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-4.1.2-08/1/A-2009-0011

G-protein-linked receptors 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-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signal transduction G-protein-linked receptors

Nomenclature G-protein-coupled receptors (GPCRs) Seven-transmembrane domain receptors 7-TM receptors Heptahelical receptors Serpentine receptor G protein-linked receptors (GPLR)

7-transmembrane-spanning receptors (7-TM) N-Glycosylation (Receptor folding, trafficking) EL1 EL2 EL3 Extracellular loops (EL1-3) Plasma membrane Interaction surface Ligand-binding Gα C-terminal tail Other Gα surfaces Helix 8 (Gβ-binding) Gα -binding TM 1 TM 2 TM 3 TM 4 TM 4 TM 5 TM 6 TM 7 Transmembrane helix (TM1-7) PKC phosphorylation (Desensitization) Intracellular loops (IL1-3) IL1 IL2 IL3 Palmitoylation (Lipid raft localization) E/DRY Motif (Receptor activity and protein-protein interactions) GRK phosphorylation (Desensitization) PKC phosphorylation (Desensitization) C

GPCR

Structure of 7-TM receptors Inactive GPCR Active GPCR Side perspective TM 1 5 6 7 Helix 8 N C 4 2 Gα 3 Ga C-terminal tail of Ga Agonist TM 1 4 5 6 7 Helix 8 IL 1 IL2 IL3 EL1 EL2 EL 3 N C 2 3 Intracellular loops Extracellular loops Intracellular persective TM 1 2 3 4 5 6 7 Helix 8 IL1 IL3 EL1 EL2 EL3 N C Ga-binding surface Non-covalent bond IL2 TM 1 2 4 Helix 8 IL1 IL2 IL3 EL1 EL2 EL3 N C 7 6 3 5 Ga

GPCR subtypes Class A (or 1) – Rhodopsin-like Class B (or 2) – Secretin receptor family Class C (or 3) – Metabotropic glutamate/pheromone Class D (or 4) – Fungal mating pheromone receptors Class E (or 5) – Cyclic AMP receptors Class F (or 6) – Frizzeled/Smoothened

Activation of G-protein-coupled receptors (GPCR) Signal molecule a g a g a g b b b GDP Inactive G-protein GDP GTP GDP GTP Plasma membrane g b Cytoplasm a g b a GTP GTP Activated G-protein subunits

G-protein coupled receptor G-proteins G-protein coupled receptor (GPCR) Plasma membrane Cytoplasm a g b GDP a g b GTP Ion channels PI3K Phospholipases Adenylyl cyclases Receptor kinases Gi Gs Gq G12/13 GTP GTP GTP GTP Adenylyl cyclase Adenylyl cyclase PLC PIP2 DAG ATP cAMP ATP cAMP Activates Rho IP3 Phospholipases Ion channels Ca2+

G-protein heterotrimer

Catecholamine hormon synthesis +H3N +H3N CO2– +H3N CO2– OH OH Tyrosine DOPA hydroxylase decarboxylase OH OH OH Tyrosine Dihydroxy phenilalanine (L-DOPA) Dopamine H2+ +H3N N H3C OH OH Dopamine HO Phenethanolamine HO β-hydroxylase N-methyltransferase OH OH Dopamine Epinephrine

Epinephrine and analogues OH OH H NH2 OH H HO N H N CH3 .HCL N CH3 CH3 .H2SO4 .HCL HO CH3 HO CH3 Tyramine HCL Pseudoephedrine HCL 2 Epinephrine (adrenaline) Ephedrine sulphate OH NH2 NH2 NH2 .H2SO4 .H2SO4 .H2SO4 CH3 CH3 2 2 2 Amfetamine sulfate Dexamfetamine sulfate Phenylethanolamine sulfate OH H OH HO N CH3 OH H HO H N CH3 .H2SO4 CH3 N CH3 .HCL HO .H2SO4 CH3 CH3 HO OH CH3 2 HO Isoprenaline HCL 2 Orciprenaline sulfate Salbutamol sulfate OH H HO N CH3 CH3 .H2SO4 CH3 OH 2 Terbutaline sulfate

cAMP-PKA pathway a a g b a P P P Gα gated channel cAMP gated channel PO4 gated channel Receptor GTP a GTP a Adenylyl cyclase GTP g b a cAMP P P ATP cAMP ADP ATP R cAMP cAMP CREB R C R C C cAMP R cAMP P Activated PKA Inactive PKA CREB P CREB Gene expression CRE Nucleus cAMP Response Element

Primary Action of Epinephrine in a Liver Cell GDP g b as Acetylcholine Epinephrine Stress signal Fuel for „fight or flight” Receptor P cAMP ATP Adenylate Cyclase Glucose Glucogen Synthase Glucose-6-Phosphatase Glucose-6-P Glucose-1-P Glycogen Fructose-2,6- bisphosphatase P Glucogen Phosphorylase Fructose-2,6-bisP Fructose-6-P P Fructose-1,6- bisphosphatase PFK-1 Phosphorylase Kinase A P Fructose-1,6-bisP Protein Kinase A PEP P Pyr Kinase Pyruvate

Glucose-6-Phosphatase Glucagon signaling GDP g b as PANCREAS Glucose low in blood Glucagon (Epinephrine) Receptor P cAMP ATP Adenylate Cyclase Glucose Glycogen Synthase Glucose-6-Phosphatase Glucose-6-P Glucose-1-P Glycogen Fructose-2,6- bisphosphatase P Glycogen Phosphorylase Fructose-2,6-bisP Fructose-6-P P Fructose-1,6- bisphosphatase PFK-1 Fructose-1,6-bisP Protein Kinase A PEP P Pyr Kinase Pyruvate

Serotonin receptor Serotonin receptor → G-protein → adenylyl cyclase → ATP → cAMP → PKA → cAMP Response Element (CRE) → Gene expression

Types of serotonin receptor Family Mechanism Potential 5-HT1 Decreasing cellular levels of cAMP Inhibitory 5-HT2 Increasing cellular levels of IP3 and DAG Excitatory 5-HT3 Depolarizing plasma membrane 5-HT4 Increasing cellular levels of cAMP 5-HT5 5-HT6 5-HT7

Receptor desensitization GRK ATP ADP Arrestin P G-protein linked receptor kinase Activated receptor Desensitized receptor