title: Roles of phosphorylation-dependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons.
abstract: When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH(PLC delta), G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G alpha(q/11). To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G alpha(q/11)-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G alpha(q/11)-binding (D110A) GRK2 mutant or the catalytically inactive (D110A,K220R)GRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of (D110A)GRK2 enhanced, whereas (D110A,K220R)GRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G alpha(q/11) binding did not affect the ability of the (D110A)GRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of (D110A,K220R)GRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, LE1 9HN, United Kingdom. jmw23@le.ac.uk
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D043643:Receptor, Muscarinic M1 / Q000737:chemistry*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D016376:Oligonucleotides, Antisense; D043643:Receptor, Muscarinic M1; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; C496569:Grk2 protein, rat; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D002118:Calcium