title: Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase.
abstract: The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-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 alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-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 beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-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 beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.
authors.affiliation: Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: J J
authors.initials: JJ
authors.lastname: Onorato
authors.forename: K
authors.initials: K
authors.lastname: Palczewski
authors.forename: J W
authors.initials: JW
authors.lastname: Regan
authors.forename: M G
authors.initials: MG
authors.lastname: Caron
authors.forename: R J
authors.initials: RJ
authors.lastname: Lefkowitz
authors.forename: J L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology; D017868:Cyclic AMP-Dependent Protein Kinases; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D005971:Glutamates; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D012243:Rhodopsin / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D051552:beta-Adrenergic Receptor Kinases
chemical_list: D005136:Eye Proteins; D005971:Glutamates; D010455:Peptides; D011943:Receptors, Adrenergic, beta; D001224:Aspartic Acid; D012243:Rhodopsin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D051552:beta-Adrenergic Receptor Kinases