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 -adrenergic receptor/adenylate cyclase pathway, in which receptor-specific phosphorylation is known to be mediated by -adrenergic receptor kinase (-ARK), the kinases responsible for phosphorylation of phospholipase C-linked receptors have yet to be identified, although a role for -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 Ser 345 -Leu 463 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) heparinSepharose. The purified protein kinase represented an ϳ18,600-fold purification and was a single polypeptide with a molecular weight of ϳ40 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, -ARK, and other members of the G-protein-linked receptor kinase family. It therefore represents a new class of receptor kinase.Many cell surface neurotransmitter and hormone receptors respond to agonist occupation by activation of phospholipase C. The subsequent hydrolysis of the lipid substrate phosphatidylinositol 4,5-bisphosphate releases the second messengers inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 1 and diacylglycerol (reviewed in Ref. 1). The second messenger response to a number of phospholipase C-linked receptors, including the m3-muscarinic receptor, is often complex, consisting of a burst of Ins(1,4,5)P 3 production reaching a peak within the first few seconds of receptor stimulation followed by a lower sustained phase of Ins(1,4,5)P 3 generation that is maintained for tens of minutes to hours (2-4). Similar patterns of Ins(1,4,5)P 3 generation are seen in response to agonist occupation of receptors for GRH (5), angiotensin (6), bombesin, and CCK (7).Little is known of the molecular mechanisms underlying these complex second messenger responses, although recent evidence suggests that some phospholipase C-linked receptors undergo a rapid partial desensitization that results in decreased phospholipase C activity within seconds of agonist occupation (3,4,7,8). Consistent with this notion are studies from our laboratory demonstrating that the early peak phase of Ins(1,4,5)P 3 production in response to m3-muscarinic receptor stimulation can be desensitized by a short pre-expos...
