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Identification of a Novel Receptor Kinase That Phosphorylates a Phospholipase C-linked Muscarinic Receptor

1996; Elsevier BV; Volume: 271; Issue: 7 Linguagem: Inglês

10.1074/jbc.271.7.3907

1083-351X

Andrew B. Tobin, Barbara Keys, Stefan R. Nahorski,

Ion channel regulation and function

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 β-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by β-adrenergic receptor kinase (β-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for β-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 ∽18,600-fold purification and was a single polypeptide with a molecular weight of ∽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, β-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase. 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 β-adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by β-adrenergic receptor kinase (β-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for β-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 ∽18,600-fold purification and was a single polypeptide with a molecular weight of ∽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, β-ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.