Cellular Communication 2 2024 PDF

ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Cellular Biology & Homeostasis CELLULAR COMMUNICATION part 2 VP 2024 Clara Camargo, DVM ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE LEARNING OBJECTIVES 1. Understand the importance of the GPCR/G proteins in cell signaling 2. Describe the G-protein coup...

ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Cellular Biology & Homeostasis CELLULAR COMMUNICATION part 2 VP 2024 Clara Camargo, DVM ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE LEARNING OBJECTIVES 1. Understand the importance of the GPCR/G proteins in cell signaling 2. Describe the G-protein coupled receptors (GPCRs) components 3. Differentiate Gs (stimulatory), Gi (inhibitory) and Gq 4. Understand the GPCR-cAMP signal pathway and the second messengers associated with it 5. Understand signal termination and list the different types discussed in class 6. Understand the mechanism of action of beta-agonists and beta-blocker drugs ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE CELL SURFACE RECEPTORS  milliseconds  seconds Muscarinic cholinergic receptors  Minutes Cytokine receptors  Minutes to hours ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE MAJOR TYPES OF RECEPTORS and SIGNAL TRANSDUCERS From: Lehninger Principles of Biochemistry ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE GPCRs are involved in many common diseases, including allergies, depression, blindness, diabetes, cardiovascular diseases  Nearly 35% of all marketed drugs currently available work by targeting different GPCRs All G proteins share a common feature: they can become activated and then, after a short time, can inactivate themselves:  they serve as molecular binary switches with built-in timers Example: β-adrenergic receptor, which mediates effects of epinephrine:  Prototype for all GPCRs, used as a model for signal transduction  target of ‘beta blockers’  Similar receptors: serotonin, angiotensin II and glucagon Nobel Prize in Physiology and Medicine in 1994 for their discovery of G-proteins and their role in signal transduction in cells ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE G-PROTEIN COUPLED RECEPTORS (GPCR) - OVERVIEW Metabotropic receptors transmembrane proteins composed of a single polypeptide chain Bind to a particular type of G protein (Gs Gi Gq)  cAMP signal pathway (Stimulatory - Gs and Inhibitory –Gi)  Phosphatidylinositol (PIP2), DAG/IP3 pathway (Gq protein) Mediate most responses to signals from the external world, as well as signals from other cells Hormones, neurotransmitters and local mediators About 900 types of GPCR have been identified in humans: >350 for detecting hormones and other endogenous ligands, >500 olfactory and gustatory receptors ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE 1. A plasma membrane receptor (GPCR) with 7 transmembrane helical segments 2. G- protein (Gs Gi Gq…) 3. An effector enzyme in the plasma membrane generating an intracellular second messenger 4. A guanosine nucleotide binding protein (GTP/GDP) which activates the G protein α -subunit Once stimulated by receptor activation → the G protein exchanges bound GDP for GTP ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Membrane associated heterotrimeric proteins that transmit the signal into the cell interior o Consist of 3 different subunits (α, β, and γ) o Bind directly to the cytoplasmic domain of GPCRs ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Are molecular switches “ON/OFF” The α subunit (Gα) is a GTPase and has a GDP bound in its inactive state Gα once activated (by the ligand/GPCR complex) exchanges GDP for GTP The activation causes the dissociation of the β/γ complex from the α subunit Both the GTP-bound Gα and the β/γ complex can interact with targets o May stimulate or inhibit effector proteins/enzymes Ex: Enzymes and ion channels which transmit the signal onward  Gα intrinsic GTPase hydrolyzes GTP to GDP and becomes inactive after certain time (build in timer) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE G-PROTEINS – Molecular switches The intrinsic GTPase activity of G proteins is increased by: → GTPase Activator Proteins (GAPs) strongly stimulate GTPase activity, causing rapid inactivation of the G protein GAP  Gα GTPase + GAP activities determine how long the switch remains ‘on’ ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Adenylate cyclase = adenylyl cyclase ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE → Activation of PKA (Protein Kinase A) ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE [cAMP] is ultimately reduced by cAMP phosphodiesterase (PDE) AC = ADENYLYL CYCLASE PDE = PHOSPHODIESTERASE ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE 1. 2. 3. 4. 5. 6. 7. Binding of ligand to receptor causes conformational change, affects interaction with Gs Gs exchanges GDP for GTP and is activated Gs α-subunit moves to Adenylyl Cyclase (AC) and activates it Adenylyl cyclase catalyzes formation of cAMP cAMP activates Phosphokinase A (PKA) Phosphorylation of cellular proteins by PKA causes cellular response Gs α is a GTPase that turns itself off by converting its bound GTP to GDP ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE  E.g., Epinephrin binds to adrenergic receptors and exerts its downstream effect through the activation of adenylyl cyclase (AC) and increase in [cAMP] cAMP allosterically activates PKA INACTIVE  PKA has catalytic and regulatory subunits PKA  PKA catalyzes the phosphorylation of other proteins: E.g., Glycogen phosphorylase kinase → mobilization of glycogen stores in liver and muscle in preparation for the need of energy Different proteins can be controlled by PKA → they all share an AA sequence that marks them for regulation by PKA (regulatory site) ACTIVE ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE RECAL: GLYCOGEN METABOLISM REGULATION REGULATION OF GLYCOGENESIS AND GLYCOGENOLYSIS (SIMPLIFIED) INSULIN  increases/stimulates glycogenesis  ANABOLIC decreases/inhibits glycogenolysis GLUCAGON (also epinephrine)  increases/stimulates glycogenolysis  CATABOLIC decreases/inhibits glycogenesis ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE 1 2 Beta Adrenergic Receptor 3 Signal transduction entails several steps that amplify the original hormone signal  Binding of hormone activates GPCR → Gs 4  Each active Gsα stimulates synthesis of many cAMP  Each PKA phosphorylates many target enzymes Net effect of cascade: the amplification of the hormone signal by several orders of magnitude 5 ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE somatostatin Some hormones inhibit AC, lowering [cAMP], and suppressing protein phosphorylation Ex.: somatostatin, adenosine, PGE1 binding to its receptor  activation of Gs and Gi are structurally homologous, but have opposite effects inhibitory G protein (G¡) FYI - Caffeine binding to Adenosine receptors https://www.youtube.com/watch?v=foLf5Bi9qXs ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE  Several mechanisms cause termination of the GPCR pathway  Signal transducing systems must be able to turn off after the stimulus has ended  Most systems also adapt to the continued presence of the signal by becoming less sensitive in a process called desensitization The β-adrenergic system can be regulated by: 1. Low concentrations of epinephrine in the blood cause its dissociation from its receptor and pathway inactivation Intrinsic GTPase activity of G protein causes hydrolysis of bound GTP to GDP, causing return of Gα to bind with β and γ subunits and consequently deactivation of the pathway GAPs (GTPase activator proteins) will speed up signal termination ↓ cAMP production ↓ active PKA 2. Desensitization via receptor sequestration ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE SIGNAL TERMINATION - GAPs GAP GTPase Activator Proteins (GAP) The ‘professional terminators’ Speed up the intrinsic GTPase activity of G protein ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE In continued presence of epinephrine: Β-adrenergic receptor kinase (βARK) is drawn to receptor and phosphorylates it, creating a binding site for β-arrestin (β-arr) It also initiates receptor sequestration: removal of receptors from membrane into intracellular vesicles by endocytosis βARK Binding of (β-arr) stops interaction between receptor and G protein Re-sensitization Receptors in endocytic vesicles dephosphorylates, β-arr dissociates, then receptors can return to membrane and resensitize the system to epinephrine β-arr ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE SIGNAL TERMINATION Desensitization reduces the cellular response even while the signal continues ROSS UNIVERSITY SCHOOL OF VETERINARY MEDICINE Β-agonists https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5480238/