title: Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation.
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.
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D019556:COS Cells; D002384:Catalysis; D002455:Cell Division; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D054774:G-Protein-Coupled Receptor Kinase 5; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D016296:Mutagenesis; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013997:Time Factors; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D008055:Lipids; D010455:Peptides; D010743:Phospholipids; D018343:Receptors, Adrenergic, beta-2; D017346:Protein Serine-Threonine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054774:G-Protein-Coupled Receptor Kinase 5; C517898:GRK5 protein, human