S85: G Protein Coupled Receptors!Main Ideas!- GPCRs are a huge fam of 7-tm receptors!- GPCRs bind ligands: from large proteins to light waves!- Binding of ligand to receptor causes a conformational change that activates a trimeric G protein with a, B, y subunits!- Activation inv. exchange of GDP for GTP, activation separates the a subunit from the By subunits!- Activated G protein subunits then activate an effector molecule or ion channel!- GPCRs rapidly inactivated by GRKs!- Some G proteins reg. adenylyl cyclase which makes cAMP, allosteric mod. of PKA= protein kinase A!- Gsa activates adenylyl cyclase!- Gis inhibits adenyl cyclase!- Activated PKA phosphorys target protteins incl. CREB (txn activator)!- Cholera toxin and pertussis toxin cause disease by disrupting signaling thru adenylyl cylase pathway!- Some GPCRs activate Gq or Go, which activates phospholipase C-B (PLC-B)!- PLC-B cleaves PIP2 into DAG + IP3!- IP3 binds to IP3-gated Ca2+ channels (IP3 receptors) in the ER outer membrane, releases Ca2+ into cytoplasm!- DAG modulattes PKC and is a precursor for arachidonic acid, a signaling molecule, can be converted into eicosanoids!- Eiconsanoids= lipid signalling molecules (incl. prostaglandins= PGs) involv. in pain and inflamma responses!- Ca2+ allosterically regs many proteins, imp.= protein kinase C (PKC) and calmodulin (CaM)!!Learning Objectives 1) Discuss G protein coupled receptors (GPCR) and G proteins, incl. their activation and inactivation.!- GPCR Membrane Complex !- Effectors: Adenylyl cyclase, phospholipases CB and Cy, K+ and Ca2+ channels, cGMP phosphodiesterase!- Examples of signals that use GPCRs: acetylcholine, cannabinoids, catecholamines, CCK, glucagon, histamine, prostaglandins, ghrelin, aMSH, GLP-1!- Peptides, proteins, nucleosides, organic compounds, fatty acids, aa derivs, light, sound, P!- All GPCRs have a sim. conformation w/7 tm domains !- Trimeric G proteins are anchored to the cytoplasmic face of pm by lipid anchors= ISOPRENOID units!- these units are in the pm, a and y subunits are linked to them!!!!!!!!!!!!!!!!!!!!!- Activation of G protein by a ligand bound GPCR (see above)!- Activation of G-protein req. exchange of GTP for the bound GDP!- Binding of GTP causes a-subunit to sep. from B-y complex!- Inactivation of G protein req. hydrolysis of GTP!- G protein is inactivated when the a-subunit hydrolyzes the bound GTP --> GDP (intrinsic GTPase activity, a-subunit= a GTPase)!- Target protein no longer active when G protein subunit isn't bound to it!- Summary of the Interaction of a GPCR w/a trimeric G protein!- GDP is released from a-sub of G protein, allows GTP to bind instead!- G protein dissoc. from receptor!- G protein dissoc. into 2 complexes: a-sub-GTP complex and By complex!- Both stay assoc. w/cytoplasmic face of pm and can move laterally to find their effector= their intracellular signaling partner!- Effector is activated and sends signal on!- Each G protein can activate many molecules of effector before GTP hydrolyzed (and turns it off)!- Signal out in response to activation of GPCRs usually involves intracellular mediators= 2nd messengers!- 2nd messengers= link between the membrane and rest of the cell!- Must be syn. or gen. by precisely reg. rxns!- Must have short half life, high specificity for targets!- Most targets are enzymes, binding of 2nd messenger --> enzymatic activation (usually)!- 2 messengers include: Ca2+, cAMP, cGMP, inositol triphosphate (IP3), and DAG (diacylglycerol)!- Signal Off req. many deactivation mechs - desensitization of GPCR!- dissoc. of ligand from GPCR!- hydrolysis of GTP--> GDP by a-sub.!- inactivation of effector!- degrading/sequester 2nd messenger!- inactivation of any activated enzymes or activation of inactivated !!2) Compare and contrast the adenylyl cylase and PIP cascades, incl. involved 2nd messengers - cAMP: 2nd Messenger!- syn. by adenylyl cyclases, hydrolyzed by phosphodiesterases!- Adenylyl Cyclase Cascade: Gs (G stimulatory) G protein activates Adenylyl Cyclase - Adenylyl cyclases= intrinsic membrane proteins!- activated by Gsa --> adenylyl cyclase syn. cAMP from ATP!- cAMP= allosteric modulator of protein kinase A (PKA, cAMP-dep. protein kinase)!- Activation of protein kinase A by cAMP!- cAMP binds to 2 reg. subunits and active subunits are then free!- activated catalytic subunits phosphorylate many target protein in cytoplasm and nucleus - PKA targets incl.: glycogen synthase (glycogen syn.), CREB (gene reg), hormone sensitive lipase (TG mobilization), pyruvate kinase (glycolysis), phosphorylase B kinase (glycogen breakdown)!- Amplification in adenylyl cyclase cascade: Glycogenolysis!- 1 activated Gsa can activate 100 adenylyl cyclase enzymes --> cAMP --> active PKA --> active phosphorylase kinase --> active glycogen phosphorylase --> glucose-1-phosphate!- Adenylyl Cyclase cascade can trigger changes in txn and in protein activity ! 3) Describe how cholera toxin and pertusis toxin impact GPCR signaling - Cholera toxin and Pertussis Toxin interfere w/the G protein signaling - ADP- ribosylation of Gsa by cholera toxin --> activates Gsa --> increases cAMP levels!- ADP-ribosylation of Gia by pertussis toxin --> inactites Gia --> increases cAMP levels!- BOTH increas cAMP thru diff. mechs!1) More of 1 - By subunits/complexes of trimeric G proteins can also function as intracellular signal transducers by opening ion channels - acetlycholine binds --> Gia loses GDP and gains GTP --> complex dissociates --> Gby subunit binds ion channel --> opens it!!- Phosphoionsitide Cascade (PI)!- analagous to adenylyl cyclase cascade !- some activators of the PI cascade: acetylcholine, epinephrine/norepinephrine, ghrelin - Major Steps in Signaling thru PI Cascade!- Ligand bind to GPCR --> conformation changes in the GPCR activate Gqa or Goa --> activated Gqa activates phospholipase C-B (PLC-B) --> hydrolyzes PIP2 (PI4,5-bisphosphate) to --> DAG (diacylglycerol) + IP3 (inositol 1,4,5-triphosphate)!- PIP2 ---(PLC-B)--> DAG + IP3!- aka PLC-B cleaves PIP2 into DAG and IP3!- phospholipases hydrolyze membrane lipids at diff. positions !- DAG helps activate protein kinase C (PKC)!- IP3 binds to IP3-gated Ca2+ channels (IP3 receptors) in ER outer membrane --> releases Ca2+ into the cytoplasm!- Ca2+ helps to activate PKC, too!- PKC phosphorylates target proteins, many involved in cell proliferation!- Synthesis of PIP2!- (phosphatidylinositol) PI + PI(4)P --> PIP2!- PI= uncommon membrane
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