The PBRl two-hybrid screen
The same target library was used as that described in Chapter 2. H'S3 reporter
gene expression was evaluated initialiy by transforming the library DNA into KGY37 cells
already containing the PHRl bait and then plating the transformations ont0 plates
containing 15 m M AT and lacking leucine, tryptophan and histidine (Gietz and Schiestl,
1995). Plates were incubated at 30°C between 5-7 days until positive colonies were
identified based on their larger size compared to background growth. From the total of
1.45 million clones screened, 90 HIS' transfomants were isolated. Each positive
transformant was picked and streaked ont0 fiesh plates (Leu-, Trp; His-, 15 mM 3-AT)
which were incubated at 30°C for two days.
The second phase of the screen in the two-hybrid system qualitatively examined
the ability of the target protein to interact with the bait and activate the lac2 reporter gene
(Bai and Elledge, 1996). To test for lac2 expression, B-galactosidase filter assays were
perfomed on ail 90 HIS' transfomüuits. From the examination of lac2 expression, 50
HIS' Lac' transfomants were identifid and were, subsequently, subdivided Uito 36 strong
and 14 weak HIS' ~ a c ' PHRl putative interactors. 1 first analyzed the 36 strong positive
cDNAs. The cDNAs encoding the PHRl putative interacting proteins were isolated fiom
the 36 HIS' ~ a c ' transformants by first selecting for yeast containing the target plasmid.
The target plasmids express the LEU2 marker and can therefore grow, by
complementation, in bacterial cells (MH6) that contain a defect in their leuB gene.
M e r isolating each target clone, specificity tests were perfonned to identify those
proteins encoded by the cDNAs capable of interacting only with the PHRl bait and not
with non-specific baits. The three non-specific baits chosen have been described in
Chapter 2. Each target was subsequently transformed into KGY37 cells expressing one of
the three non-specific baits, which were known not to cause autoactivation. Each target
plasmid was also transformed into yeast cells expressing the PHRl bait in order to re-
confirm the interaction originally identified fiom the PHRl two-hybrid screen. Specificity
was evaluated by determinhg the ability of the targets to activate the lac2 reporter gene
(P-galactosidase filter assays) in the presence of the PHRl bait only. Of the 36 HIS' Lac'
putative interactors identified, 9 were shown to interact specifically with the PHRl bait
(Tables 3-la-i).
Each cDNA was sequenced in order to identify the protein or nucleotide encoded
by the target construct. Database protein and nucleotide cornparisons were then
performed with each target cDNA (Tables 3-la-i). The first two sets of novel cDNAs
encoding PHR1-interacting proteins isolated fiom the screen are most interesting because
each one was identified twice and they each contain interesting protein homologies (Tables
3- 1 a-d).
The cDNAs of 1-3 and 29-1 encode the same protein target with the open reading
frame of the former cDNA being much larger than the latter (Fig. 3-8a) (Table 3-lc and
d). Cornparison of the proteins in the database with the proteins predicted by the open
reading m e s of cDNAs 1-3 and 29-1 revealed that they both contain a C6HC1 zinc-
binding motif, otherwise known as a RING finger domain. The RING finger domain is
considered to be required for protein-protein interactions (Borden and Freemont, 1996).
No other regions of the predicted proteins encoded by the cDNAs 1-3 and 29-1 showed
homology to known proteins in the database, indicating that they are novel molecules.
1 Protein Homology:
100% identical to the polypeptide encoded by cDNA 25-2 from amino acids 1-98 (Table 3- 1 b)
1 -4 polypeptide sequence (1 17 amino acids):
NRCSPRLPLVPGCAADHPRILQHGGRSSRTPIPP LHAPRWLPAQQDAAQSAPLFLWPPLLPLHRSL HPPQATSLWPSTSLYHQLLYHVPDILCTVQPSSR EGNPSRTESHSVPAWPEX
Table 3- la. PHRl two-hybrid target 1-4. Protein homologous to the polypeptide encoded by 1-4 is Iisted, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.
1 Protein Hamology:
168% identical to Diff33 (rat)
25-2 polypeptide sequence (3 1 1 arnino acids):
NRCSPRLPLVPGCAADHPRILQHGGRSSRTPIPPLHA , PRWLPAQQDAAQSAPLFLWPPLLPLHRSLHPPQATS LWPSTSLYHQLLYHVPDILCTVQPSPERVILQGQNH TLCLPGLSKMESHTPDTGLTVMSAGIMYACVLFAC NEAPNLAEVFGPLWTVKVY SYEFQKPSLCFCCPETG
, EPEEGECQVRIRPRGVAARPADQETSPAPPVQVQ QLSYSYSAFHFVFFLASLYVMVTLTNWFSYEG AELEKTFITGSWATFWVKVASCWACVLLYLGL LLTPFCWSPIPDPQHPILRRHCHRVLPNDKYPIX
Table 34b. PHRl two-hybrid target 25-2. Protein homologous to the polypeptide encoded by 25-2 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.
Protein Homology:
28% identical to AR1 (Drosophila) Contains a C6HC 1 RING finger domain
3- 1 polypeptide sequence (3 13 arnino acids):
GKSELSCMEGSCTCSFPASELEKVLPQTILYKYYER KAEEEVAAAYADELVRCPSCSFPALLDSDVKRFS CPNPRCRKETCRKCQGLWKEHNGLTCEELAEK DDIKYRTSIEEKMTAARIRKCHKCGTSLIKSEGCNR MSCRCGAQMCYLCRVSINGYDHFCQHPRSPGAPCQ ECSRCSLWTDPTEDDEKLIEEIQKEAEEEQKRKNGE NTFKRIGPPLEKPPEKVQRTEALPRPVPQNLHQPQIP XY AFVHPPFPLPPVRPVFNNFPLNMGPIPAPY VPALP NMRRQLRICPHPPAPGTQPAHALWPPASASX
Table 3- lc. PHR 1 two-hybnd target 3- 1. Proteins homologous to the polypeptide encoded by 3-1 are listed, with the percent identity obsewed. The amino acids in the polypeptide showing homology are in boldface.
Protein Homology :
28% identical to AR1 (Drosophila) 100% identical to the polypeptide encoded by cDNA 3-1 fiom arnino acids 1 - 1 18
Contains a C6HC1 RING finger domain
29- 1 polypeptide sequence (1 32 amino acids):
GKSELSCMEGSCTCSFPASELEKVLPQTILY KYYER KAEEEVAAAYADELVRCPSCSFPALLDSDVKRFS CPNPRCRKETCRKCQGLWKEHNGLTCEELAEK DDIKYRTSIEEKMTAAALGNATSVGPAYOIX
Table 3- Id. PHRl two-hybrid target 29- 1. Proteins homologous to the polypeptide encoded by 29-1 are listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.
Protein Homology:
( Novel I 10-2 polypeptide sequence (23 amino acids):
PPAGSRHPAELPTRRCPGPGHAPX
Table 3- 1 e. PHRl two-hybrid target 10-2. The polypeptide sequence for 10-2 is novel.
Protein Homology:
b
21-6 polypeptide sequence (23 amino acids):
1 LAOYGISSPWTPRPRTPLLRCPPX
Table 3- 1 f. PHRl two-hybrid target 2 1-6. The polypeptide sequence for 2 1-6 is novel.
Protein Homology:
97% identical to 0TX2 (human and murine) from amino acids 1-98 of 0TX2
6- 1 polypeptide sequence (1 34 amino acids):
SNPGEARPSGWVPRIWADFAPPNNLSMMSYLKQP PYAVNGLSLTTSGMDLLHPSVGYPATPRKQRR ERTTFTRAQLDVLEALFAKTRYPDIFMREEVAL KINLPESRVQVWFKNRRAKCRNKQQATGRTEV KTKX
Table 3- 1 g. PHR 1 two-hybrid target 6- 1. Protein homologous to the polypeptide encoded by 6-1 is listed, with the percent identity observed. The amino acids in the polypeptide showing homology are in boldface.
Nucleotide Homology :
86% identical to the mRNA for Spm RNA binding protein
1-3 nucleotide sequence (440 nucleotides):
GCANAGGGTGTNGTGAATACAGCTGNGTCCGCAGCAGTCC AAGCTGTTCGGGGCAGAGGAAGAGGAACTCTAACAAGGG GGGCTTTTGTTGGGGCCACAGCTGCTCCCGGCTACATAGC TCCAGGCTATGGAACACCCTATGGTTACAGCACAGCTGCCCC TGCCTATGGTTTACCCAAGAGAATGGTTCTG'ITACCCGTTATGA AATTCCCAACATATCCTGTTCCCCACTACTCArlTCTTTTAGCAAA TGACAGAAGCTAA'ITCCTATTCCAACAACAACCCAGTACATACA GAATGTTAGCGAAAAAGCCTTïTïATCCTGCTITCTTTGAACAC ATACTTGATCAAAATTAmGTAAAGAACATc-rrCCTACm T G A T T T T A A C A A A T G C A A A T T T A G T T C T C T A A A C A AACAAAAAAGAAA
Table 3- 1 h. PHR 1 two-hybnd target 1-3. Nucleotide sequence of 1-3 is homologous to the mRNA coding for Spnr RNA binding protein, the percent identity is shown. The nucleotides in 1-3 showing homology are in boldface.
Nucleotide Homology:
pACTII vector
13- 1 nucleotide sequence (440 nucleotides):
CCTTNAAANTTGATACTCGTTTGATGTATATAACTATCTA TTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAG ATCTCTATGGCTTACCCATACGATGTTCCAGATTACGCTA GCTTGGGTGGTCATATGGCCATGGAGGCCCCGGGGATCCG AATTTTTTAAATGACTAGAATTAATGCCCATCTTTTTTTT GGACCTAAATTCTTCATGAAAATATATTACGAGGGCTTAT TCAGAAGCTTTGGACTTCTTCGCCAGAGGTTTGGTCAAGT CTCCAATCAAGGTTGTCGGCTTGTCTACCTTGCCAGAAAT TTACGAAAAGATGGAAAAGGGTCAAATCGTTGGTAGATA CGTTGTTGACACTTCTAAATAAGCGAATTTCTTATGATTT ATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTG TATACAAATTTT
Table 3- li. PHRl two-hybrid target 13-1. Nucleotide sequence of 13-1 is identical to pACTII vector. The entire sequence of 13-1 listed is in boldface because it is 100% identical to pACTa vector sequence.
A RING Finger domain
CC CCCCHC
homology to 1 Diff33 311
Figure 3-8. Strong PHRl putative interactors identified in the two-hybrid screen. A. The proteins encoded by cDNAsl-3 and 29-1. Shown is the RING finger domain highlighted by the blue and orange bars which consists of 7 Cys residues (C) and 1 His residue (H). B. The proteins encoded by cDNAs 1-4 and 25-2. The polypeptide encoded by cDNA 25-2 is 68% identical to Diff33 over 66 arnino acids. This region of homology with DiM 3 is not present in the polypeptide encoded by cDNA 1-4. The proteins encoded by cDNAs 29-1 and 1-4 greatly restrict the site of the region interacting with PHRl. The open regions at the end of the polypeptides 29- 1 and 1-4 represent amino acids not present in 1-3 or 25-2, respective1 y.
The cDNA 25-2 encodes a 311 amino acid protein which contains a region of
homology, at its C-terminus, to Diff33 (Table 3-lb). Dm3 is an uncharacterized protein,
but is expressed during oligodendroblast to oligodendrocyte differentiation (Pfeiffer,
S. S.E., unpublished). The predicted protein encoded by the cDNA 1-4 is 1 17 amino acids
(Table Ma). Cornparison between the proteins predicted by the open reading frame of
the cDNAs 1-4 and 25-2 revealed that they encode the same protein, with the open
reading frame of the cDNA of the latter being much larger than the former. Analysis of
the predicted protein encoded by 1-4 also revealed that it lacked the homologous region to
Difi33 present in the predicted protein encoded by 25-2 (Fig. 3-8b). No other regions of
homology are known in the proteins encoded by the cDNAs 25-2 and 1 4 making them, as
in the case of the cDNAs 1-3 and 29- 1, novel putative PHR1-interacting proteins.
The proteins encoded by the cDNAs 1-4 and 29-1 greatly restrict the region
interacting with PHRI. It should also be noted that there are 19 amino acids at the C-
terminal end of 1-4 and 14 amino acids at the C-terminal end of 29-1 which are not
present in 25-2 or 1-3, respectively (Fig. 3-8). Because of the absence of these amino
acids in 25-2 or 1-3 both of which can interact with PHRI, it is unlikely that these amino
acids in 1-4 or 29-1 are required for the interaction of PHRI.
The final two novel cDNAs, 10-2 and 21-6, isolated in the PHRl two-hybrid
screen encode different novel polypeptides, with the sizes of each encoded protein being
small, 23 amino acids for both 10-2 and 21-6 (Tables 3-le and 0. Moreover, neither
cDNA contains a domain or motif known in the database. Because of the srnall open
reading h e s , cDNAs 21-6 and 10-2 may not encode biologically relevant PHRI-
interacting proteins.
The final three targets that are specific in their interaction with the PHRl bait
likely represent false associations. The cDNA 13-1 consists only of the pACTII vector
(Table 34 ) , and that fiom cDNA 1-3 (Table 3-lh) encodes a RNA binding protein Spnr.
Both of these cDNAs comonly yield fdse positives in two-hybnd screens. In addition,
the protein predicted by the open reading fhne of the cDNA of bovine target clone 6-1 is
97% identical to murine O W , a homeodomain containhg transcription factor (Table 3-
lg). This protein is unükely to be a physiologicaüy relevant interactor of PHRl because it
Predicted Protein Sequence
VLCCVCRALG VPWGFQVVPI 20 LSYLGTFHPP SPTALAALPA 40 APSSRRALPP ASEPGSGLLA 60 QYVQRNX 66
PPAGSRHPAE LPTRRGPGPG 20 HAPQPRPCLP LDAPALPVPA 40 LRAPGHWLLV TLPQSARAGP 60 AVAGRPLRGG WAPAGPAQAV 80 CCPVLGAAPD QATCGAGSCT 100 WWRPX 1 04
Table 3-2. The weak targets specifc in their interaction with the PHRl bait. The proteins predicted by the open reading frame of the novel cDNAs 8-1 and 24-3 are shown.
is a nuclear transcription factor whereas PHRl localizes to the plasma membrane of
ganglion cells and to the outer segments of photoreceptor cells.
The 14 weak PHRl potential interacting targets were also tested for their ability to
interact with PHRl specifically. Only 2 of the 14 weak PHRl putative interactors were
specific in their association with the PHRl bait. The cDNAs of 8-1 and 24-3 were
sequenced and compared to each other, to the strong PHR1-interacting targets, and to the
database. From these comparisons, the polypeptides encoded by cDNAs 8-1 and 24-3
were detedned to be novel. The open reading hunes of 8-1 and 24-3 encode predicted
novel polypeptides of 66 and 104 amino acids, respectively (TaMe 3-2).
The remaining 39 non-specific interacting proteins were not considered
physiologically relevant for two reasons. First, each non-specific PHR 1 -interacting
protein autoactivated transcription alone in the absence of PHRl. Second, each non-
specific PHRI-interacting protein activated transcription of the reporter gene in the
presence of three different non-specific baits to the same degree as in the presence of the
PHRl bait.
DISCUSSION
Definition of the molecular role of PHRl in the retina requires detailed expression
studies, analysis of fùnctional domains and the identification of physiologically relevant
protein associations. Cornparison of the predicted PHRl protein based on the open
reading fiame of the full-length PHRI cDNA with proteins in the database revealed the
presence of a putative pleckstrin homology (PH) domain. PH domain-containing proteins
are usuaîiy involved in signalling transduction pathways and the cytoskeleton. The
expression of PHRl in the retina occurs in the photoreceptor and ganglion celis. Both
polyclonal and monoclonal antibodies localize PHRl to the photoreceptor outer segment
and ganglion cell layer. Closer examination of the localization of PHRl in the ganglion
cells revealed that PHRl is present at the plasma membrane. The ability of PHRl to
associate with membrane components has been shown directly by its retention in
membrane fiactions prepared ftom bovine rod outer segments (personal communication S.
Xu). The regions in PHRl which may be involved in membrane association are a C-
terminal trammembrane domain, and a N-terminal PH domain. PH domaùis of several
proteins, including PLCG, (Yagisawa et al., 1998), Akt (Frech et al., 1997), and pleckstrin
(Harlan et al., 1994), have been show to interact with specific phosphatidylinositol
phosphates, indicating that at least one fiinction of this domain is to target proteins to
membranes.
PHRI is alternatively transcribed, but its expression in the retina seems to differ
from other tissues. The full-length coding PHRI product is composed of al1 nine exons
whereas exon 7, which is immediately 3' to the exons encoding the PH domain, is not
present in the altematively spliced product. The full-length product is twice as abundant
as the altematively spliced product in the retina. In the brain, kidney, and liver the
altematively spliced product is more than four times as abundant as full-length PHRI.
Further analysis is required to detedne the significance of the difference in expression
between full-length and altematively spliced PHRI. Alternative splicing is common in
genes encoding PH domain-containhg proteins such as Nltersecti~, GRBIO, Sosl,2, and
BIR (Guipponi et al., 1998; Frantz et al., 1997; Fukuda et al., 1996). Generally, the
alternative splicing events that occur for genes encoding PH domain-containing proteins
removes exons coding for part or al1 of the PH domain or sequences upstream or
downstream of the PH domain. Thus, there is no common alternative splicing pattern for
genes encoding PH domain-containing proteins.
To determine the expression and apparent mass of PHRl in the retina, Western
blot analysis was performed with lysates prepared fiom PHRl expressing HEK293 cells
and retina using PHRl polyclonal antibodies. Full-length PHRl migrated at a much
slower rate than expected (-37 Da) , based on the sue of PHRl calculated fiom its amino
acid composition (-25 kDa), in the retina and when transiently expressed in HEK293 cells,
suggesting some fonn of post-translational modification.
Identification of the specific post-translational modifications of PHRl will
contribute to Our understanding of the fùnction of the protein, and also possibly the
regdation of its function. One attractive possibüity is that PHRl is phosphorylated, since
some other PH domain-containing proteins are phosphorylated such as Akt, PLCy, and
LRS-1 (Sable et al., 1998; Falasca et al., 1998; Myers et ai., 1995). The presence of
multiple tyrosine residues in the C-terminus of PHRl is ais0 supportive of this suggestion,
and phosphorylation of tyrosine residues has been demonstrateci in the PH domain-
containing proteins PLCy, and IRS-1 (Falasca et al., 1998; Myers et al., 1995). In
addition, phosphorylation of PHRl may be required to activate PHRI, or alter its
conformation to allow the molecule to perform its function. Phosphorylation is a common
form of post-translational modification required to activate proteins containing PH
domains including pleckstrin, IRS-1, and Akt (Ma et al., 1997; Voliovitch et al., 1995;
Andjelkovic et al., 1 997). Moreover, phosphory lation generally occurs N-terminai or C-
terminal to the PH domain and can involve senne, threonine a d o r tyrosine residues.
Another possibility is that the phosphorylation state of PHRl determines or enhances
protein-protein or protein-lipid interactions it partakes in. The role of phosphorylation in
detennining or enhancing protein-protein or protein-lipid interactions has been show for
IRS-1, Akt, and PLCy (Myers et al., 1995; Sable et al., 1998; Falasca et al., 1998).
Confirming the phosphorylation of PHRl as a possible form of post-translational
modification and identifjmg the residues phosphorylated is therefore important.
Although the function of PHRl in the retina, and particularly the photoreceptor
and ganglion cell, remains to be established, it has been show in vitro that PHRl is
capable of binding to the Gay subunits of transducin and that this interaction requires the
PH domain of PHRl (personal communication S. Xu). The G protein transducin is a
GTP-binding protein which mediates the light activation signal fiom photolyzed rhodopsin
to phosphodiesterase. The association of PHRl with a molenile involved in the light
transduction pathway would be strong direct evidence that PHRl is a component of the
visual transduction cascade.
The PH domains of severai proteins, including Akt, p-spectrin, PARK, and IRS-1,
have been shown to bind to GPy subunits of G proteins (Touhara et al., 1994). This
association involves the C-terminal portion of the PH domain, and most signincantly in
terms of the differentiai splicing of exon 7 of PHRI, amho acids immediately C-terminal
to the PH domain. Recently, it has been shown that the 36 amho acids in the BM (Btk
motif) domain which are located immediately C-terminal to the PH domains of Btk and
GAPlm, are required for the interactions of these proteins with the Ga12 wbunit (Jiang et
al., 1998). The association of G a l l and Btk has functional significance, since it leads to an
increase in the kinase activity of Btk (Jiang et al., 1998). Although PHRl does not
contain a BM domain C-terminal to its PH domain, it does contain a stretch of 35 amino
acids encoded by exon 7 which is alternatively spliced. The removal of these amino acids
immediately C-terminal to the PH domain of PHRl may, therefore, have regulatory and
functional consequences for PHRI.
To begin to understand the role of PHRL in the retina, a two-hybrid screen was
performed using fùll-length PHRl as a b i t with an adult bovine retinal cDNA target
library. Four potential PHRI-interacting proteins were identified (1-4 and 25-2, 3-1 and
29- 1, 8- 1, 24-3). The two PHR1 -interacting candidates which interacted strongly with
PHRl were identified twice in the two-hybnd screen. Both cDNAs 1-4 and 25-2 encode
the sarne novel polypeptides, with cDNA 25-2 encoding a protein that is larger than the
protein encoded by cDNA 1-4 (Fig. 3-8b). In addition, the protein encoded by cDNA 25-
2 has hornology to an uncharacterized protein DiE33, which is expressed d u ~ g the
transition fiom oligodendroblast to oligodendrocyte differentiation (Table 3-1 b) (Pfeiffer,
S.S.E., unpublished). This region of homology to Diff33 is not present in cDNA 1-4. The
protein encoded by cDNA 1-4, however, greatly restricts the size of the region interacting
with PHRl in cornparison to the protein encoded by cDNA 25-2. Both cDNAs 3-1 and
29-1 encode the same novel polypeptides, with cDNA 3-1 encoding a protein that is larger
than the protein encoded by cDNA 29-1 (Fig. 3-8a). Thus, the protein encoded by cDNA
29- 1 restricts the size of the region interacting with PHRI . Interestingly, both proteins
encoded by cDNAs 3-1 and 29-1 contain a zinc binding motif referred to as the RING
finger dornain (Fig. 3-Sa). The RING finger domain present in 3-1 and 29-1 consists of 7
cysteine residues and 1 histidine residue, and is 28% identical to the RING finger domain
present in AR1 (Tables 3-lc and d). ARI is an uncharacterized Drosophila proteh. The
only information available on ARI fiom the database is that its RING fhger motif is
involved in axonal path-finding in the central nemous system of Drosophila (Oliveros, M.,
Apilera, M., Barbas, J. A., Martinez, I., Torroja, L., and Ferrus, A., unpublished). To
begin to define an axonal path-hding role for PHRI, the expression of PHRl in
Drosophila should be determined. If present, Drosophila PHRi mutants could be made
and exarnined for defects in axonal path-finding.
The RING finger mots is characterized by eight conserveci cysteine and histidine
residues which fom two 2n2+ binding sites (Brzovic et al., 1998). There are currently 80-
90 RING finger domain-containing proteins (personal communication A. Ballabio). RING
finger domain-containing proteins are found in organisms fiom yeast to humans. In
addition, proteins containing RING finger domains are involved in diverse cellular
functions and pathways including: 1) signalling pathways: TRAF2 (Cheng and Baltimore,
1996), and TRAe (Song and Donner, 199S), 2) gene transcription: SNLTRF (Moilanen et
al., 1998), and MSL proteins (Copp et al., 1998), 3) protein transpon: Vpsl lp and
Vps 1 8p (Rieder and Emr, 1997), 4) secretory pathways: RMA 1 (Matsuda and Nakano,
1998), 5) apoptosis: DIAPI (Oeda et al., 1998), and 6) embryonic patterning: XNF7
(Borden et al., 1995). The RING finger domain-containing proteins TRAF2, SNURF,
Vpsl lp, and Vpsl8p were identified using the yeast two-hybrid system to interact with
membrane associated proteins such as CD40, androgen receptor (AR), Vpsl6p and
Vps33p, respectively to fom protein complexes (Cheng and Baltimore, 1996; Moilanen et
al., 1998; Rieder and Emr, 1997). In these examples, the RING finger motif was show to
be essential for the protein-protein associations identified. Thus, it seems that the RING
finger domain is involved in protein-protein oligomerizations, acting as a convenient
scaffold for the assembly of protein complexes (Borden and Freemont, 1996). The
identification of a RING finger domain-containing protein as a candidate PHRl interacting
protein, suggests that PHRl may be a component of a membrane-associated protein
complex.
The two other cDNAs identined in the two-hybrid screen encode novel putative
PHR 1 -interacting proteins which interact weakly with PHRl (8- 1 and 24-3) (Table 3-2).
The weak association of these two different polypeptide targets with the PHRl bait may
indicate that: 1) an interaction between these proteins and PHRl occurs in vivo, but is
weak, 2) other proteins may be required to increase the strength of the interaction, 3) an
incorrect PHRl conformation allowed a spunous weak interaction, and 4) the fuiMength
target protein is required for a strong interaction with PHRI. Determinhg the expression
pattern and localization of ail four cDNAs by in situ hybridization and confirming the
interaction of PHRl and its potential interacting partners using other methods such as
affinity chromatopphy is required. The information gained from these analyses may
provide more insight into the biological role of PHRl in the retina.
CHAPTER 4
Concluding Remarks
FUTURE DIRECTIONS
The two-hybrid system was used to identlfy putative ROMI- and PHRI-
interacting proteins. From the two-hybrid screen perfomed with the ROM1 C-terminai
tail, five putative ROM 1 ginteracting proteins were identified. The PHRl two-hybnd
screen identified 11 putative PHRI-interacting proteins, two of which were each
independently isolated twice. To determine which of the ROM 1 - and PHR 1 ginteracting
proteins are capable of associating with ROMl or PHRl in vivo, the retinal expression of
the transcnpt encoding each ROMl - and PHR1 -interacting protein has to be examined. In
addition, the interaction between ROM 1 and each ROM 1 ginteracting protein and PHRl
and each PHR1 -interacting protein has to be confinned using a direct binding method such
as afEnity chromatography.
Localuing expression of PHRl and ROMl putative inteneton in the retina
To determine if the location of expression of the genes encoding PHRl and ROMl
putative interactors in the retina coincide with the expression of PHRI and ROMI,
respectively, in situ hybndization experiments have to be perfomed on adult bovine
retinal sections. Adult bovine retinal sections will be used in the in situ hybndization
experiments because the two-hybrid library used in the screens for both PHRl and ROMl
was made from adult bovine retinas. The PHRl gene is expressed in the photoreceptor
and ganglion ce11 layers. Those genes encoding PHRl putative interactors which are
expressed in the photoreceptor and/or ganglion ce11 layers will be examined further
because they are more likely to encode PHRl interactors than if they were not expressed
in the sarne regions in the retina as P H . 1 . The ROMI gene is expressed only in the
photoreceptor ce11 layer. Thus, genes encoding ROMl putative interacton which are
expressed in the photoreceptor layer wül be exarnined further for their ability to associate
with ROMl.
Using aninity chromatography to con- PBRl and ROMl interacton
To establish that the interaction of the PHRI- or ROM1-interacting proteins and
PHRl or ROMl, respectively are not artefacts of the two-hybrid system, affinity
chromatography will be used to confin the results fiom the PHRl and ROMl two-hybrid
screens. Attinity chromatography is an in vitro system which directly tests an interaction
between two proteins.
The genes encoding putative PHRl or ROMl interactors which are expressed in
the layen of retina that express PHRI or ROM, respectively will be exarnined as strong
potential PHR 1 - or ROM 1 -interacting candidates. The cDNAs encoding PHRl and
ROMl interactors will be subcloned into GSî expression vectors. Columns will be
assembled with the GST-PHR 1 or GST-ROM 1 interacting fusion proteins associated with
glutathione sepharose beads. Ce11 lysates from mamrnalian cells transiently expressing
PHRl or ROMl will be passed through the GST fusion protein columns and colurnns
containing GST alone. The association of PHRl or ROM1 with GST fused PHRl or
ROM1 interactors, resepectively can be detected by Western blot analysis with antibodies
raised against PHRl or ROM, after eluting bound proteins from the columns.
Identifying PHRl and ROMl interacting proteins using aClinity chromatography
Aanity chromatography has four advantages over the two-hybrid system to detect
protein interactions. First, affinity chromatography is a direct method in identifjmg
protein interactions whereas the two-hybrid system takes place in vivo where yeast host
proteins may interfere with protein-protein associations. Second, protein complexes,
present in the protein lysate, which bind to the protein of interest may be detected using
affinity chromatography. In the yeast two-hybrid system, interactions between protein
complexes and the bait can oniy be detected if al1 cDNAs coding for the molecules
forming the complex are present and expressed with the bait. The fact that the cDNAs
encoding the Gpy subunits of transducin were not identified in the PHRl two-hybrid
screen is not surprishg because the likelihood of both cDNAs, encoding each member of
the heterodimeric GPy subunit, would be transformed into the same yeast ceii with the bait
is low. Third, the procedure for Wty chromatography is less tirne consuming than the
screening of a yeast two-hybrid library against a protein of interest. Fourth, the abüity to
detect interacting proteins at low amounts (picograms) after atnnity chromatography is
much easier with ment technological advances in mass spectrornetry (personal
communication J. Ingles). Thus, the increase in the saisitivity and efficiency of afnnity
chromatography offers another technique to detect ROMI- and PHR1-interacting proteins
in retinal protein lysates which may not be identified in the two-hybrid system.
Future perspectives for PERl
To define the function and importance of PHRl in the retina and other tissues will
require detailed genetic and biochemical analyses. Understanding the role of PHRl in
areas of expression including the retina cm be examineci by targeting the gene in mice.
Eliminating expression of PHRl in transgenic mice may lead to an ocular phenotype. For
example, it is possible that there will be photoreceptor andlor ganglion ceIl morphological
and functional abnomalities in phri" rnice. Thus, characterizing the phenotype of phri'
mice may give insight into the biological role of PHRI.
The post-translational modification of PHRl observed in HEK293 cells transiently
expressing PHRl and in retina and brain protein lysates can be assessed by determining,
for example, the phosphorylation state of PHRl (Fig. 3-3). As discussed previously in
Chapter 3, there are clusters of tyrosine residues in PHRl located C-terrninal to the PH
domain which could be possible sites of phosphorylation. To determine if PHRl is
tyrosine phosphorylated, the protein cm be immunoprecipitated fiom HEK293 cells wit h
monoclonal or polyclonal antibodies raiseci against full-length PHRI. The
irnrnunoprecipitated PHRl can be separated by SDS-PAGE, transferred to a nitrocellulose
filter, and phosphotyrosine residues cm be detected by Western blot analysis with anit-
phosphotyrosine antibodies. To identify the tyrosine residues phosphorylated in PHRl,
the irnmunoprecipitated PHRl fkom HEK293 ceils can be cleaved into smaller peptides
and then separated by reverse phase-high performance liquid chrornatography.
Phosphorylated peptides isolated by this procedure can be microsequenced to identify the
phosphotyrosine residues in PHRI.
Future perspectives for ROM1
To idente more ROMl putative interacting proteins, other hydrophilic regions of
ROMl can be used as baits in two-hybrid screens. The two remainhg cytoplasmic regions
of ROMl, besicles the C-terminal tail, are the N-terminal tail (-23 amino acids) and the
loop between the second and third transmembrane domains (-18 arnino acids). Baits cm
be constructed with each of these short cytoplasmic regions of ROMl and used in two-
hybrid screens. One problem with using a short region of a protein in a two-hybrid screen
is that the polypeptide fused to the GALA binding domain may not fold properly. Thus, it
is possible that the two-hybrid screen will only generate cDNAs encoding spunous
interacting proteins. An alternative method to detect ROMl putative interactors is to use
affïnity chromatography. The cytoplasmic regions of ROMl mentioned above cm be
fused to GST and bound to glutathione sepharose beads. Retinai protein lysates can then
be passed through the GST fusion protein columns, and ROM1-interacting proteins can be
eluted, separated by SDS-PAGE, and identified by silver staining. ROM1-interacting
proteins will be identified by microsequencing. Non-specific binding proteins will be
identified fiom the negative control GST only column.
To begin to establish a biochemical role for ROMl in disk morphogenesis as
suggested by the formation of longer disks in the photoreceptor outer segment of ~ o m l "
mice, its involvement in membrane fision can be examined. A role for RDS in membrane
fusion has been established by the protocol designed by Boesze-Battaglia et al (1997). It
was determined that phosphorylation of serine residues in the C-terminai tail of RDS is
required for membrane fùsion (Boesze-Battaglia et al., 1997). ROM1 , however, does not
contain any senne residues in its C-terminal tail. It is possible that ROMl plays no role in
membrane fusion because of the lack of serine phosphorylation sites in the ROM1 C-
terminal tail. However, the role of ROMl in membrane fision could dEer nom RDS or is
required to support the fùnction of RDS and is, therefore, dependent on the presence of
RDS. Membrane fusion experiments designed for RDS cm be used to examine the role of
ROMl in the fusion between retinal rod outer segment membranes and mode1 membranes
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