title: Endogenous G protein-coupled receptor kinase 6 Regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells.
abstract: We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M(3) muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M(3) mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GRK6). (K215R)GRK6 expression inhibited methacholine-stimulated M(3) mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[(35)S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M(3) mACh receptor-G alpha(q/11) uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M(3) mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [(3)H]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF(4)(-). The ability of (K215R)GRK6 to inhibit agonist-mediated M(3) mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M(3) mACh receptor desensitization in the SH-SY5Y cell line.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester, LE1 9HN, United Kingdom. jmw23@le.ac.uk
authors.forename: Jonathon M
authors.initials: JM
authors.lastname: Willets
authors.forename: R A John
authors.initials: RA
authors.lastname: Challiss
authors.forename: Stefan R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D019943:Amino Acid Substitution; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D016210:Methacholine Chloride / Q000494:pharmacology; D009447:Neuroblastoma; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D007295:Inositol Phosphates; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; D016210:Methacholine Chloride; D017346:Protein Serine-Threonine Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; D019204:GTP-Binding Proteins