Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1 alpha

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist sensitive manner by a protein kinase of similar to 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 re ceptor kinase family, In the present study we propose that MRK is casein kinase 1 alpha (CK1 alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1 alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1 alpha 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 CK1 alpha 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 CK1 alpha 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 CK1 alpha, and that CK1 alpha 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.