signal transduction by g proteins discovery and structure of heterotrimeric g proteins signaling...

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 proteinsMartin 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 proteinsAl Gilman purified the first G proteins. His lab took advantage of S49 lymphoma cells that lacked Gsa (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 GaS, while pertussis toxin clearly did not act on the newly purified GaS (could use radiolabeled ADP). Using pertussis toxin to ADP-ribosylate the target, Gilman lab identified and purified Gai.

Adenylyl Cyclases as Coincidence Detectors

AC Type: I II III V

Gas GTP

Ca2+/Calmodulin

Gbg

Protein kinase C

Gai GTP

0 0

0

0

00

?

0

0 0

0

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 b and g phospates of GTP molecule in correct orientation for hydrolysis by H2O

• Hydrolysis leads to major conformation change in Gs a

• 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

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 GenomeSteve 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

GPCR desensitization mechanisms

New concepts for GPCR signalingUsing 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, b-arrestinsPDZ 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, PLC, 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



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



Make transgenic fly with C645S mutation

Do electrophysiology

(inaD2= KO, inaDwt= WT rescue)

Single photon response OK, but . . .

Light-dependent inactivation impaired



NinaD is scaffold protein that binds PKC, PLC, and TRP channel

Crystal structure of PDZ5 reveals a disulfide bond . . .

Does it occur in vivo and is it important?

WT

InaDC645S

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

g-phosphate

Ras = classical “monomeric” GTPase

Binding g-phosphate changes the conformations of two small surface elements, called “switch 1 and 2”

Swi1Swi2

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?

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


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


“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

GDPGEF

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.


Rho/Rac/Cdc42 signaling in actin assembly


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 a-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!

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 e-Kinase, PAK, WASP, S6-Kinase, MLK2,3, Borg

The GTPase switch

Schmidt & Hall, Genes & Dev. (2002)

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.

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?

signal transduction by g proteins discovery and structure of heterotrimeric g proteins signaling...

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g proteins discovery

structure of g proteins

purified g s

g s mutations

afcs slide

gs pathway http

gi pathway http

structure activation