GPCRs and their effectors: signal transduction pathways and the cardiovascular system BIOM 200AB Nov

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GPCRs and their effectors signal transduction
pathways and the cardiovascular system BIOM
200A/B November 17, 2009Joan Heller Brown
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Agonist/ligand GPCR G-protein Direct
target/effector Cellular response
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G-protein classification

• Small G-proteins, small GTPases (smgs), low
  molecular weight G-proteins
• Families Ras (Ras, Rap), Rho (Rho, Rac, cdc 42)
• Alpha subunit 20 kd
• No beta or gamma
• Activation through GEFs and inactivation via GAPs

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(e.g. Integrins, Growth Factors, Stretch)
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G-protein classification

• Heterotrimeric G-proteins
• Families Gs, Gi, Gq, G12
• Alpha subunit 40 kd
• Associated with beta and gamma subunits
• Activation through GPCRs (as GEFs) and
  inactivation via RGS proteins (as GAPs)

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Johnston and Siderovski Mol Pharm 2007.
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G-proteins are selectively activated by GPCRs
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Classification of heterotrimeric G-protein alpha
subunits
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• How do we know what G-proteins are activated?
• Agonist induced GTP loading
• GPCR/G-protein FRET or BRET
• Assay downstream responses

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Assay agonist induced G-protein activation
Receptor Agonist
a
35SGTPgS
35SGTPgS
a
35SGTPgS
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This assay would be done using a broken
cell/membrane preparation rather than intact
cells because

• A. Receptors do not activate G-proteins in the
  intact cell
• B. You could not immunoprecipitate G-proteins
  after stimulating the intact cell
• C. You could not get GTP-g-S into the cell
• D. Selective receptor coupling to a particular
  G-protein is not evident in an intact cell
• E. You can use less radioactivity

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Receptors, subtypes and their G protein
selectivity

• Adrenergic receptors
• beta 1 adrenergic to Gs
• alpha 1 adrenergic to Gq
• alpha 2 adrenergic to Gi
• Muscarinic cholinergic receptors
• M1 and M3 muscarinic to Gq
• M2 and M4 muscarinic to Gi
• Sphingosine-1-phosphate (S1P) receptors
• S1P1 to Gi
• S1P2 to G12
• S1P3 to Gq
• PAR1 thrombin receptors
• to Gq, Gi, G12

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What determines what G-protein a receptor will
couple to?

• Structure of the receptor?
• Localization of the receptor?
• Agonist induced conformational change in the
  receptor?
• Precoupling?

We dont really know
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What determines what G-protein a receptor will
couple to?

• Structure of the receptor?
• Localization of the receptor?
• Agonist induced conformational change in the
  receptor?
• Precoupling?

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Muscarinic cholinergic receptor (mAChR)
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Sequence homology in m2/m4 vs m1/m3 muscarinic
receptors
M2 and M4 couple to Gi M1, M3 and M5 couple to Gq
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Fig. 1. Structure of chimeric muscarinic
receptors composed of human m2 (?) and rat m3
sequences ().
Wess
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Wess
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What determines what G-protein a receptor will
couple to?

• Structure of the receptor?
• Localization of the receptor?
• Agonist induced conformational change in the
  receptor?
• Precoupling?

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Regulation of agonist induced interaction with
muscarinic receptors by GTP
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Functional selectivity or biased agonism in GPCR
coupling to G-proteins
From Gilchrest, Trends Pharmacol. Sci. 28
431-437, 2007.
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G-protein subunits couple to distinct
targets/effectors
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Direct G protein a subunit targets

• Gas Adenylyl cyclase (stimulation)
• Gai Adenylyl cyclase (inhibition)
• Gaq PLC-b (stimulation)
• Ga12 Rho GEFs (stimulation)

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Direct targets of G-protein beta/gamma subunits
(generally from Gi)

• Phospholipase C ß2
• Ion channels
• Rac GEFs
• PI 3-kinase
• GRKs
• Adenylyl cyclase

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Secondary Responses
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Akt
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Activation of adenylyl cyclase as a direct target
of Gs
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Gs and Gi alpha subunits antagonistically
regulate adenylyl cyclase activity
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LIGAND
LIGAND
Norepinephrine
Acetylcholine
LIGAND
LIGAND
?SGTP
?SGTP
?
?
AC
AC
ADP
ADP
?S GDP
?
cAMP
?
NAD
NAD
Pertussistoxin
PKA
Choleratoxin
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What is the receptor that norepinephrine is
binding to?
A. ß1- adrenergic receptor (ß1AdrR) B. a1-
adrenergic receptor (a1AdrR) C. a2- adrenergic
receptor (a2AdrR) D. Norepinephrine activated
receptor (NAT) E. Protease activated receptor 1
(PAR1)
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What is the receptor that acetylcholine is
binding to?
A. M2 muscarinic receptor (M2mAChR) B. Nicotinic
cholinergic receptor (nAChR) C. M3 muscarinic
receptor (M3mAChR) D. Sphingosine-1-phosphate
receptor (S1PR) E. a2- adrenergic receptor
(a2AdrR)
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LIGAND
LIGAND
Norepinephrine
Acetylcholine
LIGAND
LIGAND
?SGTP
?SGTP
?
?
AC
AC
ADP
ADP
?S GDP
?
cAMP
?
NAD
NAD
Pertussistoxin
PKA
Choleratoxin
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Pertussis toxin ribosylates the C-terminal of Gi
and blocks receptor receptor mediated Gi
activation

• Prevent acetylcholine from antagonizing
  b-adrenergic effects on cAMP formation
• Block responses mediated through bg subunits
  derived from Gi
• Prevent agonist from increasing GTP loading of Gi
  proteins
• Lower cAMP if there was constitutive activation
  of Gs
• All of the above

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Secondary Responses downstream of Gs/adenylyl
cyclase
Phosphodiesterase (PDE)
Adenylyl Cyclase (AC)
5AMP
ATP
cAMP
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G alpha subunits regulate phospholipase C
activity
q
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T Balla J.Endocrinology, 2006
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aq
aq
Smrcka et al 1991
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Secondary responses downstream of Gq/PLC
activation
Gomperts
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To prove that InsP3 induces intracellular Ca
release you could do all except

• Inject InsP3 into the cell
• B. Express the cDNA for InsP3 in the cell
• C. Permeabilze the cell with detergent to let
  InsP3 enter
• D. Express a Ca sensitive reporter in the cell
• E. Use a Ca sensitive dye that could enter the
  cell

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Gomperts
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Responses downstream of Ca/DAG elevation

• Activation of PKC
• Activation of calmodulin (CaM)
• Activation of CaM kinase
• Activation of myosin light chain kinase
• Activation of PKD
• leading to changes in contraction, secretion,
  gene expression, etc

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G12 and G13 alpha subunits directly regulate GEFs
for small Rho family G-proteins
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S1P, LPA, thrombin
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S1P, LPA, thrombin
Rho kinase ( ROCK) Phospholipase C epsilon
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PLC epsilon is activated by RhoA and functions as
a GEF for Ras family proteins and an effector of
sustained signals
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GPCRs and cardiovascular disease
GPCR
G12/13
Vascular diseases
RhoA
Gq
GPCR
PLC/Ca
Heart disease
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Pathophysiological G12/13 and Rho activation in
the vasculature
S1P, Thrombin
PLCe
Vasoconstriction
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Rho activation increases vascular endothelial
cell permeability
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Canonical pathway for Ca2 mediated smooth muscle
contraction and vasoconstriction
GPCR, Gq, PLC, InsP3
Ca2 (intracellular)
Ca2-calmodulin ( CaM)
.
MLCK-CaM
Myosin Light chain
myosin LC -PO4
(myosin LC)
Actin
Contraction
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Rho mediated smooth muscle contraction and
vasoconstriction
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• Chronic Gq activation in the heart

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Hypertrophic response to Gq signaling in the
heart
Neonatal rat ventricular myocytes GPCR
agonists (PE, ANG II, ET-1, PGF2a)

• Increased cell size
• Increased myofibrillar organization
• Fetal gene expression
• ? Immediate early genes (c-fos)
• ? Embryonic genes (ANF etc.)
• ? Constitutive contractile protein genes (MLC-2)

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GPCR agonists induced hypertrophy of neonatal rat
ventricular myocytes
Agonist treated NE, PE, ET-1, PGF2a
Control
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GPCR agonists (NE, ET-1, PE, PGF2a)
Gaq
P.Simpson, K. Chien, P. Sugden, JHB and others
PLC
Hypertrophic cardiomyocyte growth
cell size, protein synthesis, myofilament
organization, fetal gene expression
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Adenoviral gene expression induces hypertrophy
LacZ
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Pressure overload (TAC)
Gaq
Koch Dorn Offermanns
PLC
Hypertrophic cardiomyocyte growth
cell size, protein synthesis, myofilament
organization, fetal gene expression
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GPCR agonists (NE, ET-1, PE, PGF2a)
Pressure overload (TAC)
Gaq
PLC
CaMK
Hypertrophic cardiomyocyte growth
cell size, protein synthesis, myofilament
organization, fetal gene expression
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Cardiac enlargement induced by pressure overload
(TAC), or in G?q or CamKII transgenic mice
TAC or TG
WT
5.30 0.10
4.36 0.10
HW/BW
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Dot blot analysis of gene expression in
hypertrophic heart
TAC or
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Regulatory pathway for hypertrophic gene
expression
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Pathological Hypertrophy
Normal
Heart Failure
Gaq
Gaq
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Dilated cardiomyopathy in Gq or CaMKII transgenic
mice
NTG
TG
RV
LV
RV
LV
1 mm
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Transgenic mice develop edema associated with
heart failure
TG
NTG
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Early lethality in Gq transgenic mice
WT
Weeks
from Dorn, edited for effect
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Constitutive activation of G?q leads to
mitochondrial disruption in cardiac myocytes
GaqWT
GaqQ209L
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Constitutive activation of Gaq induces
cardiomyocyte apoptosis
Apoptotic nuclei
Cytochrome c release
Caspase activation
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10.0
10.0
30
7.5
7.5
of cells
of cells
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DEVD-amc cleavage
(pmoles amc/mg/min)
5.0
5.0
10
2.5
2.5
0
0.0
0.0
LacZ
Q209L
LacZ
Q209L
LacZ
Q209L
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Echocardiographic evidence for dilated
cardiomyopathy
WT
TG
EDD
ESD
EDD
ESD
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Summary Acute responses to cardiovascular GPCR
activation
Gs Gi Gq G12/13
cAMP cAMP PLC Rho
ß1AdR
a2AdR, M2AChR
a1AdR, M3AChR
S1P, LPA, PAR1
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Summary downstream consequences of
cardiovascular GPCR stimulation
Gs Gi Gq G12/13
cAMP cAMP PLC Rho
Contractility, HR, gene expression
Contractility, HR
Ca, PKC, MLCK, contraction, CaMK, hypertrophy
and heart failure
Gene expression, cytoskeletal rearrangement,
hypertension, vascular disease
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RhoA expression and activity are higher in two
models of hypertension
SHR
L-NAME
Total Rho
Active Rho
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CaMKII? deletion rescues LV chamber dilation and
contractile dysfunction in G?q-overexpressing
hearts
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CaMKII? deletion rescues arrhythmias, fibrosis
and apoptosis seen in G?q-overexpressing hearts
CaMKII? KO
TG/KO
WT
Gq TG
arrhythmia
cardiac fibrosis
apoptosis
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Pressure overload / TAC
Gaq
InsP3
PL-C
Ca2
CaMK
Ca2
RyR
Cell death
Lethality and Heart failure
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Neubig
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Harden and Sondek Regulation of Phospholipase C
Isozymes by Ras Superfamily GTPases, Annual
Review of Pharmacology and Toxicology, 2006
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GPCR agonists Thrombin, LPA, S1P, TXA2 ,
chemokines
G12/13
Rho
Rho kinase, PLCepsilon
MLC-P
p27Kip1
AP-1,NF-kB
LIM kinase


contraction
migration proliferation cytokines
atherosclerosis, cancer, inflammatory diseases
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GPCR agonists (Phe, ET1)/ Pressure overload
Gaq
DAG/PKC
PLC
InsP3
Ca2
CaMK
PKD
Hypertrophy
cell size, protein synthesis, myofilament
organization, ANF expression