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 Identification number: TÁMOP /1/A

EXTRACELLULAR RECEPTORS G-PROTEIN COUPLED RECEPTORS Tímea Berki and Ferenc Boldizsár Signal transduction 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 Identification number: TÁMOP /1/A

TÁMOP /1/A Ligand-binding Gα C-terminal tail Other Gα surfaces Helix 8 (G β  -binding) Gα -binding Interaction surface IL1IL2IL3 EL1EL3EL2 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)

TÁMOP /1/A transmembrane-spanning receptors (7-TM) Class A: Rhodopsin-like Class B: Secretin family Class C: Glutamate and GABA (metabotropic) Frizzled Adhesion family

TÁMOP /1/A transmembrane-spanning receptors (7-TM) GPCR superfamily (791 genes) GPCR superfamily (791 genes)

TÁMOP /1/A TM ligands Class A Class B Class C FrizzledAdhesion Prostaglandin s GlucagonGlutamateWnt Chondroitin- sulfate ThromboxaneGnRHGABAHedgehog SerotoninePTHSweet tastesBitter tastes DopamineCRHSecretin Histamine Catecholamine s Ach (M) Rhodopsin Melatonin Chemokines Bradykinin Somatostatin Opioid Vasopressin

TÁMOP /1/A Inactive 7-TM receptor Side perspective Intracellular perspective TM1 TM2 TM3 TM4 TM5 TM6 TM7 Helix 8 IL1 IL3 EL1 EL2 EL3 N C G  -binding surface TM1 TM4 TM5 TM6 TM7 Helix 8 IL 1 IL2 IL3 EL1 EL2 EL 3 N C TM2 TM3 Intracellular loops Extracellular loops Non-covalent bond IL2

TÁMOP /1/A TM1 TM2 TM4 Helix 8 IL1 IL2 IL3 EL1 EL2 EL3 N C TM7 TM6 TM3 TM5 GG GG TM1 TM5 TM6 TM7 Helix 8 N C TM4 TM2 GαGα GαGα TM3 GG GG C-terminal tail of G  Agonist Active 7-TM receptor Side perspective Intracellular perspective

TÁMOP /1/A GDP      GTP  GTP    GDP    Plasma membrane Cytoplasm GDPGTP G-protein coupled receptor (GPCR) Signal molecule Inactive G-protein Activated G-protein subunits GTP    7-TM receptors bind to G- proteins (G-protein-coupled receptors, GPCR)

TÁMOP /1/A G-proteins Cellular response cAMP GDP  β Gs  GDP  β Gi  Adenylyl cyclase (+) (-) Adenylyl cyclase (+) (-) GTP Gs  GTP Gi  ATP Protein kinase A Inactive Protein kinase A Inactive Protein kinase A Active Protein kinase A Active Protei n P 5’-AMP Phosphodiestera se Stimulatory hormone Inhibitory hormone Rs Ri

TÁMOP /1/A GDP  β   β GTP Gi  G-protein coupled receptor (GPCR) Phospholipases Ion channels Activates Rho Ion channels PI3K Phospholipases Adenylyl cyclases Receptor kinases GTP Gs  GTP Gq  GTP G 12/13  GTP  Ca 2+ PLC PIP 2 DAG IP 3 cAMPcAMP Adenylyl cyclase Adenylyl cyclase ATPATPcAMPcAMP Adenylyl cyclase Adenylyl cyclase ATPATP Plasma membrane Cytoplasm G-proteins

TÁMOP /1/A G-proteins GTP-binding proteins GTPase activity: they hydrolyse GTP to GDP Inactive form: GDP-bound Active form: GTP-bound Heterotrimeric: , ,  subunits Monomeric: products of ras proto- oncogens  subunit contains C terminal isoprenyl chains: anchoring in the plasma membrane

TÁMOP /1/A G-protein signaling G  signaling 1G  signaling Gs: stimulate adenylyl-cyclase → cAMP ↑ Gi: inhibit adenylyl-cyclase → cAMP ↓ Gq: stimulate PLC G12: Rho-GEF G ,  signaling 2G ,  signaling K + and Ca 2+ channels, PI3K isoforms

TÁMOP /1/A 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 /1/A 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 % of all human tumors

TÁMOP /1/A 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 /1/A 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 /1/A Ras-MAPK cascade RAS Plasma membrane SOS GRB2 Transcriptional regulation Y Y Y Y Y Y Growth factor/Hormone Receptor PTK Cytoplasm Activ eRAS Elk-1 RAF MEK1/2 ERK1/2 Sap1a Net Adaptor Guanine nucleotide exchange factor (GEF)