Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation. Export

@article{citeulike:3870885, abstract = {G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.}, author = {Noble, B. and Kallal, L. A. and Pausch, M. H. and Benovic, J. L.}, citeulike-article-id = {3870885}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/jbc.M308257200}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/14507916}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=14507916}, day = {28}, doi = {10.1074/jbc.M308257200}, issn = {0021-9258}, journal = {The Journal of biological chemistry}, keywords = {gpcr, grk, yeast}, month = {November}, number = {48}, pages = {47466--47476}, posted-at = {2009-01-09 10:03:30}, priority = {2}, title = {Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation.}, url = {http://dx.doi.org/10.1074/jbc.M308257200}, volume = {278}, year = {2003} }

TY - JOUR ID - citeulike:3870885 L3 - citeulike-article-id:3870885 N2 - G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation. IS - 48 JF - The Journal of biological chemistry SN - 0021-9258 EP - 47476 TI - Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation. VL - 278 SP - 47466 KW - gpcr KW - grk KW - yeast AU - Noble, B AU - Kallal, LA AU - Pausch, MH AU - Benovic, JL PY - 2003/11/28/ UR - http://dx.doi.org/10.1074/jbc.M308257200 ER -