GPCRs Signal Transduction & Receptor Superfamilies (2024-25) PDF

WEEK 28 MPharm Programme Signal Transduction & Receptor Superfamilies G-Protein-Coupled Receptors 1 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA115 Receptor WEEK 28...

WEEK 28 MPharm Programme Signal Transduction & Receptor Superfamilies G-Protein-Coupled Receptors 1 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA115 Receptor WEEK 28 Outline of Lectures The keys steps in receptor signalling /signal transduction Signal transduction pathways & receptor superfamilies Structure & function of G-Protein- coupled receptors (GPCRs) Structure & role of G-proteins in GPCR signalling cAMP and IP3/Ca2+ as second messengers, and their key roles in GPCR signalling Slide 2 of 60 MPharm PHA115 Receptor WEEK Learning 28 Outcomes At the end of this lecture, you should be able to: Describe the keys steps in receptor signalling or signal transduction Describe the major signal transduction pathways & receptor superfamilies Describe the structure & function of G- proteins & G-protein-coupled receptors (GPCRs) Define & name examples of second messengers, and describe their key roles in GPCR signalling Slide 3 of 60 Explain GPCR desensitisation MPharm PHA115 & Receptor WEEK 28 Signal Transduction How is Drug-Receptor binding translated into a Biological Response? Slide 4 of 60 MPharm PHA115 Receptor WEEK 28 Receptor Signalling When an agonist drug binds to its receptor A drug-receptor (D-R) complex is formed The D-R complex undergoes a conformational change This triggers a chain of biochemical processes inside the cell  a biological response This process is called ‘signal transduction’ or ‘receptor signalling’ The chain Slide 5 of 60 MPharmof biochemical PHA115 events Receptor that WEEK 28 Receptor Signalling 3 stages of the receptor signalling process Signal reception the agonist drug binds to and activates a specific ‘receptor’ on / inside the target cell Signal transduction the drug-receptor complex activates a series of relay proteins & produces 2nd messengers inside the cell Cellular Slide 6 of 60 response MPharm PHA115 Receptor WEEK 28 The 3 Stages of the Drug- Receptor Signalling Process Slide 7 of 60 MPharm PHA115 Receptor WEEK 28 Signal Transduction Pathways 4 Major Signal Transduction Pathways Activation of receptor-ion channels (Ligand-gated receptors) Activation of second messenger pathways via G-protein-coupled receptors Activation of enzyme-linked receptors (e.g. Tyrosine kinase- Slide 8 of 60linked receptors) MPharm PHA115 Receptor WEEK 28 Receptor Superfamilies Receptor Superfamily A group of receptors with a similar basic molecular structure and that use the same signal transduction pathway 4 Major Receptor Superfamilies Ligand-gated / Ion channel-linked receptors G-protein-coupled receptors Enzyme-linked Slide 9 of 60 MPharm / Kinase-linked PHA115 Receptor WEEK 28 Receptor Superfamilies Slide 10 of MPharm PHA115 Receptor 60 WEEK 28 G-protein-coupled Receptors (GPCRs) (Metabotropic Receptors) Slide 11 of MPharm PHA115 Receptor 60  G-protein-coupled WEEK 28 Receptors (GPCRs) A large & diverse superfamily of integral membrane proteins used by cells to convert extracellular signals into intracellular responses They constitute the largest receptor superfamily in humans (~800 members) They transduce a wide array of extracellular signals and regulate virtually every aspect of physiology They mediate responses to hormones, neurotransmitters and growth factors, as well as responses to vision, olfaction & Slide 12taste of signals MPharm PHA115 Receptor 60 WEEK 28 G-protein-coupled Receptors (GPCRs) Key Features & Characteristics of GPCRs They all share a common structural motif of seven transmembrane (7-TM) α-helices They couple to & activate cytoplasmic heterotrimeric G-proteins upon agonist binding, leading to modulation of downstream effector proteins  biological response They also couple to cytoplasmic Slide 13 of 60 adaptor proteins, MPharm called -arrestins, PHA115 Receptor  Structure of WEEK 28 GPCRs A GPCR typically consists of a single polypeptide chain with 3 key regions The extracellular region – the N terminus & three extracellular loops (ECL1-ECL3) modulates ligand access to the binding site on receptor The TM region – seven transmembrane (7- TM) α-helices (TM1-TM7) forms the structural core, binds to ligands & transduces this information to the intracellular regions The intracellular region – three Slide 14 of intracellular loops (ICL1-ICL3), a short MPharm PHA115 Receptor 60 WEEK 28 General Architecture of a G Protein-coupled Receptor (GPCR) Slide 15 of MPharm PHA115 Receptor 60 WEEK 28 General Architecture of a GPCR Slide 16 of MPharm PHA115 Receptor 60 WEEK 28 GPCR Signalling via G-Proteins & Arrestins Effect or enzy me Slide 17 of MPharm PHA115 Receptor 60  GPCR Signalling via WEEK 28 Heterotrimeric G Proteins The key feature of GPCRs is their interaction with heterotrimeric GTP- binding proteins (or G-proteins) Heterotrimeric G-proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation They are localised at the inner leaflet of the plasma membrane – convey signals from cell-surface GPCR to downstream intracellular effector proteins Slide 18 of 60 They act as molecular MPharm PHA115 binary switches Receptor –  Heterotrimeric G- WEEK 28 Proteins Heterotrimeric G-proteins are composed of 3 different protein subunits: ,  and  Functionally, they consist of two units: an α subunit (Gα) and a tightly associated  complex Both the G and G subunits have lipid extensions that bind & tether the G-protein complex to the plasma membrane The Gα subunit harbours a guanine nucleotide-binding site, which is occupied by GDP in the inactive resting (off) state So far, 21 G, 5 G and 12 G Slide 19 of MPharm PHA115 Receptor 60 subunits/isoforms have been identified in  Heterotrimeric G-protein in WEEK 28 the Resting GDP-bound State Gα subunit is composed of 2 domains: a Ras-like domain & an α-helical domain A nucleotide-binding pocket is located between the two domains The Ras-like domain has GTPase activity (hydrolyzes GTP to GPD) & also provides binding The N-terminus of Gα is myristoylated or sites for the Gβ palmitoylated, which results in the attachment of subunits Slide 20 of the G protein to the plasma membrane MPharm PHA115 Receptor 60 WEEK 28 Signalling via Heterotrimeric G-Proteins Interaction with agonist-bound GPCR  conformational change  exchange of GDP for GTP on the Gα subunit  dissociation of G-GTP subunit from the G dimer Both G-GTP & the freed G proceed to interact with, and regulate the activity of, unique downstream effector proteins  biological response Ultimately, the activated G-protein returns to the inactive resting (off) state: GTPase activity in the Gα subunit hydrolyses bound GTP to GDP Hydrolysis of GTP to GDP is accelerated by Slide 21 of 60 regulators MPharmof G-protein PHA115 signalling (RGS) proteins Receptor WEEK 28 Mechanism of G-Protein Activation By GPCRs Slide 22 of MPharm PHA115 Receptor 60 WEEK 28 The Guanine Nucleotide Cycle of Heterotrimeric G- Proteins Slide 23 of MPharm PHA115 Receptor 60  The Guanine Nucleotide WEEK 28 Cycle of Heterotrimeric G- Proteins Slide 24 of MPharm PHA115 Receptor 60 WEEK 28 Diversity of GPCR Signalling Mechanisms Signalling G-proteins via Gα are classified Proteins based on their Gα subunits Gα proteins are grouped into 4 families based on their peptide sequence & functional similarities – Gαs, Gαi, Gαq/11, and Gα12/13 protein families Each Gα family can relay GPCR signals to multiple downstream effectors  triggering of different signalling pathways Gαs family – stimulate adenylate cyclase   cAMP Slide 25 of Gαi family MPharm– inhibit adenylate cyclase PHA115 Receptor   cAMP 60  G-Protein Families & WEEK 28 Gα Gα family Effectors Tissue expression Effector proteins & family members Effect Gαs Gαs, Gαolf most cell types (Gαs); Activate adenylate olfactory neurons (Gαolf) cyclase Gαi Gαi1, Gαi2, Gαi3, most cell types; high in Inhibit adenylate cyclase Gαo, Gαt, Gαg, neurons (Gαo); rod & cone Activate cGMP Gαz cells of the eye (Gαt); taste phosphodiesterase receptor cells (Gαg); neuronal tissues & platelets (Gαz) Gαq Gαq, Gα11, Gα14, most cell types (Gαq); Activate phospholipase Gα16 olfactory neurons (Gαq, C-β Gα11); kidney, lung & liver (Gα14); haematopoietic cells (Gα16) Gα12/13 Gα12, Gα13 most cell types Modulate Rho & Ras- GEFs Slide 26 of MPharm PHA115 Receptor 60  GPCR-mediated Signalling WEEK 28 Pathways Slide 27 of MPharm PHA115 Receptor 60  GPCR-mediated Signalling WEEK 28 Pathways Slide 28 of MPharm PHA115 Receptor 60  GPCR-mediated Signalling WEEK 28 Pathways Slide 29 of MPharm PHA115 Receptor 60  G-mediated Signalling WEEK 28 Pathways Most G proteins mediate GPCR signalling by regulating the levels of intra­cellular regulatory molecules, called second messengers The second messengers regulate the activity of multiple downstream effector proteins  biological response Key second messengers include: cAMP, IP3 and Ca2+ The cAMP signalling pathway Gs-GTP activates Adenylyl Cyclase (AC)   cAMP Slide 30 of Gi/o-GTP inhibits PHA115 MPharm Adenylyl Cyclase (AC)   Receptor 60  G-mediated Signalling WEEK 28 Pathways IP3 cAMP PATHWAY IP3/Ca2+ PATHWAY Slide 31 of MPharm PHA115 Receptor 60 WEEK 28 MPharm Programme Receptor Superfamilies G-Protein-Coupled Receptors 2 Dr Gabriel Boachie-Ansah [email protected] Dale 113 ext. 2617 MPharm PHA115 Receptor  The cAMP Signalling WEEK 28 GPathway -GTP stimulates the activation of s Adenylyl Cyclase (AC), a membrane-bound enzyme Adenylyl Cyclase (AC) converts ATP to cAMP The increased cAMP level leads to activation of protein kinase A (PKA) Activated PKA mediates phosphorylation of multiple downstream effector targets  cellular response cAMP can also modulate the activity of several Guanine Exchange Factors (GEFs) and ion channels Slide 33 of 60 On the contrary, MPharm G -GTP inhibits PHA115 the Receptor  cAMP Signalling WEEK 28 Pathway Slide 34 of MPharm PHA115 Receptor 60  cAMP Signalling WEEK 28 Pathway Cellular Response Slide 35 of MPharm PHA115 Receptor 60  Synthesis and WEEK 28 Degradation of cAMP Slide 36 of MPharm PHA115 Receptor 60 WEEK 28 Slide 37 of MPharm PHA115 Receptor 60 WEEK 28 Slide 38 of MPharm PHA115 Receptor 60 WEEK 28 Slide 39 of MPharm PHA115 Receptor 60 WEEK 28 Slide 40 of MPharm PHA115 Receptor 60  Signal Amplification in cAMP WEEK 28 Signalling Pathway Slide 41 of MPharm PHA115 Receptor 60 WEEK 28 The IP3/Ca2+ Signalling Pathway G -GTP activates Phospholipase C- (PLC) in q/11 cell membrane Phospholipase C- (PLC) catalyzes the conversion of phosphatidylinositol 4,5- bisphosphate (PIP2) to inositol triphosphate (IP3) & diacylglycerol (DAG) IP3 acts on the endoplasmic reticulum (ER) & trigger the release of Ca2+ into the cytoplasm Ca2+ modulates the activity of Ca2+-dependent effector proteins (e.g. calmodulin, transcription factors)  cellular response DAG, in concert with the increased Ca2+ level, activatesMPharm Slide 42 of 60 protein kinase PHA115 C (PKC) Receptor in the cell  IP3/Ca Signalling WEEK 28 2+ Pathway IP3R Slide 43 of MPharm PHA115 Receptor 60  IP3/Ca Signalling WEEK 28 2+ Pathway Phosphatidylinositol-biphosphate (PIP ) is a 2 membrane phospholipid Cleaved by G-GTP activated phospholipase C- (PLC) to generate 2nd messengers: Inositol triphosphate (IP3) Diacylglycerol (DAG) IP3 diffuses through the cytosol and releases Ca2+ from the ER DAG stays in the membrane & Slide 44 of activates protein kinase C MPharm PHA115 Receptor 60  IP3/Ca Signalling WEEK 28 2+ Pathway Slide 45 of MPharm PHA115 Receptor 60  IP3/Ca Signalling WEEK 28 2+ Pathway Slide 46 of MPharm PHA115 Receptor 60  IP3/Ca2+ Signalling Pathway & SM WEEK 28 Contraction Ach (M -R) 3 Epi (1-R) Slide 47 of MPharm PHA115 Receptor 60  IP3/Ca2+ Signalling Pathway & WEEK 28 Smooth Muscle Contraction Slide 48 of MPharm PHA115 Receptor 60 WEEK 28 Diversity of GPCR Signalling Mechanisms Signalling The via G & G subunits arethe G Dimer tightly bound/associated to form an obligate functional heterodimer There are 5 G and 12 G subunits in the human genome  a large number of potential combinations of G dimers Lipid modification of the G subunit  attachment & localisation of the G dimer to the membrane GPCR activation  dissociation of G-GTP  free G dimer The Slide 49 of free G dimer plays key roles in GPCR 60 signalling by regulating MPharm PHA115 a host of downstream Receptor WEEK 28 Signalling via the G Dimer Ach-induced Lowering of Heart Rate Slide 50 of MPharm PHA115 Receptor 60  GPCR Desensitization and WEEK 28 Intracellular Trafficking Temporal and spatial signalling of activated GPCRs is controlled by desensitisation & internalisation Involves the sequential action of 2 major intracellular actors: G protein-coupled receptor kinases (GRKs) -arrestins (cytoplasmic adaptor proteins) First, GRK docks onto activated GPCR  phosphorylation of serine & threonine residues in the receptor  inhibition of G-protein activation (homologous Slide 51 of 60 desensitisation) MPharm PHA115 Receptor  GPCR Desensitization and WEEK 28 Intracellular Trafficking Next, -arrestins bind to phosphorylated GPCR & sterically hinder GPCR-G protein coupling interactions  inhibition of further activation of G protein-mediated signalling -arrestins then couple the phosphorylated GPCRs to clathrin-coated pits to facilitate receptor internalisation The internalised GPCRs are trafficked to endosomes, with one of three possible fates: They may be dephosphorylated by phosphatases & recycled back to the cell Slide 52 of surface for future MPharm signalling (recycling) PHA115 Receptor 60  Desensitization & Trafficking WEEK 28 of GPCRs Slide 53 of MPharm PHA115 Receptor 60 WEEK 28 Examples of GPCRs that Mediate the Actions of Key Chemical Messengers Slide 54 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in Adrenergic Actions Norepineph rine Epinephrin e 1 1, 2, 3 2 Slide 55 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in Acetylcholine Actions Acetylcholin e, Carbachol Slide 56 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in mediating Autonomic Nervous System Control of Heart Function Slide 57 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in Histamine Actions Histami ne H1 H2 H3, H4 Slide 58 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in Dopamine Actions Dopamine D1, D2, D3, D5 D4 Slide 59 of MPharm PHA115 Receptor 60 WEEK 28 Role of GPCRs in Serotonin (5- HT) Actions Serotonin (5-HT) 5-HT2A, 5-HT2B, 5- 5-HT4, 5-HT6, 5- HT2C 5-HT7 HT1 Slide 60 of MPharm PHA115 Receptor 60 WEEK 28 Slide 61 of MPharm PHA115 Receptor 60 WEEK 28 Slide 62 of MPharm PHA115 Receptor 60 WEEK 28 GPCR Signalling via G-Proteins & Arrestins Slide 63 of MPharm PHA115 Receptor 60 WEEK 28 Slide 64 of MPharm PHA115 Receptor 60 WEEK 28 Diversity of GPCR Signalling Mechanisms Signalling Following activation via of GPCRs, rapid -Arrestins attenuation or desensitization of receptor responsiveness is necessary to prevent uncontrolled signalling. Desensitization is initiated by phosphorylation of the receptor by GPCR kinases followed by uncoupling of GPCR-G protein interactions mediated by members of the β-arrestin protein family. In addition to terminating G protein signalling, β-arrestins also play a role in promoting GPCR signalling by internalizing the receptor and acting as a molecular scaffold to recruit Slide 65signalling of proteins.PHA115 MPharm Receptor 60 WEEK 28 Slide 66 of MPharm PHA115 Receptor 60 WEEK 28 Slide 67 of MPharm PHA115 Receptor 60 WEEK 28 Slide 68 of MPharm PHA115 Receptor 60 WEEK 28 Arrestins and Receptor Regulation Slide 69 of MPharm PHA115 Receptor 60 WEEK 28 an L i g d GPCR Cellular Substr Cellular Response ate Response Slide 70 of MPharm PHA115 Receptor 60 WEEK 28 Slide 71 of MPharm PHA115 Receptor 60 WEEK 28 ? Slide 72 of MPharm PHA115 Receptor 60  The Calcium Signalling WEEK 28 Pathway Slide 73 of MPharm PHA115 Receptor 60  The Calcium Signalling WEEK 28 Pathway Ach (M -R) 3 Epi (1-R) Slide 74 of MPharm PHA115 Receptor 60  The Calcium Signalling WEEK 28 Pathway Slide 75 of MPharm PHA115 Receptor 60 WEEK 28 Slide 76 of MPharm PHA115 Receptor 60 WEEK 28 Slide 77 of MPharm PHA115 Receptor 60 WEEK 28 Slide 78 of MPharm PHA115 Receptor 60 WEEK 28 Slide 79 of MPharm PHA115 Receptor 60 WEEK 28 Slide 80 of MPharm PHA115 Receptor 60 WEEK Signal Transduction 28 Pathways Activation of Receptor-Ion Channels Slide 81 of MPharm PHA115 Receptor 60 WEEK Signal Transduction 28 Pathways Activation of G-protein coupled Receptors Slide 82 of MPharm PHA115 Receptor 60 WEEK Signal Transduction 28 Pathways Activation of G-protein Coupled Receptors Slide 83 of MPharm PHA115 Receptor 60 WEEK Signal Transduction 28 Pathways Activation of Tyrosine Kinase- linked Receptors Slide 84 of MPharm PHA115 Receptor 60 WEEK Signal Transduction 28 Pathways Direct Activation of Gene Transcription Slide 85 of MPharm PHA115 Receptor 60