Upload
vanthien
View
220
Download
0
Embed Size (px)
Citation preview
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
Discovery of G proteins Martin Rodbell first proposed the concept of “discriminator-transducer-amplifier” to address the problem: “How can many hormones (epinephrine, ACTH, TSH, LH, secretin, and glucagon) activate lipolysis and cAMP production in adipocytes through presumably a single cyclase? He called this problem “too many angels on a pinhead.” His work identified GTP as important for the “transducer”.
His work was not initially received well by the scientific community:
Nobel prize, 1994
Discovery of G proteins Al Gilman purified the first G proteins. His lab took advantage of S49 lymphoma cells that lacked Gsα (although at the time, the cells were thought to lack adenylate cyclase, thus the name cyc-). Reconstitution experiment rationale: Isolate membranes from cyc- cells, then add back fractions from donor wt membranes that restore adenylate cyclase activity. Nobel prize, 1994
Donor membranes were incubated for increasing time at 30oC, which inactivates the adenylate cyclase activity (- - - - -). Fortunately, G proteins are relatively heat stable. Addition of NaF, Gpp(NH)p, GTP, or GTP and isoproterenol restored activity in the cyc- membranes.
Ross, et al. JBC (1978)
Gs and Gi have opposing actions on adenylyl cyclases
Toxins help identify a second G protein. Both toxins result in increased cAMP production, but by different mechanisms. Cholera toxin ADP-ribosylates GαS, while pertussis toxin clearly did not act on the newly purified GαS (could use radiolabeled ADP). Using pertussis toxin to ADP-ribosylate the target, Gilman lab identified and purified Gαi.
Adenylyl Cyclases as Coincidence Detectors
AC Type: I II III V
Gαs GTP
Ca2+/Calmodulin
Gβγ
Protein kinase C
Gαi GTP
0 0
0
0 0 0
? 0
0 0 0
Trimeric G Proteins: GTPase CycleAdded complexity
GTPGDP R* !"#e
#GTP
#GDP
R*
!"
R*
!"
Pi
E1
2E
RGS
GEF function requires cooperation between GPCR (R*) and !"GTPase is faster (2-6/min) than for small GTPasesBut RGS (Regulators of G Signaling) proteins accelerate GTPase even more (>1,000/sec)
TWO effectors, #-GTP and !"
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
Hydrolysis of GTP
• Arg & Gln stabilize the β and γ phospates of GTP molecule in correct orientation for hydrolysis by H2O
• Hydrolysis leads to major conformation change in Gs α
• Mutations in the Gln or Arg (or ADP ribosylation by cholera toxin) blocks the ability to stabilize transition state, and therefore locks G protein in the “on” position.
• Examples include adenomas of pituitary and thyroid glands (GH secreting tumors, acromegaly), and McCune-Albright syndrome. Iiri, et al. NEJM (1999)
Canonical Gs Signaling Pathway
For interactive pathways at STKE: Gs pathway http://stke.sciencemag.org/cgi/cm/CMP_6634 Gi pathway http://stke.sciencemag.org/cgi/cm/CMP_7430 Gq pathway http://stke.sciencemag.org/cgi/cm/CMP_6680 G12 pathway http://stke.sciencemag.org/cgi/cm/CMP_8022
Neves, Ram, Iyengar, Science 2002
McCune-Albright Syndrome
• Polyostotic fibrous dysplasia
• Café au lait skin lesions • Autonomous
hyperfunction of one or more endocrine glands
• Gonadotropin-independent precocious puberty
• Cushing’s syndrome • Acromegaly
The constellation of symptoms varies from one individual to the next. How can a single mutation present in patches?
Testotoxicosis and PHP, 1a • Two unrelated boys with both gain-of function and loss-of
function diseases associated with Gs. • Testotoxicosis=inappropriate secretion of testosterone.
Usually under the control of LH (luteinizing hormone) secretion by the pituitary. LH receptors in the testes activate Gs.
• Pseudohypoparathyroidism=lack of PTH (parathyroid hormone) signaling resulting in impaired calcium homeostasis and bone abnormalities (Albright’s osteodystrophy). PTH receptors in bone activate Gs.
Mechanism?
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
G protein signaling
• Many ligands • Robust switches • Multiple effectors • Conserved 7 TM
architecture • More than 50%
of drugs target GPCRs
Bockaert & Pin, EMBO J (1999)
GPCRs in the Human Genome Steve Foord, GlaxoWelcome
Rhodopsin Secretin Metabotropic
Liganded 163 25 11Orphan 140 34 4Olfactory 350 6Taste 15 3
Identifying Ligands for Orphan GPCRS
Big Pharm approach: set up individual stable cell lines expressing each orphan GPCR. Fractionate peptides, tissue factors, etc. and apply to each cell line. Example: Orexin receptors
Cottage industry approach: expression cloning strategy in Xenopus oocytes. Use sib selection to identify cDNAs that encode desired receptor. Example: Thrombin receptor
10 seconds is too long! !t-GTP
must be inactivated in < 1 sec
Many variations: eg, effectors with RGS activity
eg, phospholipase C" acts on !q
EE*
EPi
EFFECT
Regulators of G Signaling (= RGS1-~RGS16; RGS9 in ROS)
GTP
RGSRGS
RGSPi
GDP!t GTP
!t !t
Most RGSs act on !i or !q families
RGSSwi1
Swi2
GTPAccelerate GTPase from < 1/sec to
>103/sec
GTP GDP!q GTP
!q !q
eg, # subunit of cGMP PDE enhances
effect of retinal RGS on !t
New concepts for GPCR signaling Using mainly two-hybrid screening approaches, many proteins have been found to interact with portions of the GPCRs. Non-PDZ scaffolds: AKAPs (A-Kinase Anchoring Proteins, JAK2 (Janus Activated Kinase), homer, β-arrestins PDZ scaffolds: InaD, PSD-95 (Post-Synaptic Density), NHERF (Na/H Exchanger Regulatory Factor).
The arrestins have been found to bind to other signaling proteins and activate downstream effectors: Examples: src, Raf & ERK, ASK1 & JUNK3
Lefkowitz reviews
Bonus material--Dynamic scaffolding
Visual system in the fly
NinaD is scaffold protein that binds PKC, PLCβ, and TRP channel
Crystal structure of PDZ5 reveals a disulfide bond . . .
Does it occur in vivo and is it important? Mishra et al Cell 2007
Bonus material--Dynamic scaffolding
Visual system in the fly
Titrate the disulfide bond with increasing concentration of DTT
Redox Potential of the disulfide in InaD is very strong
Most cytosolic proteins are -0.23 to -0.30
Mishra et al Cell 2007
Bonus material--Dynamic scaffolding
Visual system in the fly
Make transgenic fly with C645S mutation
Do electrophysiology
(inaD2= KO, inaDwt= WT rescue)
Single photon response OK, but . . .
Light-dependent inactivation impaired
Bonus material--Dynamic scaffolding
Visual system in the fly
NinaD is scaffold protein that binds PKC, PLCβ, and TRP channel
Crystal structure of PDZ5 reveals a disulfide bond . . .
Does it occur in vivo and is it important?
WT
InaDC645S
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
Discovery of Small G proteins
Ras genes first identified in ‘60’s as transforming genes of rat sarcoma viruses. Weinberg, Varmus, Bishop and others in the early ‘80’s showed that many cancer cells have mutated versions of ras. Activated form of ras found in 90% of pancreatic carcinomas, 50% of colon adenocarcinomas, and 20% of malignant melanomas.
Ras-GTP vs. Ras-GDP
Signaling GTPases are
Allosteric Switches
γ -phosphate
Ras = classical “monomeric” GTPase
Binding γ-phosphate changes the conformations of two small surface elements, called ! “switch 1 and 2”
Swi1 Swi2
Rho/Rac/Cdc42 In early ‘90’s, Alan Hall discovered that newly characterized ras homologs (rho, rac, cdc42) induced cytoskeletal changes.
Reviewed by Hall, Science 1998
GTPases: How to use reverse genetics to identify their roles in cell regulation
Depends on understanding how the machines work
Epistasis question: Where in a pathway does a specific protein convey its particular message?
A B
C D E
Response
M N Q
Idea: 1. Inhibit activity of the protein of interest
2. Increase activity of the protein of interest
How to do this? Drugs, genetic diseases, mouse KOs, and . . .
Reverse genetics: express one or two mutant versions of the protein of interest
Depends on understanding how the machines work
1. Inhibit activity of the protein with a “dominant-negative” interfering mutant of that protein
2. Increase activity of the protein with a “dominant-positive” or “constitutively active” interfering mutant of the protein
The mutant titrates (binds up) a limiting component to block the normal protein’s signal
The mutant exerts the same effect as the normal protein would, if it were activated in the cell
Reverse genetics: small GTPases as examples Depends on understanding how the machines work
“Dominant-negative” mutation “Dominant-positive”
mutation
The mutant titrates (binds up) a limiting component to block the normal protein’s signal
The mutant exerts the same effect as the normal protein would, if it were activated
GAP
GTP
Pi
GDP GEF
GEF GDP
Binds GEF but cannot replace GDP by GTP; so GEF not available for activating normal protein
Cannot hydrolyze GTP, so remains always active
Reverse genetics: advantages/pitfalls of using dominant-interfering mutants
Pro: Quick-and-dirty; no biochem
Many different families of signaling proteins amenable . . . once we understand how one of them works
Examples:
RTKs? Other kinases? Adaptors?
Con: Dominant-negatives
Dominant positives
Therefore . . . Still need biochemistry Hard to apply to complex networks
Over-expression can titrate too many proteins (or the wrong proteins
Not always precise mimics of the normal protein (e.g., may be in the wrong place))
Can induce adaptation, turn-off mechanisms
Hierachy of small G protein activation
Ras
Use of constitutively active or dominant negative mutant small G proteins revealed that ras and cdc42 can activate rac. Rac, in addition to inducing lamellipodia, also activates Rho.
Takai, et al. Physiological Reviews, 2001
Identification of RasGAP
McCormick injected Xenopus oocytes with oncogenic ras (V12) versus wt ras (G12) and monitored germinal vesicle breakdown (GVB) (top panel)
% G
VB
[ras] (ng)
V12
G12
Time (min)
% R
as-G
TP
V12
G12
Then loaded ras with α-32P GTP, injected into oocytes, did immppt at increasing times and determined if GTP or GDP was bound (bottom panel)
Purified the factor that promoted GTPase activity, cloned and named it GAP (or ras-GAP). Another ras-GAP later identified is NF1 (the gene mutated in neurofibromatosis, i.e., Elephant Man Syndrome).
Rate of GTP hydrolysis is 300-fold faster in oocytes than in vitro!
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
Small G proteins “turn off” mechanisms
RhoGAPs outnumber the small G proteins Rho/Rac/Cdc42 by nearly 5-fold. Why so much redundancy? Luo group did RNAi against 17 of the 20 RhoGAPs in fly. Six caused lethality when expressed ubiquitously. Tissue specific expression of RNAi revealed unique phenotypes. P190RhoGAP implicated in axon withdrawal. Increasing amounts of RNAi caused more axon withdrawal (panels C-G). Why so many RhoGAPs?
Billuart, et al. Cell (2001)
Small G protein “turn on” mechanisms
First mammalian GEF, Dbl, isolated in 1985 as an oncogene in NIH 3T3 focus forming assay. It had an 180 amino acid domain with homology to yeast CDC24. This domain, named DH (Dbl homology) is necessary for GEF activity. In 1991, Dbl shown to catalyze nucleotide exchange on Cdc42. Schmidt & Hall, Genes & Dev. (2002) Dbl= Diffuse B-cell lymphoma
Rho/Rac/CDC42 activation of downstream effectors
Rho Effectors: PI 3-Kinase, PLD, Rho Kinase, Rhophilin, and others. Rac-interacts via a CRIB domain in downstream effectors. CRIB
(Cdc42/Rac interacting binding) Effectors: NADPH oxidase, PAK, PI 3-Kinase, MLK2,3, POSH, DGK Cdc42 Effectors: PI ε-Kinase, PAK, WASP, S6-Kinase, MLK2,3, Borg
Mechanism of GDI-rab association
Does this interaction really happen in cells? Probably--mutations in domain II cleft abolish ability of RabGDI to remove Ypt1 from PM.
Rak, et al.
Ypt1 is a small G protein (rab family). Rab-GDI binds the GDP-Ypt and removes it from the PM. Recent co-crystal structure reveals possible mechanism.
Signal Transduction by G proteins
• Discovery and Structure of Heterotrimeric G proteins
• Signaling pathways of G proteins • Receptors that activate G proteins • Small G proteins-discovery and
structure • Activation and inactivation mechanisms • Alliance for Cell Signaling (AfCS)
Central Questions of the AfCS: I
Question 1: How complex is signal processing in cells? The set of ligands for cellular receptors is the potential combinatorial code of inputs. How much of this input complexity can a cell uniquely decode as outputs?
Experiment: Systematic single- and double- (multi?) ligand screens. Classify output responses; determine degree of crosstalk; identify “hotspots” for later quantitative analysis.
New Technologies: Analytic methods to classify and compare multi-dimensional data for different ligand combinations
Central Questions of the AfCS: II
Question 2: What is the structure of the whole signaling network? Is the connectivity sparse or dense?
Experiment: Wholesale mapping of relevant protein-protein and small molecule-protein interactions.
New Technologies: High-throughput assays for intermolecular interactions in vivo, especially in response to ligand stimulation.
Central Questions of the AfCS: III
Question 3: How much does network topology constrain signal processing capability? How much function is specified by the nature of the connections, rather than by the specific biochemical constants of individual activities.
Experiment: Perturbation methods; gain and loss of function, coupled with functional assays.
New Technologies: Perturbations in vivo, singly and in combinations.
Central Questions of the AfCS: IV
Question 4: What are the dynamics of the signaling network? Can we visualize how information propagates through the network and emerges as functional activities?
Question 5: Can functional modules be abstracted mathematically? Can we make physical models and predict input-output relationships
Question 6: Why is the network the way it is? Why have the observed solutions been chosen? What is being optimized?