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)

TÁMOP-4.1.2-08/1/A-2009-0011

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)

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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

TÁMOP-4.1.2-08/1/A-2009-0011

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)

TÁMOP-4.1.2-08/1/A-2009-0011

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)