title: Peptide substrates for G protein-coupled receptor kinase 2.
abstract: G protein-coupled receptor kinases (GRKs) control the signaling and activation of G protein-coupled receptors through phosphorylation. In this study, consensus substrate motifs for GRK2 were identified from the sequences of GRK2 protein substrates, and 17 candidate peptides were synthesized to identify peptide substrates with high affinity for GRK2. GRK2 appears to require an acidic amino acid at the -2, -3, or -4 positions and its consensus phosphorylation site motifs were identified as (D/E)X1-3(S/T), (D/E)X1-3(S/T)(D/E), or (D/E)X0-2(D/E)(S/T). Among the 17 peptide substrates examined, a 13-amino-acid peptide fragment of β-tubulin (DEMEFTEAESNMN) showed the highest affinity for GRK2 (Km, 33.9 μM; Vmax, 0.35 pmol min(-1) mg(-1)), but very low affinity for GRK5. This peptide may be a useful tool for investigating cellular signaling pathways regulated by GRK2.
authors.affiliation: Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki 216-8511, Japan. Electronic address: asai@marianna-u.ac.jp.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Asai
authors.affiliation: Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan.
authors.forename: Riki
authors.initials: R
authors.lastname: Toita
authors.affiliation: Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Murata
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Moto-oka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Katayama
authors.affiliation: Department of Microbiology, St. Marianna University School of Medicine, Sugao 2-16-1 Miyamae, Kawasaki 216-8511, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Nakashima
authors.affiliation: Division of Biopharmaceutics and Pharmacokinetics, Department of Biomedical Engineering, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Electronic address: jrjhkang@ncvc.go.jp.
authors.forename: Jeong-Hun
authors.initials: JH
authors.lastname: Kang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D054774:G-Protein-Coupled Receptor Kinase 5 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D014404:Tubulin; D054769:G-Protein-Coupled Receptor Kinase 2; D054774:G-Protein-Coupled Receptor Kinase 5
keywords: Cellular signal transduction pathway; Consensus sequence; G protein-coupled receptor kinase; Peptide substrate; Phosphorylation