Role of Acidic Amino Acids in Peptide Substrates of the β-Adrenergic Receptor Kinase and Rhodopsin Kinase

The β-adrenergic receptor kinase (β-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by β-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet α₂-adrenergic receptor is shown to serve as a substrate for β-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this α₂-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of β-ARK. By kinetic analyses of the phosphorylation reactions, β-ARK exhibited a marked preference for negatively charged amino acids localized to the NH₂-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike β-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to β-ARK and RK was a much greater K_m for peptide substrates as compared to that for intact receptor substrates.