title: Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha.
abstract: We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, University Road, Leicester LE1 9HN, United Kingdom. TBA@le.ac.uk
authors.forename: A B
authors.initials: AB
authors.lastname: Tobin
authors.forename: N F
authors.initials: NF
authors.lastname: Totty
authors.forename: A E
authors.initials: AE
authors.lastname: Sterlin
authors.forename: S R
authors.initials: SR
authors.lastname: Nahorski
mesh_terms: D000818:Animals; D016466:CHO Cells; D047388:Casein Kinases; D002417:Cattle; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D019204:GTP-Binding Proteins / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000502:physiology*; D020794:Receptor Protein-Tyrosine Kinases / Q000302:isolation & purification; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D018411:Spodoptera; D013552:Swine; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D012243:Rhodopsin; D011494:Protein Kinases; D020794:Receptor Protein-Tyrosine Kinases; D047388:Casein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; C080577:muscarinic receptor kinase; D054769:G-Protein-Coupled Receptor Kinase 2; D019204:GTP-Binding Proteins