GPCR Lectures 1-3 PCL302 2024 PDF

Introduction to G Protein-Coupled Receptor Signal transduction  G Protein-Coupled Receptors and their Signaling Systems Become familiar with the components of a GPCR system Understand how the receptor becomes activated Major cell signaling systems regulated by GPCRs Physiologi...

Introduction to G Protein-Coupled Receptor Signal transduction  G Protein-Coupled Receptors and their Signaling Systems Become familiar with the components of a GPCR system Understand how the receptor becomes activated Major cell signaling systems regulated by GPCRs Physiological and Pharmacological examples  Major players covered in PCL302 G-protein Zhang and Eggert Mol. BioSyst., 2013, 9, 586 GPCRs are encoded by hundreds of human genes GPCR Superfamily  GPCR Ligands Neurotransmitters: adrenaline, dopamine, serotonin, glutamate, GABA Peptide hormones: Thyroid-stimulating hormone, Parathyroid hormone, vasopressin, glucagon Sensory stimuli: light, odours, salt, sugar Prostaglandins: Prostaglandin E, F Glycoprotein Growth-Differentiation factors  GRAFS classification of vertebrate GPCR Family Characteristics Example Receptor Large N-terminus with Cys- Secretin Cys bridges Glucagon Receptor Very large N-terminus 200- Adhesion 2800 aa, EGF-like and CD97 Receptor mucin-like motifs.Highly glycosylated Large bi-lobed N-terminus Glutamate Ligand binding closes the Metabotrophic Glutamate Receptor bi-lobular domain Obligate heterodimers 200 aa N-terminus binds Frizzled ligands, Receptors complex Frizzled 1 with LRP proteins Ligands bind to TM domains Rhodopsin Or to ec N-termini Adrenergic receptors Family Equivalence Class-A (7TM1) Rhodopsin Class-B (7TM2) Secretin Class-C (7TM3) Metabotropic Class-D Fungal Non-vertebrate pheromones Class-E Cyclic AMP Non-vertebrate Class-F Frizzled Still Controversial (may not be GPCRs) Nomenclature GPCR: G-Proteins Coupled Receptors Problem: GPCR are not only coupled to G-Proteins 7TM: Seven Transmembrane Domains Receptors Problem: Not all 7TM proteins are GPCRs GPCRs are major drug targets Santos et al., 2017 Structural Features of GPCRs All GPCRs have the following structural features 7 lypophylic transmembrane helical domains Extracellular N-terminus and 3 ec loops Intracellular C-terminus and 3 ic loops Ligands bind to the outside or within the membrane helices G proteins couple to the inside surface of the receptors Secondary Structure (Class-A) Glycosylation sites. Usually Asparagine Palmitoylation site- Cysteine Serine and Threonines in Third loop and C-tail Can be sites for Phosphorylation Secondary Structure (Class-A) Class-A: Beta-Adrenergic Receptor Class-B: Secretin Receptor Laurence J. Miller, in Handbook of Biologically Active Peptides (Second Edition), 2013 Class-C: Metabotropic Glutamate Receptor IUPHAR  Fig. 1 Structural model and schematic representation of class C GPCRs. Class C GPCRs are composed of a Venus flytrap (VFT) domain, a cysteine-rich domain (CRD) and a transmembrane (7TM) domain. This class of receptors forms obligatory dimers, either homodimers (e.g. mGlu) or heterodimers (e.g. GABAB) Moller et al. Biophys Rep 2017, 3(4–6):57–63 Class-C Obligates dimers Ex: mGluR and GABA-B G-Proteins  Receptor Outside Plasma membrane Inside G Proteins a subunit Composed of three different subunits 1a 1b and 1g. g subunit The a subunit binds the guanine nucleotide b subunit Heterotrimeric G protein Receptor Activation of G Proteins Agonist+ Ga + bg Receptor* GTP Ga + Effector GTP Gabg Second messengers GDP Gbg + Effector RGS proteins act on Ga Ga-GDP +Pi Gbg  The Family of Heterotrimeric G Proteins 4 Subfamilies of Gs, Gi, Gq, G12 Gs proteins (Gs,Golf) stimulate adenylyl cyclase to increase cAMP Gi proteins(Gi,Go,Gt,Gz) inhibit adenylyl cyclase to decrease cAMP (Gi) or stimulate cGMP-PDE (Gt) Gq proteins (Gq,G11,G14, G15) stimulate phospholipase C proteins to increase IP3, DAG and Ca2+ G12 proteins (G12, G13) stimulate Rho proteins  Inactivation of G Proteins a a bg GTP GDP PiPi Gq + PLC Gq Mayeenuddin et al. 2001, J, Neurochem. 78,1350-1358  Regulators of G protein Signaling (RGS Proteins) RGS a a bg GTP GDP Pi  G protein deactivation by RGS Neubig and Siderovski 2002, Nat Rev Drug Dis. INTRODUCTION TO GPCRs PART 2 Functions of G Proteins and Arrestins  Effectors Regulated by G proteins Adenylyl cyclase enzymes 2 groups of 6 TM domains Linked by C1 domain and C2 domain at the C-terminus C1 C2  Adenylyl Cyclase Isoforms Group Isoform Activators Inhibitors 1 I, III, VIII Gsa, Fsk, Ca2+ Gia, bg, /Calm CamK 2 II, IV, VII Gsa, Fsk, bg, PKC (ACIV) PKC (ACII, ACVII) 3 V, VI Gsa, Fsk, PKC Gia, PKA, Ca2+ 4 IX Gsa Calcinurin  Gs and Gi Regulation of Adenylyl Cyclase Gsa-GTP binds to C1 & C2 Stabilize the active conformation Forskolin binds to C1 & C2 Activates ACI-VII Gia-GTP binds only C1 to Inhibit ACI, III, V, VI, VIII C. Dessauer, UTHSC  cAMP Signaling Much of the signaling from cAMP is mediated by activation of PKA that in turn phosphorylates (Ser and Thr) proteins Such as metabolic enzymes and transcription factors Rapid changes mins R PKA P R PKA G-6-Pase Increased glucose output PhosphorylaseP PKA R cAMP kinase PKA R cAMP Glycogen P synthase Gene transcription P P Increased proteins, slow hrs CREB CREB Examples of cAMP-regulated systems Cardiac output - b-adrenergic receptors Bronchodilation - b-adrenergic receptors Extracellular calcium homeostasis – Parathyroid hormone and Calcitonin Motor control – Dopamine receptors  Effectors Regulated by G proteins PLC-b PLC proteins are cytoplasmic proteins that when activated hydrolyze membrane inositol phospholipids. GPCRs regulate PLC-b proteins that belong to a larger family of phospholipase enzymes EF-hand PLC-d PH X Y C2 PLC-b PH X Y C2 PLC-g PH X C2 Y SH2 SH3 PLC-e X Y C2 RasGEF PH, pleckstring homology; XY, catalytic; C2, Ca2+ binding; SH2/SH3 Src-homology; RasGEF, Ras GTP exchange factor  Inositol Phosphates Primary PLC substrate Phosphatidic Acid Inositol phosphate cycle  Stimulation of PLC by Gq Gqa-GTP binds PLC-b Stimulates X-Y catalytic domains PIP2 hydrolyzed to IP3 and DAG PLC Initial activation requires recruitment to the PM through Ga-GTP and Gbg  Down-stream of PLC Increases in Calcium stimulate CAM kinases (CaMK). Increases in Calcium and DAG stimulate PKC (a,b,g,d,e,h,q) Regulation by phosphorylation of many enzymes and transcription factors Activation of Calcineurin PKC family Activators Ca2+, DAG,PS DAG PI3K Adenylyl Cyclase R R Gs AC Gi/o + - adenylate cyclase ATP AMPc PKA PLC R PIP2 DAG G PLC PKC + IP3 Ca++ activation Ion Channels R G +  GPCR Desensitization Following activation, GPCRs are inactivated by multi-step mechanisms Following activation G proteins are inactivated by increasing GTPase activity Activation of 2nd-messenger regulated kinases (such as PKA) can inhibit receptors All of these processes contribute to inactivation and desensitization mechanisms- making the cell temporarily less responsive to stimulation Homologous Desensitization and Down-regulation of GPCRs GRKs Arrestins (Desensitization) Clatherin-coated pits Internalization(Down-regulation) Time to recovery GPCR Kinases: (GRKs) Gurevich et al., 2012 Pharmacol Ther. Putative domain architecture of the {beta}-arrestins Luttrell, L. M. et al. J Cell Sci 2002;115:455-465 Heterologous Desensitization Dopamine R Beta adrenergic P P The many roles of beta arrestins Beta Arrestin as signaling effectors Multiplicité d’action des GPCRs Example of Receptor signaling network The Dopamine D2 receptor Beaulieu and Gainetdinov 2011, Pharm Rev. Multidimensional Drug efficacy Galandrin et al. TiPS 2007 End of Second class INTRODUCTION TO GPCRs PART 3 INTRODUCTION TO GPCRs PART 3 Examples of GPCR based Pharmacological Interventions  GPCRs That Stimulate Adenylyl Cyclase: b-Adrenergic Receptors 1. b1 receptors: §Expressed in the heart §Stimulation of these receptors results in increased force and velocity of heart contraction and increased stroke volume. 2. b2 Receptors: §These receptors are found in the smooth muscle of the lungs. §Their stimulation leads to bronchodilation. 3. b3 Receptors: §These receptors are expressed in adipocytes. §Stimulation causes increased lipolysis. b1 Adrenergic receptors mechanisms of action: Heart Adrenergic Stimulation ↑ Heart rate ↑ Conduction velocity ↑ Conractility ↑ Rate of myocyte relaxation  b-Adrenergic Receptor Antagonists Drug Receptor(s) Use Propranolol b1 and b2 Antihypertensive Antiarrhythmic Nadolol b1 and b2 Angina prophylactic Metoprolol b1 selective Antiarrhythmic Carvediol b1 and a1 Heart failure  b2 Adrenergic Receptors Mechanism of Action: Blood Vessels and Lungs Mechanism: § The b2 receptor associated with smooth muscle also utilizes the cAMP signaling system. § Stimulation of the b2 receptor leads to smooth muscle relaxation. §Production of cAMP inhibits processes linked to contraction Terasaki et al. Clin. Invest. (2014) 4(5), 429–439  Asthma Asthma stimulated by exposure to irritants Pollen Moulds Cigarette smoke  b2- Selective Receptor Agonists Salbutamol Taken by inhalation Effective in 3-5 mins Max effect 60-90 mins Lasts 3-6 hours Salmeterol Longer duration of action, 12 h b3 Adrenergic Receptors Regulation of lipolysis: A. In the basal state, §nonphosphorylated hormone- stimulated lipase (HSL) is in the cytosol bound to lipotransin. §nonphosphorylated perilipin is bound to the lipid droplet. B. When catecholamine interacts with ß3AR, §ß3AR couples to Gs. §Gs leads to the sequential stimulation of adenylyl cyclase and PKA. §The catalytic subunits of PKA phosphorylates both HSL and perilipin. §Release of free fattyacids and glycerol Molecular Endocrinology 18: 2123, 2004  b3-Selective Agonist BRL37344 increases lipolysis Potential use in the treatment of obesity Cardiovascular effects profound hypotension Uterine relaxation Decreased cardiac contractility  GPCRs That Stimulate Phospholipase C Activity: a1-Adrenergic Receptors §These receptors are present in the vascular smooth muscle, respiratory mucosa and radial muscles of the eye. §Their stimulation results in vasoconstriction. 1. Agonist binds to the vascular smooth muscle a1-receptor. 2. G-protein is in complex with GDP. 3. The receptor promotes exchange of GTP for GDP and release of Gα-GTP. 4. The G-protein activates phospholipase C leading to an increase of both inositol trisphosphate (IP3) and diacylglycerol (DAG). 5. IP3 binds to specific sites on the SR and stimulates the release of intracellular Ca2+. 6. Ca2+ influx is activated. 7. Like the b-receptors, a receptors can also be desensitized and down regulated via phosphorylation of the receptor. Agonists of Adrenergic Receptors Second Receptor Agonists G protein Messenger alpha1 (a1) E>NE IP3/Ca2+; DAG Gq alpha2 (a2) NE>E ¯ cyclic AMP Gi beta1 (b1) E=NE ­ cyclic AMP Gs beta2 (b2) E>>NE ­ cyclic AMP Gs beta3 (b3) E