MCB LECTURE 110807

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MCB LECTURE 11/08/07

• Signal Transduction by G proteins
• Tom Baranski

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Big G proteins and Little G proteins
Katie Baranski
Elizabeth Baranski
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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)

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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
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Discovery of G proteins
Al 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)
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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.
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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)

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G protein signal transduction
Neves, Ram, Iyengar, Science 2002
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Structure of G proteins
Iiri, et al. NEJM (1999)
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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)
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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
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McCune-Albright Syndrome

• Polyostotic fibrous dysplasia
• Café au lait skin lesions
• Autonomous hyperfunction of one or more endocrine
  glands
• Gonadotropin-independent precocious puberty
• Cushings syndrome
• Acromegaly

The constellation of symptoms varies from one
individual to the next. How can a single mutation
present in patches?
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Testotoxicosis and PHP, 1a

• Two unrelated boys with both gain-of function and
  loss-of function diseases associated with Gs.
• Testotoxicosisinappropriate secretion of
  testosterone. Usually under the control of LH
  (luteinizing hormone) secretion by the pituitary.
  LH receptors in the testes activate Gs.
• Pseudohypoparathyroidismlack of PTH (parathyroid
  hormone) signaling resulting in impaired calcium
  homeostasis and bone abnormalities (Albrights
  osteodystrophy). PTH receptors in bone activate
  Gs.
• Mechanism?

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Human Genome Sequencing
More added complexity
Human
Fly
Worm
Yeast
Plant
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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)

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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)
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G protein-coupled receptors

• 5 main families
• Conserved 7 TM architecture

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GPCRs in the Human GenomeSteve Foord,
GlaxoWelcome
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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
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GPCR desensitization mechanisms
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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, b-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
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Arrestins act as scaffolds for ERK and JNK
signaling pathways
Lefkowitz reviews
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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
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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
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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
. . .
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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?
WT
InaDC645S
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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)

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Discovery of Small G proteins
Ras genes first identified in 60s as
transforming genes of rat sarcoma viruses.
Weinberg, Varmus, Bishop and others in the
early 80s 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.
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Rho/Rac/Cdc42
In early 90s, Alan Hall discovered that newly
characterized ras homologs (rho, rac, cdc42)
induced cytoskeletal changes.
Reviewed by Hall, Science 1998
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Ras superfamily of small G proteins
Takai, et al. Physiological Reviews, 2001
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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 . . .
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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 proteins
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
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Reverse genetics small GTPases as examples
Depends on understanding how the machines work
Dominant-negative mutation
GEF
Dominant-positive mutation
GDP
Binds GEF but cannot replace GDP by GTP so
GEF not available for activating normal protein
Cannot hydrolyze GTP, so remains always active
Pi
The mutant titrates (binds up) a limiting
component to block the normal proteins signal
The mutant exerts the same effect as the
normal protein would, if it were activated
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Reverse genetics advantages/pitfalls of using
dominant-interfering mutants
Pro
Con
Quick-and-dirty no biochem
Dominant-negatives
Over-expression can titrate too many
proteins (or the wrong proteins
Many different families of signaling proteins
amenable . . . once we understand how one
of them works

Dominant positives
Not always precise mimics of the normal
protein (e.g., may be in the wrong
place))
Examples
RTKs? Other kinases? Adaptors?
Can induce adaptation, turn-off mechanisms
Hard to apply to complex networks
Therefore . . .
Still need biochemistry
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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.
Takai, et al. Physiological Reviews, 2001
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Rho/Rac/Cdc42 signaling in actin assembly
Takai, et al. Physiological Reviews, 2001
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Identification of RasGAP
V12
McCormick injected Xenopus oocytes with oncogenic
ras (V12) versus wt ras (G12) and monitored
germinal vesicle breakdown (GVB) (top panel)
GVB
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)
ras (ng)
Rate of GTP hydrolysis is 300-fold faster in
oocytes than in vitro!
V12
Ras-GTP
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).
G12
Time (min)
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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)

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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)
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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
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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
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The GTPase switch
Schmidt Hall, Genes Dev. (2002)
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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.
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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)

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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
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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.
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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.
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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?
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2.5
5.0
30
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BLC-ELC less than additive
Ca2
cAMP
BLC B-lymphocyte chemoattractant ELCEBV-induced
molecule-1 Ligand Chemokine
S
Time (seconds)
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Potential Integrative Pathways to Ca
AIG-low
AIG-high
M3A
S1P




IFB



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LPS