Cell Signaling 3 2020 PDF

Cell Signaling (3): the G-protein coupled receptor (Cont.) Stuart Cruickshank  Signalling Pathways Some G Proteins activate the inositol phospholipid signaling pathway by activating phospholipase C  G protei...

Cell Signaling (3): the G-protein coupled receptor (Cont.) Stuart Cruickshank  Signalling Pathways Some G Proteins activate the inositol phospholipid signaling pathway by activating phospholipase C  G proteins and Phospholipase C phospholipase C (PLC) linked to receptor by Gq protein (stimulatory) from the membrane phospholipid PLC produces the second messengers: – inositol trisphosphate (IP3) – diacylglycerol (DAG) IP3 moves into cytosol, DAG stays in membrane – this leads to a possible 2-limbed signal transduction system  Phosphatidylinositol (PI) Second Messenger System Drug PIP2 IP3 + DAG Receptor G protein Ca2+ Endoplasmic reticulum CELL’S RESPONSE CELL’S RESPONSE  The sequence of events by which Receptor binding activates DAG / IP3 signalling: 1. Initially Gα has bound GDP, and α, β, & γ subunits are complexed together. 2. Activation of a 7-helix receptor (GPCR ) causes a conformational change that is transmitted to the G protein. The nucleotide-binding site on Ga becomes more accessible to the cytosol, where [GTP] > [GDP]. Ga releases GDP & binds GTP (GDP-GTP exchange). 3. Substitution of GTP for GDP causes another conformational change in Ga. Ga-GTP dissociates from the inhibitory bg complex & can now bind to and activate PLC. 4. Membrane bound PIP2, hydrolysed by PLC, DAG and IP3  S1P outside GPCR plasma membrane  cytosol PIP2 GDP GTP GTP GDP PLC DAG + IP3 5. Protein Kinase C catalyzes phosphorylation of various cellular proteins, altering their activity. Receptor mediated activation of PLC PLC hydrolysis of PIP2 Formation of DAG and IP3 IP3 releases Ca2+ from SR IP3 receptors DAG activates PKC PKC catalyses phosphorylation of intracellular proteins  Acetylcholine signalling via PLC / IP3 / Ca2+ in acetylcholine 1 pancreas cells Gq PLC DAG C a 2+ IP 3 -receptor IP 3 2 6 3 5 Ca 2+ 4 C a 2+ ER  IP3 and Ca2+ IP3 acts by increasing free [Ca2+] in cells [Ca2+] inside cell about 0.1 µM [Ca2+] outside cell about 2 mM Ca2+ stored in endoplasmic reticulum tightly bound to certain proteins (calsequestrin) IP3 releases Ca2+ from ER by interacting with specific IP3- receptors – increased [Ca2+] in cytosol – can lead to opening of Ca2+-channels in plasma membrane  Role of Ca2+ in cellular mechanisms Ca2+ signalling as impt as phosphorylation Ca2+ binding to proteins is non-covalent (cf. phosphorylation) Ca2+ binding to protein causes structural changes – DIRECTLY can activate enzymes, ion channels, cytoskeletal proteins – INDIRECTLY via calcium-binding proteins (e.g. calmodulin, CaM)) which activate protein kinases (CaM-protein kinases)  Termination of Ca2+ response: 1. Dephosphorylation by IP3ase : IP2 2. Phosphorylation by Kinase: IP4 3. Ca2+ export to the exterior of cells 4. Ca2+ sequestration to the ER  DAG / Ca2+ activation of protein kinase C protein kinase C (PKC) - “C” for Ca2+ activated by both DAG and Ca2+ PKC inactive without DAG or Ca2+ and is soluble in cytoplasm binding of DAG and Ca2+ activates PKC and moves it to membrane (translocation) – PKC phosphorylates proteins on the serine and threonine residues – PKC impt in cellular growth and division  SHELLFISH POISONING & CELL SIGNALLING some shellfish contain dinoflagellates dinoflagellates contain okadaic acid – a serine / threonine phosphatase inhibitor this keeps the PKC phosphorylated proteins longer than needed – Diarrhoea from Na+ -channel in intestinal cells disturbed influencing fluid balance. severe poisoning leads to non-regulated cell growth (tumours)  TYROSINE KINASE RECEPTOR S these receptors traverse the membrane only once receptor has intrinsic enzyme activty – i.e. the receptor itself is an enzyme respond exclusively to protein stimuli – cytokines – mitogenic growth factors: platelet derived growth factor epidermal growth factor  TYROSINE KINASE: (phosphorylation & dephosphorylation) kinase enzymes add a phosphate (Pi) group tyrosine kinase specifically to tyrosine residue phosphatases remove Pi phosphorylation state alters shape (conformation) of protein and changes its function – enzyme activity; solute transport; gene expression covalent modification by phosphorylation is extremely important in regulation biological responses  ATP ADP protein K ina se P pho sph ata se change in s hape by addition of phos phate Pi ph os ph orylatio n dephosphorlyation  PLATELET DERIVED GROWTH FACTOR SIGNAL TRANSDUCTION 2 PDGF receptors come together (dimerize) and phosphorylate each other by tyrosine kinase activity phosphorylated receptors activate GTPase Ras ras leads to phosphorylation of the mitogen activated protein kinases (MAPKs) MAPKs phosphorylate 1000’s of proteins – growth & development - “cell proliferation” Mechanism of PDGF signal transduction PDGF dim erize & phosphorylate eac h P other ras MAPK MAPK P CELL phosphorlyates multiple G R O W TH target proteins  Regulation of receptor numbers adaptive mechanism responding to ligand exposure Desensitization (down regulation) 1) modify receptor by phosphorylation 2) receptor internalization 3) receptor degradation Note: 1 to 3 from normal to extreme  Receptor Desensitization (normal) within seconds to minutes of receptor occupation receptor phosphorylated by receptor kinases (RKs) – RKs act only on occupied receptors arrestins bind to phosphorylated receptors – e.g. arrestins disrupt interaction of receptor and G-protein  1st messenger receptor G proteins      amplifier  GDP GTP GTP GDP   P  GRK GDP GTP a rrestin  Receptor Endocytosis (moderate) prolonged exposure to high [agonist] leads to internalization/endocytosis receptors congregate and membrane invaginates and pinches off vesicles form that contain receptors in time, receptors transported back to membrane and are functional – resensitzation  Receptor Degradation (extreme) in very prolonged/high concentration exposure receptors internalized and vesicle form lysosomes receptors digested by proteases  Additional Information. Rang, Dale, Ritter and Moore. Chapter 3