FFP1-62 GPCRs STS 2023.pptx

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-62 Introduction to Receptors: G protein-coupled receptors Prof Steve Safrany 341 [email protected] Dr. Roger Preston Learning Outcomes • Outline the concept and nature of receptor signalling • Explain the struc...

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1-62 Introduction to Receptors: G protein-coupled receptors Prof Steve Safrany 341 [email protected] Dr. Roger Preston Learning Outcomes • Outline the concept and nature of receptor signalling • Explain the structure of G-protein-coupled receptors • Explain the nature of the signalling cascades G- protein coupled proteins can generate • Describe the mechanisms by which Gproteins regulate the effector enzymes Receptor response theory Four types of receptor – others are available G-protein coupled receptor structure • Monomeric proteins MW 35K70K • Pass through the membrane 7 times • At least 500 different receptors • Include light, taste and smell Key points about G-proteins 1. An enzyme composed of 3 subunits:  , , 2. Bind to and hydrolyse GTP to GDP 3. Inactive when GDP bound 4. Active when GTP bound 5. Acquires high What does each part of the Gprotein do? • The activity status of G proteins is determined by the subunit • 4 families of Gproteins based on structural similarities – Gs, Gi, Gq and G12 • Main purpose is to regulate amplifier or effector protein activity – βγ exist as dimers – 6 different β and 11 different γ – Can also exert signalling activity GPCR signalling • • • The and of the G-protein anchor it to the membrane in its “inactive” or “unbound” state G-protein is not linked to the receptor G-protein has GDP-bound Lodish et al. Molecular Cell Biology Ligand binding and activation • Receptor ligand binds to GPCR Lodish et al. Molecular Cell Biology Ligand binding and activation • Receptor ligand binds to GPCR • The occupied receptor couples with the  subunit Lodish et al. Molecular Cell Biology Signal transduction begins • GDP is replaced with GTP • GTP-Gα dissociates from Gβγ Lodish et al. Molecular Cell Biology Second messengers are produced • GTP-Gα and / or Gβγ activate the effector • Meanwhile the agonist has dissociated Lodish et al. Molecular Cell Biology The system resets • GTPase activity returns the system to resting • The cycle can start again Lodish et al. Molecular Cell Biology Regulatory control of GPCRs • Guanine-nucleotide exchange factors (GEF) • Ligand-bound receptor acts GEF (Accelerates signalling) • Guanine nucleotide dissociation inhibitor (GDI) • βγ acts as GDI preventing GDP release (Inhibits signalling) • GTPase-accelerating proteins (GAPs) • Stimulate GTPase activity (Turn off signalling) GDI Activation of adenylyl cyclase • AC generates cAMP, an important ‘second messenger’ in cells • Activated by Gs • Inhibited by Gi Cell Signalling Biology - Michael J. Berridge - www.cellsignallingbiology.org - 2009 Ga subunits control AC activation Gαs activates adenylyl cyclase Activated by cholera toxin Inhibited by pertussis toxin Gai inhibits adenylyl cyclase Receptors regulating adenylyl cyclase Lodish et al. Molecular Cell Biology Chapter 13 Ga subunits control AC activation <4 million cases, 143,000 deaths / year. 240 million cases, >160,00 deaths / year Role of cyclic adenosine monophosphate • • • cAMP activates protein kinases A (PKAs) Protein kinases phosphorylate proteins* cAMP-dependent kinases have many substrates ‐ ion channels and ‐ metabolic pathways • cAMP is metabolised When AC activity is disrupted • E. coli toxin – E. coli ‘traveller’s diarrhoea’ toxin • Covalent modification of Gαs - can’t hydrolyse GTP (locked ‘ON’) • Elevated cAMP levels in colonic epithelium cause efflux of water and ions • Severe diarrhea and dehydration Treatment for this disruption • Loperamide/Imodium acts as a μ-opioid receptor agonist in large intestine • Treatment – opiate receptor coupled to Gi • Another example of functional antagonism E. coli toxin Loperamide* Activation of phospholipase C • An alternative ‘effector protein’ • Classically activated by Gαq/11 • Hydrolyses the phosphoinositide (PIP2) from the membrane • Produces second messengers 1. Diacylglycerol (DAG) 2. Inositol 1,4,5- PLC-generated second messengers • IP3 is water soluble • Binds to the IP3 receptor in the ER • Results in Ca2+ release (smooth muscle contraction, hormone release, neuron transmitter release) • Signal terminated PLC-generated second messengers • Calmodulin is a major Ca2+ target • Binds 4 Ca2+ ions • Kd 1 μM • Change in shape allows new interactions • Change in shape destroys other interactions PLC-generated second messengers • Ca2+ also binds some Ca2+dependent protein kinases C (PKCs) • Causes translocation • PKC is activated by DAG and PS • As a kinase… Diversity of GPCRs Gs • 5-HT4, 5-HT7 • Adenosine A2 • Adrenergic β1, 2, 3 • Dopamine D1, D5 • Histamine H2 • Vasopressin V Gi 5-HT1, 5-HT5 ACh M2, M4 Adenosine A1, A3 Adrenergic α2 Cannabinoid CB Dopamine D2, 3, 4 Glutamate mGlu2, 3, 4, 6, 7, 8 • Histamine H3, 4 • Opioid δ, κ, μ • Prostanoid EP3 • • • • • • • NO ! Gq/11 • 5-HT2 • ACh M1, M3, M5 • Adrenergic α1 • Glutamate mGlu1, 5 • Histamine H1 • Vasopressin V1 Diversity of GPCR signalling NO ! Further reading and viewing • https://www.nature.com/scitable/topicpage/gpcr-1404747 1 • Katzung • Rang • Medical Pharmacology at a glance • https://portlandpress.com/pages/cell_signalling_biology What we have learned • The concept and nature of receptor signalling • The structure of G-protein-coupled receptors • The nature of the signalling cascades Gprotein coupled proteins can generate • The mechanisms by which G-proteins regulate the effector enzymes – adenylate cyclase and phospholipase C