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The variable C‐terminal extension of G‐protein‐coupled receptor kinase 6 constitutes an accessorial autoregulatory domain
Author(s) -
Vatter Petra,
Stoesser Claudia,
Samel Ines,
Gierschik Peter,
Moepps Barbara
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04995.x
Subject(s) - palmitoylation , gene isoform , biology , microbiology and biotechnology , c terminus , g protein coupled receptor kinase , kinase , transfection , g protein coupled receptor , alternative splicing , receptor , phosphorylation , biochemistry , chemistry , gene , signal transduction , amino acid , enzyme , cysteine
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.