Identification of a novel receptor kinase that phosphorylates a phospholipase C-linked muscarinic receptor.
Phosphorylation of G-protein-linked receptors is thought to play a central role in receptor regulation and desensitization. Unlike the case of the extensively studied beta-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by beta-adrenergic receptor kinase ( beta-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for beta-ARK has been implicated. This study describes the purification of a novel 40-kDa receptor kinase from porcine cerebellum that is able to phosphorylate the phospholipase C-linked m3-muscarinic receptor in an agonist-dependent manner. The assay for kinase activity was based on the ability of the kinase to phosphorylate a bacterial fusion protein, Ex-m3, containing amino acids Ser345-Leu463 of the third intracellular loop of the m3-muscarinic receptor. Purification of the muscarinic receptor kinase from a high speed supernatant fraction of porcine cerebellum was achieved using the following steps: (i) 30-60% ammonium sulfate cut and successive chromatography on (ii) butyl-Sepharose (iii) Resource Q, (iv) Resource S, and (v) heparin-Sepharose. The purified protein kinase represented an approximately 18,600-fold purification and was a single polypeptide with a molecular weight of approximately 40 kDa. Based on the chromatographic mobility, molecular weight, and kinase inhibitor studies, the kinase, designated MRK, was shown to be distinct from previously characterized second messenger regulated protein kinases, beta-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.
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Leicester University, Department of Cell Physiology and Pharmacology, P.O. Box 138, Medical Sciences Building, University Road, Leicester, LE1 9HN, United Kingdom.
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Nahorski
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D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001483:Base Sequence; D016466:CHO Cells; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000201:enzymology; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers; D054769:G-Protein-Coupled Receptor Kinase 2; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D017931:DNA Primers; D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D001285:Atropine; D002217:Carbachol; D020794:Receptor Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; C080577:muscarinic receptor kinase; D054769:G-Protein-Coupled Receptor Kinase 2; D010738:Type C Phospholipases