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 “discriminatortransducer-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 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.
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)
Nobel prize, 1994
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
0
0
?
0
0
0
0
Gas GTP
Ca2+/Calmodulin
Gbg
Protein kinase C
Gai GTP
0
0
0
0
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 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
• Gonadotropinindependent 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
Orphan
Olfactory
Taste
163
140
350
15
25
34
11
4
6
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 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 (PostSynaptic 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, PLCb, 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, PLCb, 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.
Signaling GTPases are
Allosteric Switches
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.
g-phosphate
Swi1
Ras-GTPvs. Ras-GDP
Swi2
Binding g-phosphate changes the conformations
of two small surface elements, called
“switch 1 and 2”
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
A
D
E
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”
mutation
GDP
Binds GEF but cannot
GEF
replace GDP by GTP;
so GEF not available for
activating normal protein
GDP
Pi
The mutant titrates (binds up)
a limiting component to block
the normal protein’s signal
GTP
Cannot hydrolyze GTP,
so remains always active
GAP
The mutant exerts the same
effect as the normal protein
would, if it were activated
Reverse genetics: advantages/pitfalls of using
dominant-interfering mutants
Pro:
Con:
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?
Therefore . . .
Dominant-negatives
Over-expression can titrate
too many proteins (or
the wrong proteins
Dominant positives
Not always precise mimics of
the normal protein (e.g.,
may be in the wrong place))
Can induce adaptation,
turn-off mechanisms
Hard to apply to complex networks
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
Rate of GTP hydrolysis is 300-fold
faster in oocytes than in vitro!
Purified the factor that promoted GTPase
activity, cloned and named it GAP (or rasGAP). Another ras-GAP later identified is
NF1 (the gene mutated in neurofibromatosis,
i.e., Elephant Man Syndrome).
% GVB
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)
V12
G12
[ras] (ng)
% Ras-GTP
McCormick injected Xenopus oocytes
with oncogenic ras (V12) versus wt ras
(G12) and monitored germinal vesicle
breakdown (GVB) (top panel)
V12
G12
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 e-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 proteinprotein 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?