title: The variable C-terminal extension of G-protein-coupled receptor kinase 6 constitutes an accessorial autoregulatory domain.
abstract: G-protein-coupled receptor kinases (GRK) are known to phosphorylate agonist-occupied G-protein-coupled receptors. We expressed and functionally characterized mouse GRK6 proteins encoded by four distinct mRNAs generated by alternative RNA splicing from a single gene, mGRK6-A to mGRK6-D. Three isoforms, mGRK6-A to mGRK6-C differ in their C-terminal-most portion, which is known to mediate membrane and/or receptor interaction and regulate the activity of GRK4-like kinases. One isoform, mGRK6-D, is identical to the other mGRK6 variants in the N-terminal region, but carries an incomplete catalytical domain. Mouse GRK6-D was catalytically inactive and specifically present in the nucleus of transfected cells. Recombinant mouse GRK6-A to mGRK6-C were found to be membrane-associated in cell-free systems and in transfected COS-7 cells, suggesting that the very C-terminus of GRK6-A, lacking in GRK6-B and mGRK6-C and carrying consensus sites for palmitoylation, is not required for membrane interaction. Interestingly, the shortest catalytically active variant, mGRK6-C, was conspicuously more active in phosphorylating light-activated rhodopsin than mGRK6-A and mGRK6-B, implying that the C-terminus of the latter two variants may fulfil an autoinhibitory function. Mutation and removal of C-terminal-most region of mGRK6-A by site-directed mutagenesis revealed that this region contains three autoregulatory elements: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element. The results suggest that mGRK6-C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension.
authors.affiliation: Department of Pharmacology and Toxicology, University of Ulm, Germany.
authors.forename: Petra
authors.initials: P
authors.lastname: Vatter
authors.forename: Claudia
authors.initials: C
authors.lastname: Stoesser
authors.forename: Ines
authors.initials: I
authors.lastname: Samel
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
authors.forename: Barbara
authors.initials: B
authors.lastname: Moepps
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D054768:G-Protein-Coupled Receptor Kinases; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
chemical_list: D010743:Phospholipids; D020033:Protein Isoforms; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6