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”. Nobel prize, 1994

His work was not initially received well by the scientific community:

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 0 0 ? Gβγ 0 0 Protein kinase C 0 0 0 Gαi GTP 0 0 0 Trimeric G Proteins: GTPase Cycle Added complexity

GDP R* #e !" GTP R* R* !" !" # # GDP GTP E1

E2

Pi RGS

GEF function requires cooperation between GPCR (R*) and !" GTPase is faster (2-6/min) than for small GTPases But 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) G protein signal transduction

Neves, Ram, Iyengar, Science 2002 Structure of G proteins

Iiri, et al. NEJM (1999) 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? Human Genome Sequencing

More added complexity:

Human Fly Worm Yeast Plant 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) G protein-coupled receptors

• 5 main families • Conserved 7 TM architecture GPCRs in the Human Genome Steve Foord, GlaxoWelcome

Rhodopsin Secretin Metabotropic

Liganded 163 25 11 Orphan 140 34 4 Olfactory 350 6 Taste 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 GPCR desensitization mechanisms 10 seconds is too long! !t-GTP must be inactivated in < 1 sec Regulators of G Signaling (= RGS1-~RGS16; RGS9 in ROS)

RGS Pi RGS RGS !tGTP !tGTP !tGDP

GTP Accelerate GTPase from < 1/sec to >103/sec Most RGSs act on Swi2 !i or !q families Swi1 RGS

Many variations: eg, effectors with RGS activity eg, # subunit of cGMP PDE enhances effect of retinal RGS on !t eg, phospholipase C" acts on !q E Pi E

!q GTP !q GTPE* !q GDP EFFECT 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 Arrestins act as scaffolds for ERK and JNK signaling pathways

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 WT PKC, PLCβ, and TRP channel

Crystal structure C645S of PDZ5 reveals a InaD disulfide bond . . . Does it occur in vivo and is it important? 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 Signaling GTPases are ‘60’s as transforming genes of Allosteric Switches rat sarcoma viruses. Ras = classical “monomeric” GTPase 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. Swi2 -phosphate γ Swi1 Ras-GTP vs. Ras-GDP Binding γ-phosphate changes the conformations of two small surface elements, called ! “switch 1 and 2” G!t-GTP vs. Ras-GTP

Swi3

Swi2 Swi1

!-helical domain G!

Ras 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 Ras superfamily of small G proteins

Takai, et al. Physiological Reviews, 2001 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?

C D E A B 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

The mutant titrates (binds up) a limiting component to block the normal protein’s signal

2. Increase activity of the protein with a “dominant-positive” or “constitutively active” interfering mutant of the protein

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 GEF “Dominant-positive” GDP mutation

Binds GEF but cannot GEF Cannot hydrolyze GTP, replace GDP by GTP; GDP GTP so GEF not available for so remains always active activating normal protein

Pi GAP

The mutant titrates (binds up) The mutant exerts the same a limiting component to block effect as the normal protein the normal protein’s signal would, if it were activated Reverse genetics: advantages/pitfalls of using dominant-interfering mutants

Pro: Con: Quick-and-dirty; no biochem Dominant-negatives Over-expression can titrate Many different families of too many proteins (or signaling proteins amenable the wrong proteins . . . once we understand how one of them works Dominant positives Not always precise mimics of Examples: the normal protein (e.g., may be in the wrong place)) RTKs? Other kinases? Can induce adaptation, Adaptors? turn-off mechanisms

Hard to apply to complex networks Therefore . . . Still need biochemistry Hierachy of small G protein activation

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.

Ras

Takai, et al. Physiological Reviews, 2001 Rho/Rac/Cdc42 signaling in actin assembly

Takai, et al. Physiological Reviews, 2001 Identification of RasGAP

McCormick injected Xenopus oocytes V12 with oncogenic ras (V12) versus wt ras (G12) and monitored germinal vesicle breakdown (GVB) (top panel) Then loaded ras with α-32P GTP, injected % GVB G12 into oocytes, did immppt at increasing times and determined if GTP or GDP was bound (bottom panel) [ras] (ng) Rate of GTP hydrolysis is 300-fold faster in oocytes than in vitro! V12

Purified the factor that promoted GTPase activity, cloned and named it GAP (or ras- % Ras-GTP GAP). Another ras-GAP later identified is G12 NF1 (the gene mutated in neurofibromatosis, i.e., Elephant Man Syndrome). Time (min) 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 The GTPase switch

Schmidt & Hall, Genes & Dev. (2002) Mechanism of GDI-rab association 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.

Rak, et al. Does this interaction really happen in cells? Probably--mutations in domain II cleft abolish ability of RabGDI to remove Ypt1 from PM.

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?