Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: TÁMOP-4.1.2-08/1/A-2009-0011

EXTRACELLULAR RECEPTORS

G-PROTEIN COUPLED RECEPTORS

Tímea Berki and Ferenc Boldizsár Signal transduction

Medical Biotechnology Master’s Programmes

at the University of Pécs and at the University of Debrecen

Identification number: 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 EL2 EL3 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

TM 1

TM 2

TM 3

TM 4

TM 5

TM 6

TM 7

Transmembrane helix (TM1-7)

TM 4

7-transmembrane-spanning

receptors (7-TM)

7-transmembrane-spanning receptors (7-TM)

• Class A: Rhodopsin-like

• Class B: Secretin family

• Class C: Glutamate and GABA (metabotropic)

• Frizzled

• Adhesion family

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)

7-TM ligands

Class A Class B Class C Frizzled Adhesion

Prostaglandins Glucagon Glutamate Wnt Chondroitin-sulfate

Thromboxane GnRH GABA Hedgehog

Serotonine PTH Sweet tastes Bitter tastes

Dopamine CRH Secretin

Histamine

Catecholamines Ach (M)

Rhodopsin Melatonin Chemokines Bradykinin Somatostatin Opioid

Vasopressin

Inactive 7-TM receptor

Side perspective

Intracellular perspective

TM 1

TM 2

TM

3 TM

4 TM

5 TM

6 TM

7

IL1

IL3

EL1 EL2

EL3 N

C

Ga-binding surface

TM 1

TM 4

TM 5 TM

6 TM

7

IL 1

IL2

IL3 EL1

EL2 EL 3

N

C ^TM ₂

TM 3

Intracellular loops

Extracellular loops

Non-covalent bond

IL2

TM 1

TM

2 TM

4 IL1

IL2

IL3

EL1

EL2 N EL3

C

TM 7

TM 6

TM 3

TM 5 Ga

TM 1

TM 5 TM TM 6

7

N

C

TM 4 TM

2 Gα

TM 3

C-terminal tail Ga of Ga

Agonist

Active 7-TM receptor

Side perspective

Intracellular perspective

GDP b  a

b  GTP

a GTP

b  a

GDP b  a

Plasma membrane

Cytoplasm

GDP GTP G-protein coupled receptor

(GPCR)

Signal molecule

Inactive G-protein

Activated G-protein subunits

GTP b  a

7-TM receptors bind to G-proteins

(G-protein-coupled receptors, GPCR)

G-proteins

Cellular response

cAMP GDP

 β

Gsa

GDP β 

Gia Adenylyl

cyclase (+) (-)

GTP

Gsa

GTP

Gia

ATP

Protein kinase A Inactive

Protein kinase A Active

Protein Protein

OH P

5’-AMP

Phosphodiesterase Stimulatory

hormone

Inhibitory hormone

Rs Ri

GDP β  a

β 

GTP Gia

G-protein coupled receptor (GPCR)

Phospholipases Ion channels

Activates Rho

Ion channels PI3K Phospholipases Adenylyl cyclases Receptor kinases GTP

Gsa

GTP Gqa

GTP G_12/13a GTP

a

Ca²⁺

PLC

PIP₂ DAG

IP₃ cAMP

Adenylyl cyclase ATP

cAMP Adenylyl

cyclase ATP

Plasma membrane

Cytoplasm

G-proteins

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

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 Ca ²⁺ channels, PI3K isoforms

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

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

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

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)

Ras-MAPK cascade

RAS

Plasma membrane

SOS GRB2

Transcriptional regulation

Y Y Y

Y Y Y

Growth factor/Hormone

Receptor PTK

Cytoplasm Active

RAS

Elk-1

RAF

MEK1/2

ERK1/2

Sap1a Net

Adaptor

Guanine nucleotide exchange factor

(GEF)