Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
EXTRACELLULAR RECEPTORSG-PROTEIN COUPLED RECEPTORS
Tímea Berki and Ferenc BoldizsárSignal transduction
Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat the University of Pécs and at the University of DebrecenIdentification number: TÁMOP-4.1.2-08/1/A-2009-0011
TÁMOP-4.1.2-08/1/A-2009-0011
Ligand-binding
Gα C-terminal tail
Other Gα surfaces
Helix 8 (Gβ-binding)
Gα -binding
Interaction surface
IL1 IL2 IL3
EL1 EL3EL2 Extracellular loops (EL1-3)
Intracellular loops (IL1-3)
N
C
GRK phosphorylation(Desensitization)
PKC phosphorylation(Desensitization)
PKC phosphorylation(Desensitization)
Palmitoylation(Lipid raft localization)
N-Glycosylation(Receptor folding, trafficking)
E/DRY Motif(Receptor activity and
protein-protein interactions)
Plasma membrane
TM1
TM2
TM3
TM4
TM5
TM6
TM7
Transmembrane helix(TM1-7)
TM4
7-transmembrane-spanning receptors (7-TM)
TÁMOP-4.1.2-08/1/A-2009-0011
7-transmembrane-spanning receptors (7-TM)• Class A: Rhodopsin-like• Class B: Secretin family• Class C: Glutamate and GABA
(metabotropic)• Frizzled• Adhesion family
TÁMOP-4.1.2-08/1/A-2009-0011
7-transmembrane-spanning receptors (7-TM)
GPCR superfamily(791 genes)
Class A(662)
Class B(15)
Class C(22)
Others(92)
Endogenous ligand/Orphan(271)
Olfactory/Pheromone(391)
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7-TM ligandsClass A Class B Class C Frizzled Adhesion
Prostaglandins Glucagon Glutamate Wnt Chondroitin-sulfate
Thromboxane GnRH GABA HedgehogSerotonine PTH Sweet tastes Bitter tastesDopamine CRH Secretin HistamineCatecholaminesAch (M)RhodopsinMelatoninChemokinesBradykininSomatostatinOpioidVasopressin
TÁMOP-4.1.2-08/1/A-2009-0011
Inactive 7-TM receptorSide perspective
Intracellular perspective
TM1
TM2
TM3 TM
4
TM5
TM6
TM7
Helix 8
IL1
IL3
EL1 EL2
EL3N
C
Ga-binding surface
TM1
TM4
TM5
TM6
TM7
Helix 8
IL 1IL2
IL3
EL1EL2
EL 3N
C TM2
TM3
Intracellular loops
Extracellular loops
Non-covalent bond
IL2
TÁMOP-4.1.2-08/1/A-2009-0011
TM1
TM2 TM
4
Helix 8
IL1
IL2
IL3
EL1
EL2
EL3NC
TM7
TM6
TM3
TM5
Ga
TM1
TM5
TM6TM
7
Helix 8
N
CTM4
TM2 Gα
TM3
GaC-terminal tailof Ga
Agonist
Active 7-TM receptorSide perspective
Intracellular perspective
TÁMOP-4.1.2-08/1/A-2009-0011
GDP
ba
b
GTP
a
GTP
ba
GDP
ba
Plasma membrane
Cytoplasm
GDP GTP
G-protein coupled receptor(GPCR)
Signal molecule
Inactive G-protein
Activated G-protein subunits
GTP
ba
7-TM receptors bind to G-proteins(G-protein-coupled receptors, GPCR)
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G-proteins
Cellular response
cAMP
GDP
β Gsa
GDP
βGia
Adenylylcyclase(+) (-)
GTP
GsaGTP
Gia
ATP
Protein kinase AInactive
Protein kinase AActive
ProteinProtein
OH P
5’-AMP
Phosphodiesterase
Stimulatoryhormone
Inhibitoryhormone
Rs Ri
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GDP
βa
β
GTP
Gia
G-protein coupled receptor(GPCR)
PhospholipasesIon channels
Activates Rho
Ion channelsPI3K
PhospholipasesAdenylyl cyclasesReceptor kinases
GTP
GsaGTP
Gqa
GTP
G12/13a
GTP
a
Ca2+
PLCPIP2 DAG
IP3
cAMP
Adenylylcyclase
ATPcAMP
Adenylylcyclase
ATP
Plasma membrane
Cytoplasm
G-proteins
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G-proteins• GTP-binding proteins• GTPase activity: they hydrolyse GTP to GDP• Inactive form: GDP-bound• Active form: GTP-bound• Heterotrimeric: a, b, subunits• Monomeric: products of ras proto-oncogens• subunit contains C terminal isoprenyl
chains: anchoring in the plasma membrane
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G-protein signaling
1 Ga signaling• Gs: stimulate adenylyl-cyclase → cAMP
↑• Gi: inhibit adenylyl-cyclase → cAMP ↓• Gq: stimulate PLC• G12: Rho-GEF 2 Gb, signaling• K+ and Ca2+ channels, PI3K isoforms
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GPCR regulation• PKA feedback phosphorylation• G-protein receptor kinases (GRK1-7): regulate 7TM
activation by phosphorylation of the C terminus of the receptors
• Translocation: the active receptor with the surrounding membrane is internalized – dephosphorylated in acidic vesicles and recycled to the surface
• Arrestin linking: binding of arrestin molecules inhibit the binding of Gs proteins to the receptors (eg. rhodopsin in retina); + activation of alternative pathways: MAPK, PI3-K, PKB/Akt, Src
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Monomeric G-proteins (Ras family)• First found as transforming oncogenes: Harvey (H-
Ras) and Kirsten (K-Ras) sarcoma viruses; Rat sarcoma
• N-Ras found in human neuroblastoma• 189 AA• Anchored to the membrane through lipid chains• Mutations in the Ras family is found in 20-30% of all
human tumors
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Ras regulation• Guanine-nucleotide exchange factors (GEFs):
catalyse the GDP-GTP exchange of Ras → Activation
• GTPase activating protein (GAP): the intrinsic GTPase activity of Ras is weak, enhanced by GAP → Inactivation
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Ras function – MAPK cascade• Signaling through growth factor receptors• Ras-Raf-MEK-ERK (Mitogen-activated protein
kinase = MAPK cascade)• Increased activity (= “constitutively active
Ras”) promotes tumor transformation– G-nucleotide exchange ↑ (point mutations)– GTPase activity ↓ (point mutations or lack of
GAP)
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Ras-MAPK cascade
RAS
Plasma membrane
SOSGRB2
Transcriptional regulation
YY
YYY
Y
Growth factor/Hormone
Receptor PTK
CytoplasmActiveRAS
Elk-1
RAF
MEK1/2
ERK1/2
Sap1a Net
AdaptorGuanine nucleotide
exchange factor(GEF)