Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
An N-terminal region of C. elegans RGS proteins EGL-10 and EAT-16 directs inhibition of
Gαo versus Gαq signaling*
Georgia A. Patikoglou and Michael R. Koelle!
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520
Running title: An RGS N-terminal region directs inhibition of Gα signaling
Correspondence: Michael R. Koelle
Department of Molecular Biophysics and Biochemistry
Yale University School of Medicine
333 Cedar Street, SHM C-E30
New Haven, CT 06520-8024
Tel: 203-737-5808
Fax: 203-785-6404
E-mail: [email protected]
1
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 26, 2002 as Manuscript M208186200 by guest on A
ugust 24, 2020http://w
ww
.jbc.org/D
ownloaded from
SUMMARY
Regulators of G protein signaling (RGS proteins) contain an RGS domain that inhibits
Gα signaling by activating Gα GTPase activity. Certain RGS proteins also contain a G gamma-like
(GGL) domain and a poorly-characterized but conserved N-terminal region. We assessed the
functions of these subregions in the C. elegans RGS proteins EGL-10 and EAT-16, which
selectively inhibit GOA-1 (Gαo) and EGL-30 (Gαq), respectively. Using transgenes in C.
elegans, we expressed EGL-10, EAT-16, their subregions, or EGL-10/EAT-16 chimeras. The
chimeras showed that the GGL/RGS region of either protein can act on either GOA-1 or EGL-
30 and that a key factor determining Gα target selectivity is the manner in which the N-terminal
and GGL/RGS regions are linked. We also found that coexpressing N-terminal and GGL/RGS
fragments of EGL-10 gave full EGL-10 activity, while either fragment alone gave little activity.
Biochemical analysis showed that coexpressing the two fragments caused both to increase in
abundance and also caused the GGL/RGS fragment to move to the membrane, where the N-
terminal fragment is localized. By coimmunoprecipitation we found that the N-terminal
fragment complexes with the C-terminal fragment and its associated Gβ subunit, GPB-2. We
conclude that the N-terminal region directs inhibition of Gα signaling by forming a complex
with the GGL/RGS region and affecting its stability, membrane localization and Gα target
specificity.
2
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Many hormones and neurotransmitters bind and activate heptahelical transmembrane
receptors which in turn catalyze exchange of bound GDP for GTP on G protein α subunits. GTP
binding induces dissociation of Gα from Gβγ subunits and enables these activated proteins to
signal downstream effectors. RGS1 proteins (regulators of G protein signaling) help terminate
signaling by greatly enhancing the weak intrinsic GTPase activity of Gα proteins, thus driving
reassembly of the inactive, GDP-bound Gαβγ heterotrimer (1). All RGS proteins contain a ~120
amino acid region known as the RGS domain that contacts Gα subunits and functions as the
GTPase activation domain (2). To date more than 20 mammalian RGS proteins and about 20
mammalian Gα proteins have been identified (1).
Although RGS proteins have been extensively studied in vitro and by overexpression in
cultured cells, their in vivo Gα targets and physiological functions remain largely unclear. For
example, the RGS proteins RGS4 and GAIP act similarly on both Gαi and Gαo in vitro (3), but
show strong and opposing selectivity between these targets when assayed in cultured chick
sensory neurons (4). However, when overexpressed in HEK293 cells, both RGS proteins acted
similarly to block Gαi signaling (5). Another dilemma is illustrated by the fact that the RGS2
protein shows different preferences for Gαi versus Gαq depending on the specific in vitro assay
system used (6, 7). An additional complication arises from the fact that many studies have been
carried out on small RGS domain-containing fragments of RGS proteins. RGS proteins typically
contain additional conserved regions outside the RGS domain that appear to affect their functions
and target specificities (1).
Genetic studies have the potential to conclusively demonstrate the true physiological
3
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
functions and Gα target specificities of RGS proteins. To date only a few RGS proteins have
been genetically characterized. A mouse knockout of RGS9 shows that it functions in the retina
to limit the duration of visual signaling (8). RGS9 is the only abundant RGS protein in rod outer
segments (9), which also contain only one Gα protein, the visual G protein Gαt. Thus the
selectivity of the RGS-Gα interaction in this case appears to be a relatively simple issue. A more
complex, and likely more typical case, is revealed by genetic studies of the C. elegans RGS
proteins EGL-10 and EAT-16. Although both are widely expressed in the nervous system, they
select different Gα targets and thus have opposite effects on C. elegans behavior (Fig. 1C).
Genetic experiments have shown that EGL-10 inhibits signaling by the C. elegans Gαo homolog
GOA-1, which in turn inhibits C. elegans egg-laying behavior (10). EAT-16, on the other hand,
inhibits signaling by the C. elegans Gαq homolog EGL-30, which in turn activates egg-laying
behavior (11). EGL-10 and EAT-16 constitute the clearest example of RGS proteins that have
been shown through rigorous genetic experiments to have distinct Gα target specificities.
EGL-10 and EAT-16 are members of a subfamily of RGS proteins that includes RGS9
as well as the mammalian RGS6, RGS7, and RGS11 proteins. Just N-terminal to their RGS
domains, these proteins contain a ~60 amino acid G gamma-like (GGL) domain (Fig. 1A and
1B) that mediates binding to a divergent Gβ subunit, Gβ5 (12). The exact role of Gβ5 in signaling is
unclear, but genetic and molecular studies in C. elegans show that EGL-10 and EAT-16 require
association with the C. elegans Gβ5 ortholog, GPB-2, for their stability and function (13). All
GGL-containing RGS proteins also contain a conserved N-terminal region of ~220 amino acids
(Fig. 1A) that has been termed the RGS-N domain (9). The significance of this region is
indicated by its extraordinary conservation (e.g. 69% identity comparing EGL-10 and RGS7,
4
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Fig. 1B).
The function of the RGS-N region is not clearly understood. It contains an 80-100
amino acid subregion known as the DEP domain, named for its weak sequence similarity (10-
19% identity) comparing the Dishevelled, EGL-10, and pleckstrin proteins (14). Two studies
analyzed the stimulation of Gαt GTPase activity by N-terminal truncation mutants of RGS9
deleted for the RGS-N region. One study saw little effect of the RGS-N region (15). The other
(16), using a different method of kinetic analysis, found that the RGS-N region contributed to
the ability of RGS9 to act preferentially on Gαt when Gαt is complexed with the gamma subunit
of its effector, phosphodiesterase. Recent studies of RGS9 have also shown that its RGS-N
region binds an anchoring protein that tethers it to the membrane (17, 18).
In this study, we show that the RGS-N regions of EGL-10 and EAT-16 contribute to the
functions and distinct Gα target specificities of these RGS proteins in vivo. Using EGL-
10/EAT-16 chimeras, we found that the manner in which the RGS-N and GGL/RGS regions are
linked influences whether Gαo or Gαq is selected for inhibition. Most intriguingly, we found
that the RGS-N region can direct Gα inhibition by the GGL/RGS region even when these
regions are expressed as separate polypeptides. This observation can be explained by our finding
that the RGS-N region directly or indirectly binds to the GGL/RGS region and its associated Gβ
subunit GPB-2, and the complex thus formed appears to be the functional unit that acts on Gα
targets in vivo.
EXPERIMENTAL PROCEDURES
Plasmids for Neural Expression of RGS proteins--A 2.2 kb PstI/PflMI fragment of the
5
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
rgs-1 promoter (19) was inserted into pPD49.26 (20) to construct pGP3, a vector that directs
expression of inserted open reading frames in all neurons of C. elegans. cDNA fragments coding
full-length EGL-10 or EAT-16 were inserted into pGP3 to generate plasmids pGP4 and
pMK340, respectively. Conserved protein subregions were identified in alignments of EGL-10
and EAT-16 with each other and their human homologs (Fig. 1B), and cDNA fragments coding
some of these regions were inserted into pGP3. For expression of C-terminal fragments of
EGL-10 or EAT-16, an artificial AUG codon was added to initiate translation. The extents of
the EGL-10 subregions, the names designating them, and the corresponding expression plasmids
were: residues 1-223, EGL-N, pGP39; residues 224-334, EGL-L, (not expressed
individually); and residues 335-555, EGL-C, pGP40. The subregions, designations, and
plasmids for EAT-16 were: residues 1-201, EAT-N, pGP51; residues 202-211, EAT-L, (not
expressed individually); and residues 212-473, EAT-C, pGP52. An alternative EGL-N
fragment, consisting of residues 1-243 of EGL-10, was also tested: it behaved in all respects
like the smaller 223 residue EGL-N fragment (data not shown). The FL-N construct pGP91 was
made by adding sequences encoding DYKDDDDKDYKDDDDK (containing two FLAG tags)
immediately after the initiating AUG codon of the EGL-N construct pGP39. Similarly, the HA-
C construct pGP92 was made by adding sequences encoding GYPYDVPDYAGYPYDVPDYA
(containing two HA tags) after the AUG codon of the EGL-C construct pGP40.
Derivatives of pGP3 were also constructed to express EGL-10/EAT-16 chimeras
composed of the N, L, and C subregions described above. The chimeras used and the
corresponding expression plasmids were: EGL-N/EGL-L/EAT-C, pGP55; EGL-N/EAT-
L/EAT-C, pGP58; EGL-N/EAT-L/EGL-C, pGP59.
Transgenic animals--Test plasmids were coinjected with a marker plasmid into the
6
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
gonad of C. elegans to generate extrachromosomal transgene arrays (20). Animals from
transgenic lines were recognized among the F2 progeny because they showed the phenotype
induced by the marker plasmid. Rescue of egl-10 was tested by injection into animals of
genotype egl-10(md176); lin-15(n765). The lin-15 rescuing plasmid pL15EK (11) was
coinjected at 50 ng/µl as the marker. Rescue of eat-16 was tested by injection into animals of
genotype eat-16(ad702), and the marker plasmid pRF4 (20), which induces a dominant Rol
phenotype, was coinjected at 80 ng/µl as the marker. To test for dominant negative effects,
injections were into lin-15(n765) animals carrying no RGS mutations, and pL15EK was used as
the marker plasmid. Each RGS expression plasmid tested was injected at 80 ng/µl, and pGP3
(empty vector) DNA was included in certain injections so that the total expression plasmid DNA
concentration was identical for every injection.
Extrachromosomal transgenes expressing FLAG and HA epitope-tagged EGL-10
fragments were chromosomally integrated by irradiating transgenic animals with γ-rays. The
resulting strains were outcrossed to egl-10(md176); lin-15(n765) animals at least two times to
produce clean genetic backgrounds. The integrated FL-N and HA-C transgenes shown in this
work have the allele designations vsIs20 and vsIs18, respectively. vsIs18 male animals were
mated to vsIs20 hermaphrodites and the resulting cross-progeny were allowed to self-fertilize to
produce animals homozygous for both vsIs20 and vsIs18. The genotypes of all these strains were
verified by PCR amplification of the transgenes.
Behavioral Assays--Unlaid eggs were counted by dissolving adult animals in bleach and
counting the bleach-resistant fertilized eggs under a microscope (10). All assays were on
animals selected as late L4 larvae and aged at 20° C for 30 hours to produce precisely-staged
adults. For each extrachromosomal transgene analyzed, at least 50 animals were assayed (e10
7
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
animals from at least five independent transgenic lines). For integrated transgenes, e30 animals
were assayed. In certain cases we also carried out a second egg-laying assay in which the
developmental stages of freshly laid eggs were determined (10). In each case, this verified that
the transgenes affected egg-laying behavior, not egg production, indicating that the transgenes
generated normal EGL-10 and EAT-16 activities (data not shown).
C. elegans Protein Extracts--Worm strains carrying the egl-10(md176) null mutation as
well as the integrated transgene(s) vsIs18 and/or vsIs20 were grown in liquid culture at 20º C as
mixed-stage populations. Worms were purified by flotation on 30% sucrose and transferred to
lysis buffer (50 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 150 mM NaCl,
protease inhibitors, and in certain experiments, 1% Triton X-100). Lysis was by three passages
through a French Press followed by 60 seconds of sonication with a microtip probe (Fisher
Scientific Model 550 Sonic Dismembrator). Debris and unlysed worms were removed by
centrifugation at 2,000 RPM in a clinical centrifuge. The resulting “total lysates” were flash-
frozen in liquid nitrogen and stored at –80º C. Protein concentrations were determined by
Bradford analysis. When required, total lysates were fractionated into soluble and pellet fractions
by centrifugation at 100,000Xg for 30 minutes. To assess the levels of FLAG- and HA-tagged
proteins in total lysates (Fig. 6), 50 and 160 µg total protein, respectively, were fractionated by
SDS-PAGE. We further analyzed total lysates by sucrose density gradient centrifugation.
Sucrose gradients were formed by successively overlaying a 49% (w/v) sucrose cushion with
equal volumes of 20% sucrose and total lysate. Centrifugation was carried out in a TLS-55
swinging-bucket rotor at 55,000 RPM for 2 hours at 4º C. Twelve equal volume fractions were
collected with fraction 1 at the top and fraction 12 (including any pellet fraction) at the bottom.
8
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
The results shown in Fig. 6 and Fig. 7 are representative of those obtained in multiple trials
carried out with independently prepared lysates.
Western Blotting-- Proteins were fractionated by SDS-PAGE and then transferred to
nitrocellulose filters. The primary antibodies used to probe Western blots were: mouse anti-
FLAG (M2; Sigma); rat anti-HA High Affinity (3F10; Roche Molecular Biochemicals); mouse
anti-beta tubulin (E7; developed by Michael Klymkowsky and obtained from the Developmental
Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological
Sciences); rabbit anti-GPB-2 (13); and rabbit anti-GOA-1 (13). The secondary antibodies were
horseradish peroxidase-coupled goat anti-mouse (Bio-Rad), goat anti-rabbit (Bio-Rad) or goat
anti-rat (Pierce). Protein bands were visualized using chemiluminescence detection reagents
(Pierce) and Kodak BioMax MR film. The proteins studied had mobilities on SDS-PAGE
analysis approximately as follows: HA-C, 28 kDa; FL-N, 32 kDa; beta tubulin, 55 kDa; GPB-
2 isoforms, 42 and 44 kDa; and GOA-1, 40 kDa.
Immunoprecipitation--Soluble fractions of extracts made in lysis buffer with 1% Triton
X-100 were incubated with anti-FLAG M2 antibodies coupled to agarose beads (Sigma),
tumbling for 2 hours at 4º C. The beads were washed three times in lysis buffer containing 1%
Triton X-100 and pelleted by centrifugation. Proteins bound to the pelleted beads were eluted in
100 mM glycine, pH 3.5 for 5 minutes with gentle shaking at room temperature and neutralized
with 10% elution volume of 0.5 M Tris, pH 7.4, 1.5 M NaCl. The results shown in Fig. 8 are
representative of those obtained in >3 trials.
RESULTS
EGL-10 and EAT-16 Have Opposite Effects Even When Expressed from the Same
9
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Promoter--EGL-10 and EAT-16 have precisely opposite mutant phenotypes because EGL-10
specifically inhibits Gαo and EAT-16 specifically inhibits Gαq. In principle, these RGS proteins
might achieve such specificity by being expressed in different cells that also express different Gα
proteins. Alternatively, the RGS and Gα proteins might all be found in the same cells, but each
RGS protein could have the ability to selectively act on only one of the Gα targets available to it.
This latter possibility is supported by the observation that both RGS and both Gα proteins are
expressed in all the neurons of C. elegans, although each RGS and Gα protein is also
additionally expressed in muscle cells that may differ for each protein (10, 11, 21-23).
To distinguish between these alternative models, we constructed transgenes in which the
same promoter was used to direct expression of either the EGL-10 or the EAT-16 cDNA. We
used the rgs-1 promoter, which is active in all neurons of C. elegans but in no other cells (19).
The constructs were transformed into egl-10 or eat-16 null mutants and tested for their ability to
rescue the opposite defects in egg-laying behavior seen in the two mutants.
egl-10 mutants are lethargic in their egg-laying behavior and thus accumulate excess
unlaid eggs compared to control animals (compare Fig. 2 A and B). Transgenic expression of
EGL-10 rescued the egl-10 egg-laying defect (Fig. 2C). In contrast, expression of EAT-16
from the same heterologous promoter did not affect the egl-10 mutant (Fig. 2D). We quantitated
our results by counting the number of unlaid eggs retained inside animals carrying the different
transgenes (Fig. 2E). The egl-10 transgene rescued the egl-10 defect fully, and even resulted in
slightly hyperactive egg laying, seen as a decrease in the accumulation of unlaid eggs relative to
the wild-type control (Fig. 2E, compare bars 1 and 3). Overexpressing EGL-10 has previously
been shown to cause a gain-of-function effect that results in hyperactive egg laying (10). The
10
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
EAT-16 protein had no detectable EGL-10 activity, since expression of EAT-16 did not reduce
the accumulation of unlaid eggs (Fig. 2E, bar 4).
eat-16 mutants are hyperactive in their egg-laying behavior (11). Whereas wild-type
animals retain their fertilized eggs for two or more hours before laying them, eat-16 mutants lay
their eggs very soon after fertilization, resulting in a decreased accumulation of unlaid eggs (Fig.
2F, compare bars 1 and 2). Transgenic expression of EAT-16 resulted in substantial rescue of
the eat-16 egg-laying defect (Fig. 2F, bar 4), whereas expression of EGL-10 using the same
promoter resulted in no detectable rescue of the eat-16 defect (Fig. 2F, bar 3). The rescue
resulting from EAT-16 expression may have been incomplete either due to insufficient levels of
expression from the heterologous promoter, or because EAT-16 expression may be required
outside of the nervous system to achieve full rescue.
These experiments demonstrate that, even when expressed from the same heterologous
promoter, EGL-10 and EAT-16 have opposite effects on egg laying. Neural expression of either
RGS protein can fully (EGL-10) or substantially (EAT-16) rescue loss of endogenous
expression of the same RGS protein, but cannot rescue loss of the other RGS protein at all. We
conclude that EGL-10 and EAT-16 have distinct effects on egg laying due to distinct properties
of these RGS proteins themselves, and that any small differences in their endogenous expression
patterns do not account for their different functions.
The EGL-10 N- and C-Terminal Fragments Need Not Be Covalently Attached for
EGL-10 Function--We tested the ability of subregions of EGL-10 and EAT-16 to function in
vivo. The fact that the linker between the RGS-N region and the C-terminal GGL/RGS region is
variable in length and sequence among different RGS proteins (Fig. 1B) suggested that a precise
attachment between these two regions may not be required, and encouraged us to try expressing
11
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
the two regions as separate polypeptides in transgenic animals to test for their function. The same
neural-specific promoter used above for expressing full-length RGS proteins was also used to
express the protein subfragments. In addition, we tested coexpression of these fragments by
cotransforming expression constructs for each. The expression constructs were transformed into
egl-10 or eat-16 null mutants and tested for their ability to rescue the egg-laying defects of these
mutants. The constructs were also transformed into animals with no RGS mutations to test for
dominant-negative effects on egg laying.
Experiments in which protein fragments are expressed suffer from the problem that such
fragments may not be properly folded or stable, making negative results difficult to interpret. We
set a stringent criterion for dealing with this issue: we do not present or interpret results from
constructs that give only negative results. However, if a construct gives strong rescuing activity
in one experiment, we do interpret negative results it may give in other experiments because the
positive result demonstrates that the construct successfully expresses an active protein fragment.
Figure 3A shows the effects of EGL-10 fragment expression in an egl-10 null mutant
background. We refer to the N- and C-terminal fragments of EGL-10 (indicated in Fig. 1A) as
EGL-N and EGL-C, respectively. Expression of either EGL-N or EGL-C only weakly rescued
the egl-10 egg-laying defect (Fig. 3A, bars 3 and 4). Surprisingly, coexpression of both
fragments gave full rescue of the egl-10 mutant (Fig. 3A, bar 5), equivalent to the strong rescue
previously seen by expressing full-length EGL-10 (Fig. 2E, bar 3). This result demonstrates that
EGL-N and EGL-C act together to inhibit Gαo signaling, and that EGL-N need not be
covalently attached to EGL-C for full EGL-10 function.
We tested the EGL-10 fragments for EAT-16 activity by expressing them in the eat-16
12
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
null mutant, but found that neither fragment nor the combination of both showed much activity
(Fig. 3B). We also tested the EGL-10 fragments in a background carrying no RGS mutations to
test the fragments for dominant-negative effects (Fig. 3C). Interference with endogenous EGL-
10 function would be seen as an increased accumulation of unlaid eggs. Expression of EGL-N
alone (Fig 3C, bar 2) gave a result similar to the control (bar 1). Expression of either EGL-C
alone or coexpression of EGL-C and EGL-N also did not show dominant-negative effects.
Rather, expression of these fragments resulted in small decreases in the accumulation of unlaid
eggs ( Fig. 3C, bars 3 and 4) showing that these constructs gave positive EGL-10 activity, just as
they did in the egl-10 mutant background (Fig 3A, bars 4 and 5).
We also tested constructs expressing N- and C-terminal fragments of EAT-16 (termed
EAT-N and EAT-C, respectively) in egl-10, eat-16, and wild-type RGS backgrounds. Neither
fragment nor the combination of both showed strong effects in any background tested (data not
shown). According to our criterion for interpretability outlined above, we therefore do not
interpret these experiments. Although we know EAT-C is active (see below), EAT-N may
simply not be folded or stable.
The RGS-N Region of EGL-10 Can Direct the GGL/RGS Region of EAT-16 to Have
Full EGL-10 Activity--To identify the regions of EGL-10 and EAT-16 responsible for their
distinct Gα target specificities, we generated transgenes to express EGL-10/EAT-16 chimeras,
transformed them into egl-10 and eat-16 null mutants, and tested for their ability to rescue the
egg-laying defects of these mutants. In these experiments we hoped to identify discrete protein
subregion(s) that determine EGL-10 activity versus EAT-16 activity.
Our first strategy for generating chimeras was based on the observation that the N- and
C-terminal fragments of EGL-10, when coexpressed, give full EGL-10 function. We
13
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
coexpressed combinations of N- and C-terminal fragments from EGL-10 and EAT-16 to see if
these combinations, which we term "noncovalent chimeras", could give EGL-10 or EAT-16
function.
We found that coexpression of the EGL-N fragment with the C-terminal GGL/RGS
fragment of EAT-16 (EAT-C) gave full EGL-10 function. As shown in Fig. 4A, expression of
either fragment alone in an egl-10 null background gave little rescue of the egl-10 egg-laying
defect (bars 3 and 4). When coexpressed, however, the EGL-N and EAT-C fragments fully
rescued the egl-10 mutant (bar 5). Indeed, the number of unlaid eggs in the animals
coexpressing the fragments was below that of the wild-type control (bar 1), indicating that
animals coexpressing EGL-N and EAT-C have excess EGL-10 activity and thus are slightly
hyperactive for egg laying. Expression of EGL-N, EAT-C, or both gave no rescue of the eat-16
egg-laying defect (Fig. 4B, bars 3-5), showing that these fragments have no detectable eat-16
activity.
We also coexpressed the EAT-N fragment with the C-terminal fragment of EGL-10, but
found this noncovalent chimera was unable to rescue either egl-10 or eat-16 mutants (data not
shown). As discussed above, we have no evidence that the EAT-N fragment was successfully
expressed and folded in these experiments and thus do not interpret these negative results.
Our main finding from the use of noncovalent chimeras was that expression of the EGL-
N and EAT-C fragments resulted in full EGL-10 activity and gave results essentially identical
to those seen when EGL-N and EGL-C were coexpressed (Fig. 3). In the context of these
experiments, the EGL-C and EAT-C fragments are thus equivalent and interchangeable.
Whereas the GGL/RGS region of EAT-16 would normally act to inhibit Gαq signaling, when
14
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
this region of EAT-16 is coexpressed with the RGS-N region of EGL-10, it is instead
apparently directed to inhibit Gαo signaling.
The Relationship between the RGS-N and GGL/RGS Regions Directs Gα Target
Specificity--To refine our understanding of Gα target specification, we generated transgenes
that express full-length chimeric RGS proteins as single polypeptides. We term these "covalent
chimeras" to distinguish them from the noncovalent chimeras described above. In designing
these chimeras, we divided EGL-10 and EAT-16 into three subregions: 1) the N-terminal
RGS-N domain; 2) the linker region between the RGS-N domain and the GGL domain; 3) a C-
terminal region consisting of the GGL and RGS domains and residues C-terminal to them (see
Fig. 1A). We generated the complete set of six chimeras in which each of the three subregions
was derived from either EGL-10 or EAT-16 in every possible combination. For example, the
"EGL-N/EAT-L/ EAT-C" chimera contains the RGS-N domain of EGL-10, followed by the
linker from EAT-16, and finally the C-terminal GGL/RGS region from EAT-16. The chimeras
were expressed using the same neural-specific promoter employed in the experiments described
above. As before, we present data only from chimeras that gave strong rescuing activity in either
the egl-10 or eat-16 backgrounds. Purely negative results were obtained from the three chimeras
containing the EAT-N region, and these results were considered uninterpretable.
A key factor determining whether a chimera had EGL-10 activity or EAT-16 activity
was the manner in which the RGS-N and C-terminal regions were linked. For example, the
EGL-N/EGL-L/EAT-C chimera gave strong EGL-10 activity (Fig. 4A, bar 6) and little EAT-
16 activity (Fig. 4B, bar 6). In this chimera, the EGL-N region apparently directs the EAT-C
region to have EGL-10 activity, just as occurred when these two regions were coexpressed as
15
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
separate polypeptides (Fig. 4 A and B, bars 5). However, swapping from the EGL-10 to the
EAT-16 linker caused a switch from EGL-10 activity to EAT-16 activity. This is seen in the
EGL-N/EAT-L/EAT-C chimera, which has strong eat-16 rescuing activity (Fig. 4B, bar 7) but
little egl-10 rescuing activity (Fig. 4A, bar 7).
The linker region is not the sole determinant of Gα target specificity. If this were true, the
EGL-N/EAT-L/EGL-C chimera would be expected to show EAT-16 activity rather than EGL-
10 activity. Instead, this chimera shows strong EGL-10 activity (Fig. 4A, bar 8) and little EAT-
16 activity (Fig. 4B, bar 8). This chimera demonstrates that the C-terminal GGL/RGS region
also contributes to Gα target specificity, since the EGL-10 activity of this chimera can be
converted to EAT-16 activity by swapping the C-terminal region to that of EAT-16 (Fig. 4 A
and B, compare bars 7 and 8).
In summary, analysis of EGL-10 and EAT-16 transgenes shows that the C-terminal
GGL/RGS regions of these proteins are not sufficient for in vivo function, but require a N-
terminal conserved region to gain full function in vivo. Surprisingly, the two protein regions
need not be covalently linked to function together. However, if they are covalently linked, the
manner in which they are attached can determine which Gα protein is selected as a target.
Functional Epitope-Tagged EGL-10 N- and C-Terminal Fragments for Biochemical
Analysis of Their Interactions--Perhaps the most intriguing result from our transgenic
experiments is our finding that neither the EGL-N nor EGL-C fragments are able to rescue egl-
10 mutant animals, but that coexpression of these fragments as separate polypeptides does give
full EGL-10 function (Fig. 3A). For the remainder of this work, we present a biochemical
analysis of these two protein fragments and their interactions in extracts of transgenic animals.
We modified our original EGL-10 N- and C-terminal fragments to include FLAG and
16
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
HA epitope tags, respectively. We refer to these modified fragments as FL-N and HA-C. We
generated transgenes expressing these fragments in C. elegans and chromosomally integrated the
transgenes to produce stable transgenic lines that could be grown in biochemical quantities. An
additional benefit of the integrated transgenes is that we could genetically cross the two strains
carrying the individual FL-N and HA-C transgenes to generate a strain carrying both
transgenes. Thus proteins expressed from the exact same FL-N or HA-C transgenes could be
compared when expressed alone or in combination.
Before analyzing the tagged proteins biochemically, we checked their function in vivo by
testing their ability to rescue egl-10 mutant animals. We found that the tagged fragments (Fig. 5)
behaved similarly to their untagged counterparts (Fig. 3A). We note one subtle but interesting
difference: while the untagged N transgene appeared to have a small amount of egl-10 rescuing
activity (Fig. 3A, bar 3), the FL-N transgene (perhaps due to a lower expression level) had no
such detectable activity (Fig. 5, bar 3). Nevertheless, the FL-N transgene, when combined with a
partially rescuing C-terminal transgene, could give rise to full egl-10 rescuing activity (Fig. 5,
bar 5). These results suggest that the cooperation between N- and C-terminal fragments may be
synergistic rather than merely additive.
FL-N and HA-C Proteins Show Increased Abundance When Coexpressed--We carried
out Western analyses of total worm extracts from strains carrying the same FL-N and HA-C
transgenes separately or together. Our results showed that the FL-N and HA-C proteins were
both increased in abundance when coexpressed with each other (Fig. 6). Blots carrying different
loadings of the samples to achieve equivalent signals revealed about a two-fold enhancement in
the levels of FL-N and HA-C when coexpressed (data not shown). Since the HA-C protein by
itself showed partial rescue of the egl-10 mutant (Fig. 5, bar 4), the increase in HA-C protein
17
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
upon coexpression with FL-N could, at least in part, explain the full rescue observed when these
proteins were coexpressed (Fig. 5, bar 5). One way the FL-N and HA-C proteins might increase
each others’ abundance is by forming a complex and thus stabilizing each other. In order to test
this hypothesis by coimmunoprecipitation, we first searched for conditions that could solubilize
the FL-N and HA-C proteins for precipitation.
Coexpression of FL-N with HA-C Decreases the Solubility of HA-C, and Both Proteins
Can Be Solubilized with Triton X-100--We fractionated total worm extracts by 100,000Xg
centrifugation into soluble and pellet fractions in the presence or absence of detergent. We used
Western blots to determine the solubility of the FL-N and HA-C proteins when expressed
separately or together (Fig. 7A). In the absence of detergent, HA-C expressed alone was more
than 50% soluble. However, when coexpressed with FL-N, almost all of the HA-C protein
moved to the pellet fraction. This insoluble HA-C could be substantially solubilized by addition
of 1% Triton X-100 (compare top two panels in Fig. 7A). In contrast to HA-C, FL-N, whether
expressed alone or in the presence of HA-C, was almost entirely in the pellet fraction. Addition
of 1% Triton X-100 also resulted in solubilization of a significant amount of FL-N (compare
bottom two panels in Fig. 7A).
To distinguish whether the insolubility of FL-N and HA-C observed in the absence of
detergent was due to membrane association or due to the formation of particulate/aggregate
structures, we carried out sucrose density gradient centrifugation on total lysates containing these
proteins. Our results revealed that the bulk of the insoluble FL-N and HA-C floated in fractions
8 and 9 of these gradients, which comprised the 20%/49% sucrose interface where membrane-
associated proteins typically reside (top two panels in Fig. 7B). No FL-N or HA-C were
detected in fraction 12, which included the pellet where any aggregates should be found. Western
18
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
analysis of the same sucrose gradients showed that fractions 8 and 9, in addition to containing
FL-N and HA-C, also contained endogenous membrane-associated GOA-1, the Gα target of
EGL-10 (lower panel in Fig. 7B). FL-N and HA-C floated rather than pelleted in sucrose
gradients regardless of whether these two proteins were coexpressed (Fig. 7B), or expressed
individually (data not shown). FL-N and HA-C therefore appear to be membrane-associated,
and their insolubility is not due to aggregation.
Since the RGS domain found in the EGL-10 C terminus is believed to act directly on the
membrane-associated Gα protein GOA-1, the apparent increase in HA-C membrane
localization upon coexpression with FL-N could explain, at least in part, the increase from
partial to full egl-10 rescuing activity that occurs when HA-C is coexpressed with FL-N. The
effect of FL-N on the solubility of HA-C suggests that FL-N may form a complex with HA-C
and thereby affect not only its stability, but also its membrane localization. Using the 1% Triton
solubilization conditions shown in Fig. 7A, we have tested this hypothesis by
coimmunoprecipitation.
FL-N forms a complex with HA-C and its associated Gβ subunit--To determine if FL-
N and HA-C form a complex, we immunoprecipitated FL-N from Triton-solubilized extracts of
worms expressing both FL-N and HA-C and tested for coprecipitation of HA-C. As controls,
we used extracts of worms expressing only FL-N or HA-C, which should not show any
coprecipitated signal. We found that HA-C could be immunoprecipitated by the FLAG antibody
only in the presence of FL-N, demonstrating that HA-C forms a complex with FL-N in worm
extracts (Fig. 8). We expected that the Gβ subunit, GPB-2, should also be in this complex since
it had previously been shown to be an obligate subunit of EGL-10, presumably via an interaction
with the GGL domain (13). Indeed we found that GPB-2 coimmunoprecipitates with FL-N (Fig.
19
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
8, lower panel). Interestingly, GPB-2 forms a complex with FL-N even in the absence of HA-
C, suggesting that both the RGS-N and GGL regions of EGL-10 may each have an independent
ability to bind the Gβ subunit.
DISCUSSION
An Experimental Approach Focused on the Physiologically Relevant Determinants of
RGS-Gα Specificity--The RGS domains of a number of RGS proteins have been found to
promiscuously activate the GTPase activities of many Gα proteins in vitro (1). These puzzling
results are at odds with the expectation that different RGS proteins might achieve distinct
functions in vivo at least in part by targeting different Gα proteins. The Gα target specificity of
RGS proteins might be increased in vivo by other cellular proteins or by non-RGS domain
regions of RGS proteins themselves. Several studies have compared purified full-length and
deleted versions of RGS proteins in in vitro GTPase activation assays to test for effects of
regions outside the RGS domain on Gα target specificity (12, 15, 16, 24). These studies might
not identify functions crucial to RGS protein activity in vivo, such as membrane localization or
interaction with cellular proteins other than Gα. An additional problem is that, in such studies,
RGS9-1 (the retinal-specific isoform of RGS9) was the only RGS protein tested that had a
known, physiologically relevant Gα target identified by genetic studies. RGS9-1 is atypical in
that it acts in rod outer segments where Gαt is the only Gα protein present at a significant level.
Thus RGS9-1 does not physiologically face the challenge of selecting a Gα target as do other
RGS proteins that are typically expressed in cells containing multiple Gα proteins. Another
20
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
experimental approach used to analyze the basis of Gα target selection by RGS proteins involves
expressing full-length or deleted RGS proteins in cultured cells or Xenopus oocytes and using
assays that indirectly measure signaling by Gα targets (reviewed in Ref. 1). In these studies, the
physiologically relevant Gα targets of the RGS proteins used, again, are typically not known,
and the signaling readouts arranged for purposes of the experiments may have no relation to the
normal physiological functions of the RGS proteins studied.
In contrast, our studies analyze two RGS proteins, EGL-10 and EAT-16, that genetic
studies have shown to target two distinct Gα proteins, Gαo and Gαq, respectively. Previous
studies showed that these RGS and Gα proteins are all expressed in the same cells, suggesting
that the RGS proteins must actively select from at least two accessible Gα targets. We
demonstrated this more clearly by using transgenes to express EGL-10 and EAT-16 from the
same heterologous promoter and showing that they retained their proper Gα target specificities.
Using this same promoter, we have also expressed subregions or chimeras of EGL-10 and EAT-
16 and measured their functions in vivo using assays of egg-laying behavior. Importantly, this
readout of RGS function measures the normal physiological actions of EGL-10 and EAT-16 on
their genetically identified Gα targets. Our experimental approach also allows us to use extracts
of the transgenic animals to biochemically analyze the RGS proteins expressed. Thus, our
experimental system is designed to analyze RGS function and Gα target selectivity in a
physiological setting, and enables us to correlate in vivo and in vitro data.
The RGS-N Region Is Essential for Activity of EGL-10 and EAT-16, and Has a
Membrane Targeting Function--Studies of mammalian RGS proteins have shown that their
21
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
GGL/RGS regions can function in vitro as efficient Gα GTPase activators even when the RGS-
N domain has been removed (15, 16). In contrast, our results show that the GGL/RGS regions of
either EGL-10 or EAT-16 have relatively little function in vivo unless attached to or
coexpressed with an RGS-N region. The differences between these results can be explained, at
least in part, by the fact that the in vitro assays were carried out in the absence of membranes,
while in vivo the RGS-N region functions to target RGS proteins to the membrane, the location
of their Gα targets.
By analyzing soluble and membrane fractions of C. elegans extracts, we found that the
RGS-N and GGL/RGS regions are each independently targeted to membrane fractions, although
less than 50% of the GGL/RGS region, when expressed alone, ended up in the membrane
fraction. When complexed with an RGS-N region, however, the GGL/RGS region was almost
entirely targeted to the membrane. Our results correlate with studies of RGS9, which also
identified membrane targeting functions in both the N- and C-termini of this protein. A portion
of the RGS-N region contains weak similarity to a region of Dishevelled (the "DEP domain")
that serves as a membrane anchor (25). In RGS9, the RGS-N domain binds the protein R9AP,
which anchors it to the rod outer segment membrane (18). Lishko et al. (17) showed that this
membrane association results in a ~70-fold increase in the activity of RGS9 on its Gα target. In
C. elegans there is no clear homolog of R9AP that could serve as a membrane anchor for EGL-10
and/or EAT-16, and the nature of the membrane attachment of RGS-N domains in C. elegans
remains to be elucidated. It is possible that there is a distant homolog of R9AP that does not
stand out as statistically significant in homology searches of the C. elegans genome, but may
serve as a membrane anchor for EGL-10 and/or EAT-16. It is also possible that such a
membrane anchor will be uncovered in the future by cloning additional genetically-identified
22
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
genes that affect C. elegans egg-laying behavior.
The RGS-N Region Directs Gα Target Specificity--How are RGS proteins directed to a
specific Gα target? Our studies of EGL-10 and EAT-16, as well as recent work on the
mammalian members of the same RGS protein subfamily (RGS6, 7, 9, and 11), are beginning to
answer this question. Studies of mammalian RGS proteins have shown that the RGS domain is
sufficient on its own to act as an efficient Gα GTPase activator (26). Thus the RGS domain
might have been expected to fully specify the Gα target. However, addition of the GGL domain
and the Gβ5 subunit appeared to restrict the specificity of the RGS domain in vitro, making it
more selective for Gαo (12, 24, 27). RGS-Gβ5 complexes need not always be Gαo-selective in
vivo since EAT-16 is in a complex with the Gβ5-like protein GPB-2 but is a Gαq selective
regulator (11, 13). Thus, at least in the case of EGL-10 and EAT-16, which both individually
bind to GPB-2, specificity appears to be achieved by some means other than association with a
Gβ subunit.
Our in vivo experiments show that an interaction between the RGS-N region and the
GGL/RGS region specifies the Gα protein targets of EGL-10 and EAT-16. We were able to
direct the GGL/RGS regions of either EGL-10 or EAT-16 to act on either GOA-1 or EGL-30,
depending on how the GGL/RGS regions were attached to the RGS-N region of EGL-10. For
example, EAT-16 normally acts on the Gαq protein EGL-30, but the EAT-16 GGL/RGS
region can be made to act on the Gαo protein GOA-1 when coexpressed with the RGS-N
domain of EGL-10 (Fig. 4). Our results showing a role for the RGS-N region in recognition of
Gα targets are consistent with in vitro studies of RGS9. These studies showed that the RGS-N
23
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
region of RGS9 helps allow it to act preferentially on a complex of Gαt with the gamma subunit
of phosphodiesterase versus on Gαt alone (15, 16). More recent work has shown that RGS9 can
form a stable complex with the GDP-bound form of Gαt using contacts other than those
previously characterized between the RGS domain of RGS9 and the switch regions of Gαt (18).
Our studies and the work on RGS9 together suggest that the RGS-N region may contact Gα
proteins and contribute to Gα target specificity. The RGS-N region is very large (~220 amino
acids). However, neither our work nor the studies of RGS9 have yet identified the features within
this region that allow it to specify Gα targets, nor have any RGS-N binding proteins been
identified other than the membrane anchor R9AP described above.
The RGS-N and GGL/RGS Regions Are Associated by Both a Noncovalent Interaction
as well as a Covalent Linker--A surprising aspect of our results is that the RGS-N and
GGL/RGS regions need not be covalently attached to function together. For example,
coexpression of these fragments of EGL-10 as separate polypeptides is sufficient to provide full
EGL-10 activity. Our results explain how this can occur, since we observed that the separately
expressed RGS-N and GGL/RGS fragments form a complex (Fig. 8), which appears to be the
functional unit. This identification of a noncovalent interaction between the RGS-N and
GGL/RGS regions is a novel observation. The binding interaction could be direct or indirect,
perhaps via the Gβ5-like GPB-2 subunit that is bound to the GGL region (13). The latter
possibility is suggested by our observation that the RGS-N fragment of EGL-10
coimmunoprecipitates with GPB-2 even in extracts lacking the GGL/RGS fragment of EGL-10.
Since GPB-2 binds to the GGL domains of both EGL-10 and EAT-16 (13), our observation
24
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
that the RGS-N fragment of EGL-10 can function with the GGL/RGS fragments of either
protein (Fig. 3A and Fig. 4A) could be explained if the RGS-N fragment binds directly to GPB-
2.
In our experiments, we transgenically expressed RGS-N and GGL/RGS regions as
separate protein fragments and induced them to bind and function together. In endogenous RGS
proteins these regions are covalently attached by a linker, and the linker may simply allow the
two regions to more efficiently find each other and form a functional unit. Alternatively,
complexes between the RGS-N region of one molecule and the GGL/RGS region of another
might occur normally, so that higher order complexes containing one or more type of RGS
protein might exist.
Studies of the yeast RGS protein Sst2p have given results intriguingly analogous to those
presented here for EGL-10 and EAT-16. Coexpression of N- and C-terminal fragments of
Sst2p gave more function in vivo than expression of either fragment alone (28). Sst2p is only
distantly related to EGL-10: it has no recognizable GGL domain, although its N-terminus has
weak similarity to the DEP domain of the RGS-N region. Sst2p is endoproteolytically processed
so that it can naturally exist as separate N- and C-terminal fragments (28). There is currently no
evidence, however, that the N- and C-terminal fragments of Sst2p form a complex with each
other, or that EGL-10, EAT-16, or any of their mammalian counterparts (RGS6, 7, 9, and 11)
are proteolytically processed.
The noncovalent linkage between the RGS-N and GGL/RGS regions cannot exist merely
for the purpose of attaching the GGL/RGS region to the membrane via the RGS-N region. The
covalent linkage that also exists between these regions would be sufficient for this purpose. The
noncovalent association between the regions is likely to have additional functional consequences,
25
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
including influencing Gα target specificity, as discussed above.
The covalent linkage between the RGS-N and GGL/RGS regions is not absolutely
required for RGS protein function, as shown by our functional coexpression of these regions as
separate polypeptides. However, the nature of the covalent linker can influence Gα target
specificity as demonstrated in our experiments with EGL-10/EAT-16 chimeras. The linker
regions of EGL-10 and EAT-16 are very different. The EGL-10 linker is ~110 residues long,
shows no conservation with the linker of the human EGL-10 ortholog RGS7, and is very rich in
proline, serine, alanine, and glycine (Fig. 1B). This linker may not fold into an ordered structure,
and thus may provide a long and flexible attachment between the RGS-N and GGL/RGS
regions. The ten residue EAT-16 linker, in contrast, would provide a short, rigid attachment that
might alter the geometry of the complex between the RGS-N and GGL/RGS regions. We note
that coexpression of RGS-N and GGL/RGS regions using the long EGL-10 linker gave results
similar to coexpressing these regions as separate polypeptides: in every case where activity was
seen, it was EGL-10 activity, not EAT-16 activity. These cases include full-length EGL-10,
coexpression of EGL-N with either EGL-C or EAT-C, and the EGL-N/EGL-L/EAT-C
chimera. In contrast, when the short EAT-16 linker was used, any activity observed was usually
EAT-16 activity, not EGL-10 activity. These cases include full-length EAT-16, and the EGL-
N/EAT-L/EAT-C chimera. We speculate that the geometry of the complex allowed by a
flexible linker, or no linker at all, between the RGS-N and GGL/RGS regions promotes EGL-10
activity (targeting Gαo), while a rigid attachment between these regions promotes EAT-16
activity (targeting Gαq).
26
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Acknowledgements--This paper is dedicated to the memory of our late colleague Paul
Sigler who provided inspiration and insight during the early stages of this work.
27
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
REFERENCES
1. Ross, E. M., and Wilkie, T. M. (2000) Annu. Rev. Biochem. 69, 795-827
2. Tesmer, J. J. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261
3. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452
4. Diversé-Pierluissi, M. A., Fischer, T., Jordan, J. D., Schiff, M., Ortiz, D. F., Farquhar, M. G.,
and De Vries, L. (1999) J. Biol. Chem. 274, 14490-14494
5. Huang, C., Hepler, J. R., Gilman, A. G., and Mumby, S. M. (1997) Proc. Natl. Acad. Sci. U. S.
A. 94, 6159-6163
6. Heximer, S. P., Watson, N., Linder, M. E., Blumer, K. J., and Hepler, J. R.
(1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14389-14393
7. Ingi, T., Krumins, A. M., Chidiac, P., Brothers, G. M., Chung, S., Snow, B. E., Barnes, C. A.,
Lanahan, A. A., Siderovski, D. P., Ross, E. M., Gilman, A. G., and Worley, P. F. (1998)
J. Neurosci. 18, 7178-7188
8. Chen, C.-K., Burns, M. E., He, W., Wensel, T. G., Baylor, D. A., and Simon, M. I. (2000)
Nature 403, 557-560
9. He, W., Cowan, C. W., and Wensel, T. G. (1998) Neuron 20, 95-102
10. Koelle, M. R. and Horvitz, H. R. (1996) Cell 84, 115-125
11. Hajdu-Cronin, Y. M., Chen, W. J., Patikoglou, G., Koelle, M. R., and Sternberg, P. W.
(1999) Genes Dev. 13, 1780-1793
12. Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S.-F., Wall, M. A., Chung, S., Mangion,
J., Arya, S., Gilman, A. G., and Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. U. S. A.
95, 13307-13312
13. Chase, D. L., Patikoglou, G. A., and Koelle, M. R. (2001) Current Biol. 11, 222-
28
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
231
14. Ponting, C. P. and Bork, P. (1996) Trends Biochem. Sci. 21, 245-246
15. He, W., Lu, L., Zhang, X., El-Hodiri, H. M., Chen, C.-K., Slep, K. C., Simon,
M. I., Jamrich, M., and Wensel, T. G. (2000) J. Biol. Chem. 275, 37093-
37100
16. Skiba, N. P., Martemyanov, K. A., Elfenbein, A., Hopp, J. A., Bohm, A, Simonds, W. F., and
Arshavsky, V. Y. (2001) J. Biol. Chem. 276, 37365-37372
17. Lishko, P. V., Martemyanov, K. A., Hopp, J. A., and Arshavsky, V. Y. (2002) J. Biol. Chem.
277, 24376-24381
18. Hu, G., and Wensel, T. G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 9755-9760
19. Dong, M.-Q., Chase, D., Patikoglou, G. A., and Koelle, M. R. (2000) Genes Dev. 14, 2003-
2014
20. Mello, C. and Fire, A. (1995) in Methods in Cell Biology (Caenorhabditis elegans: Modern
Biological Analysis of an Organism), eds. Epstein, H. F. and Shakes, D. C. (Academic
Press, Inc., San Diego) 48, 451-482
21. Ségalat, L, Elkes, D. A., and Kaplan, J. M. (1995) Science 267, 1648-1651
22. Mendel, J. E., Korswagen, H. C., Liu, K. S., Hajdu-Cronin, Y. M., Simon, M. I., Plasterk, R.
H., and Sternberg, P. W. (1995) Science 267,1652-1655
23. Brundage, L., Avery, L., Katz, A., Kim, U. J., Mendel, J. E., Sternberg, P. W., and Simon, M.
I. (1996) Neuron 16, 999-1009
24. Posner, B. A., Gilman, A. G., and Harris, B. A. (1999) J. Biol. Chem. 274,
31087-
29
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
31093
25. Axelrod, J. D., Miller, J. R., Shulman, J. M., Moon, R. T., and Perrimon, N.
(1998) Genes Dev. 12, 2610-2622
26. Popov, S., Yu, K., Kozasa, T., and Wilkie, T. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94,
7216-7220
27. Shuey, D. J., Betty, M., Jones, P. G., Khawaja, X. Z., and Cockett, M. I. (1998) J.
Neurochem. 70, 1964-1972
28. Hoffman, G. A., Garrison, T. R., and Dohlman, H. G. (2000) J. Biol. Chem. 275, 37533-
37541
30
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Footnotes:
* This work was supported by a National Science Foundation predoctoral fellowship to G.A.P.,
by a Leukemia and Lymphoma Society Scholar award to M.R.K., and by grants from the
National Institutes of Health and the Robert Leet & Clara Guthrie Patterson Trust.
‡ To whom correspondence should be addressed: Dept. of Molecular Biophysics and
Biochemistry, Yale University School of Medicine, SHM C-E30, 333 Cedar Street, New Haven,
CT 06520-8024. Tel.: 203-737-5808; Fax: 203-785-6404; E-mail: [email protected].
1The abbreviations used are: RGS, regulator of G protein signaling; GGL, G gamma-like; DEP,
Dishevelled/EGL-10/pleckstrin homology; RGS-N, conserved N-terminal region found in
GGL-containing RGS proteins; EAT–(N, L, or C), N-terminal, linker, or C-terminal region of
EAT-16, respectively; EGL–(N, L, or C), N-terminal, linker, or C-terminal region of EGL-10,
respectively; FL-N, FLAG epitope-tagged EGL-10 N-terminal fragment; HA-C, HA epitope-
tagged EGL-10 C-terminal fragment.
31
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
FIGURE LEGENDS
FIG. 1. RGS proteins with opposing effects on C. elegans egg laying share several conserved
regions. A, schematic representation of the 555 amino acid EGL-10 protein. An N-terminal
conserved region of unknown function (RGS-N; gray box) contains a subregion known as the
DEP domain. A poorly conserved linker region (white box) is followed by a G gamma-like
domain (GGL, hatched box) and an RGS domain (black box). In this work we analyze N- and
C-terminal fragments of EGL-10 indicated by bars under the schematic. B, alignment of the
EGL-10, human RGS7 (hRGS7), and EAT-16 RGS proteins. Gray, hatched, and black shaded
bars (top) overlie sequences corresponding to the gray, hatched, and black shaded regions in the
EGL-10 schematic in A. Amino acids identical in two or three of the sequences are shaded
black. C, schematic representation of the opposing G protein signaling pathways that control
egg-laying behavior in C. elegans. Signaling through cell surface receptors activates the Gαο
and Gαq proteins (known in C. elegans as GOA-1 and EGL-30, respectively). GOA-1 inhibits
egg laying whereas EGL-30 activates this behavior. The RGS proteins EGL-10 and EAT-16
each exist as obligate dimers with the Gβ5 ortholog, GPB-2. EGL-10 inhibits GOA-1 activity,
while EAT-16 inhibits EGL-30. Thus EGL-10 and EAT-16 have opposite effects on egg-
laying behavior.
FIG. 2. Effects of transgenic expression of EGL-10 and EAT-16, using a heterologous neural
32
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
promoter, in egl-10 and eat-16 mutant animals. A, control adult hermaphrodite that carries an
empty vector transgene. B, egl-10 mutant carrying an empty vector transgene. C, egl-10 mutant
carrying a transgene expressing EGL-10 in all neurons using a heterologous promoter. D, egl-
10 mutant carrying a transgene expressing EAT-16 using the same promoter. Arrows in A-D
indicate individual unlaid fertilized eggs inside the adults. E, phenotypes shown in panels A-D
were quantitated by counting the number of unlaid eggs inside >50 animals for each genotype.
RGS mutant backgrounds and transgenes are indicated below the graph, and bars 1-4 correspond
to the genotypes shown in A-D, respectively. The egl-10 mutant phenotype, seen as an
accumulation of unlaid eggs, is rescued by expression of EGL-10 (bar 3), but not by expression
of EAT-16 (bar 4). Error bars indicate the 95% confidence interval of the mean. F, quantitation
of an experiment analogous to that shown in panel E, but examining rescue of eat-16 rather than
of egl-10. The eat-16 mutant phenotype, seen as a decrease in the number of unlaid eggs
retained in adults, is substantially rescued by expression of EAT-16 (bar 4), but not by
expression of EGL-10 (bar 3). Note the change of scale from panel E, and that the control (bar
1) is the same data as shown in panel E (bar 1). Control data from this figure are also replotted in
subsequent figures for purposes of comparison. The mutations used in this and subsequent
figures were egl-10(md176) and eat-16(ad702). Each is an apparent null mutation (10, 11).
FIG. 3. Effects of expressing EGL-10 protein fragments in egl-10, eat-16, and wild-type
animals. The fragments expressed were as indicated in Figure 1A: EGL-N, an N-terminal
fragment consisting of the RGS-N domain (indicated in figure as "N"); EGL-C, a C-terminal
fragment containing the GGL and RGS domains ("C"); or coexpression of both fragments
("N+C"). A, effects of EGL-10 protein fragment expression in the egl-10 null mutant
33
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
background. Individual expression of EGL-N (bar 3) or EGL-C (bar 4) showed only partial
rescue of the egl-10 egg-laying defect, while coexpression of both fragments showed full rescue
(bar 5). B, expression of the same EGL-10 fragments as shown in panel A, but in an eat-16
mutant background. None of the EGL-10 fragments showed significant rescue of the eat-16
egg-laying defect. C, effects of EGL-10 protein fragment expression in a wild-type RGS
background. No significant dominant-negative effects were observed. The EGL-C transgene
(bar 3) and the coexpression of EGL-N and EGL-C (bar 4) both showed some positive egl-10
activity as evidenced by decreases in the number of unlaid eggs relative to the control (bar 1).
FIG. 4. Effects of expressing EGL-10/EAT-16 chimeric proteins in egl-10 and eat-16 mutant
animals. A, effects of chimera expression in the egl-10 null mutant background. B, effects of
chimera expression in the eat-16 mutant background. Egg-laying behavior was quantitated by
counting unlaid eggs. The three components of the RGS proteins were as indicated in Figure 1A:
an N-terminal subregion consisting of the RGS-N domain (denoted as "N"); a linker subregion
("L"); and a C-terminal subregion containing the GGL and RGS domains, ("C"). The label for
each subregion is printed under the graph on a gray or black background to indicate that it is
derived from EGL-10 or EAT-16, respectively. Bars 3-5 in panels A and B show the results of
expressing the EGL-10 N-terminal fragment and EAT-16 C-terminal fragment as separate
polypeptides in the absence of any linker region. Bars 6-8 show the results of expressing
individual chimeric polypeptides in which regions from the two proteins were covalently linked.
Coexpression of the EGL-10 N-terminal fragment and EAT-16 C-terminal fragment (bars 5)
gave strong EGL-10 activity, while the EGL-N/EAT-L/EAT-C chimeric protein (bars 7) gave
substantial EAT-16 activity. Other covalent chimeras (bars 6 and 8) showed partial EGL-10
34
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
activity.
FIG. 5. Effects of chromosomally-integrated transgenes expressing epitope-tagged EGL-10
fragments in egl-10 mutant animals. The fragments expressed were: FL-N, a FLAG epitope-
tagged N-terminal fragment consisting of the RGS-N region; HA-C, an HA epitope-tagged C-
terminal fragment containing the GGL and RGS domains. The two transgenes expressing FL-N
or HA-C were chromosomally integrated to give stable expression, and the strain expressing
both FL-N and HA-C was generated by genetically crossing strains carrying the individual
transgenes (FL-N X HA-C). The results shown are similar to those in Figure 3A, indicating that
the epitope tags used do not interfere with the function of the EGL-10 fragments, and
reproducing the result that coexpression of both N- and C-terminal fragments is required for full
EGL-10 rescuing activity.
FIG. 6. Western blot analysis of EGL-10 protein fragment expression in transgenic animals.
Total worm lysates were separated by SDS-PAGE, transferred to nitrocellulose filters, and
immunoblotted with anti-HA, anti-FLAG, or anti-tubulin antibodies. Combining the HA-C and
FL-N transgenes by genetically crossing the strains carrying them resulted in increases in the
levels of both the HA-C and FL-N proteins. To control for loading, the same blots probed with
anti-FLAG and anti-HA antibodies were also probed with an anti-tubulin antibody.
FIG. 7. Solubility and effect of detergent on HA-C and FL-N expressed in transgenic animals.
A, total worm lysates (T), soluble fractions generated as supernatants from 100,000Xg
centrifugation of the same lysates (S), and the insoluble pellet fractions (P) were separated by
35
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-HA and anti-
FLAG antibodies. This experiment was conducted either in the presence or absence of 1% Triton
X-100 in the lysis buffer. The HA-C protein was largely soluble when expressed alone, but
moved almost entirely to the pellet when coexpressed with the FL-N protein. In contrast, the
distribution of the FL-N protein remained unchanged in the presence or absence of the HA-C
protein, remaining predominantly in the pellet fraction. The use of 1% Triton resulted in
significant solubilization of both the HA-C and FL-N proteins. B, sucrose density gradient
fractionation of total lysates prepared in the absence of detergent from worms carrying integrated
transgenes expressing HA-C and FL-N. The bulk of these proteins floated in fractions 8 and 9,
as did endogenous membrane-associated GOA-1.
FIG. 8. Coimmunoprecipitation of the HA-C and FL-N proteins. Triton X-100 solubilized
protein extracts from worm strains carrying integrated transgene(s) expressing HA-C and/or
FL-N were subjected to immunoprecipitation (IP) with anti-FLAG antibodies. The pellets,
along with control extracts representing 5% of the material used for the IPs, were fractionated by
SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted with anti-HA, anti-FLAG,
or anti-GPB-2 antibodies. Anti-FLAG antibody coprecipitates HA-C only in the presence of
FL-N, indicating that the HA-C and FL-N proteins form a complex.
36
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from
Georgia A. Patikoglou and Michael R. Koelleq signalingαo versus Gαinhibition of G
An N-terminal region of C. elegans RGS proteins EGL-10 and EAT-16 directs
published online September 26, 2002J. Biol. Chem.
10.1074/jbc.M208186200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on August 24, 2020
http://ww
w.jbc.org/
Dow
nloaded from