Amino Acids

, Volume 48, Issue 12, pp 2875–2880 | Cite as

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2)

  • Daisuke AsaiEmail author
  • Masaharu Murata
  • Riki Toita
  • Takahito Kawano
  • Hideki Nakashima
  • Jeong-Hun KangEmail author
Short Communication


A series of amino acid substitutions was made in a previously identified β-tubulin-derived GRK2 substrate peptide (404DEMEFTEAESNMN416) to examine the role of amino acid residues surrounding the phosphorylation site. Anionic amino acid residues surrounding the phosphorylation site played an important role in the affinity for GRK2. Compared to the original peptide, a modified peptide (Ac-EEMEFSEAEANMN-NH2) exhibited markedly higher affinity for GRK2, but very low affinity for GRK5, suggesting that it can be a sensitive and selective peptide for GRK2.


G protein-coupled receptor kinase Amino acid residue Phosphorylation Cellular signal transduction pathway Consensus sequence 



The authors thank Ms. Sigemi Terakubo and Ms. Niño Okamura (St. Marianna University School of Medicine) for technical assistance. This work was supported by a Grant-in-Aid for Challenging Exploratory Research (KAKENHI Grant Number 15K12531) and for Scientific Research (C) (15K01319) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


  1. Asai D, Toita R, Murata M, Katayama Y, Nakashima H, Kang JH (2014) Peptide substrates for G protein-coupled receptor kinase 2. FEBS Lett 588:2129–2132CrossRefPubMedGoogle Scholar
  2. Brinks H, Koch WJ (2010) βARKct: a therapeutic approach for improved adrenergic signaling and function in heart disease. J Cardiovasc Trans Res 3:499–506CrossRefGoogle Scholar
  3. Cannavo A, Liccardo D, Koch WJ (2013) Targeting cardiac β-adrenergic signaling via GRK2 inhibition for heart failure therapy. Front Physiol 4:264CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cant SH, Pitcher JA (2005) G protein-coupled receptor kinase 2-mediated phosphorylation of ezrin is required for G protein-coupled receptor-dependent reorganization of the actin cytoskeleton. Mol Biol Cell 16:3088–3099CrossRefPubMedPubMedCentralGoogle Scholar
  5. Dinudom A, Fotia AB, Lefkowitz RJ, Young JA, Kumar S, Cook DI (2004) The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels. Proc Natl Acad Sci USA 101:11886–11890CrossRefPubMedPubMedCentralGoogle Scholar
  6. Fredericks ZL, Pitcher JA, Lefkowitz RJ (1996) Identification of the G protein-coupled receptor kinase phosphorylation sites in the human β2-adrenergic receptor. J Biol Chem 271:13796–13803CrossRefPubMedGoogle Scholar
  7. Freeman JL, Gonzalo P, Pitcher JA, Claing A, Lavergne JP, Reboud JP, Lefkowitz RJ (2002) β2-Adrenergic receptor stimulated, G protein-coupled receptor kinase 2 mediated, phosphorylation of ribosomal protein P2. Biochemistry 41:12850–12857CrossRefPubMedGoogle Scholar
  8. Gainetdinov RR, Premont RT, Bohn LM, Lefkowitz RJ, Caron MG (2004) Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev Neurosci 27:107–144CrossRefPubMedGoogle Scholar
  9. Gurevich EV, Tesmer JJ, Mushegian A, Gurevich VV (2012) G protein-coupled receptor kinases: more than just kinases and not only for GPCRs. Pharmacol Ther 133:40–69CrossRefPubMedGoogle Scholar
  10. Haga K, Ogawa H, Murofushi H (1998) GTP-binding-protein-coupled receptor kinase 2 (GRK2) binds and phosphorylates tubulin. Eur J Biochem 255:363–368CrossRefPubMedGoogle Scholar
  11. Hildreth KL, Wu JH, Barak LS, Exum ST, Kim LK, Peppel K, Freedman NJ (2004) Phosphorylation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-2 reduces receptor signaling and interaction with the Na+/H+ exchanger regulatory factor. J Biol Chem 279:41775–41782CrossRefPubMedGoogle Scholar
  12. Ho J, Cocolakis E, Duman VM, Posner BI, Laporte SA, Lebrun JJ (2005) The G protein-coupled receptor kinase-2 is a TGFβ-inducible antagonist of TGFβ signal transduction. EMBO J 24:3247–3258CrossRefPubMedPubMedCentralGoogle Scholar
  13. Kahsai AW, Zhu S, Fenteany G (2010) G protein-coupled receptor kinase 2 activates radixin, regulating membrane protrusion and motility in epithelial cells. Biochem Biophys Acta 1803:300–310CrossRefPubMedGoogle Scholar
  14. Kang JH, Jiang Y, Toita R, Oishi J, Kawamura K, Han A, Mori T, Niidome T, Ishida M, Tatematsu K, Tanizawa K, Katayama Y (2007) Phosphorylation of Rho-associated kinase (Rho-kinase/ROCK/ROK) substrates by protein kinases A and C. Biochimie 89:39–47CrossRefPubMedGoogle Scholar
  15. Kang JH, Asai D, Yamada S, Riki T, Oishi J, Mori T, Niidome T, Katayama Y (2008) A short peptide is a protein kinase C (PKC)α-specific substrate. Proteomics 10:2006–2011CrossRefGoogle Scholar
  16. Kang JH, Asai D, Tsuchiya A, Mori T, Niidome T, Katayama Y (2011) Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2). PLoS One 6:e22699CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kang JH, Toita R, Kim CW, Katayama Y (2012) Protein kinase C (PKC) isozyme-specific substrates and their design. Biotechnol Adv 30:1662–1672CrossRefPubMedGoogle Scholar
  18. Kang JH, Asai D, Toita R, Kawano T, Murata M (2015) Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption–ionization time-of-flight mass spectrometry. Amino Acids 47:2377–2383CrossRefPubMedGoogle Scholar
  19. Ohguro H, Johnson RS, Ericsson LH, Walsh KA, Palczewski K (1994) Control of rhodopsin multiple phosphorylation. Biochemistry 33:1023–1028CrossRefPubMedGoogle Scholar
  20. Onorato JJ, Palczewski K, Regan JW, Caron MG, Lefkowitz RJ, Benovic JL (1991) Role of acidic amino acids in peptide substrates of the beta-adrenergic receptor kinase and rhodopsin kinase. Biochemistry 30:5118–5125CrossRefPubMedGoogle Scholar
  21. Penela P, Murga C, Ribas C, Lafarga V, Mayor F Jr (2010) The complex G protein-coupled receptor kinase 2 (GRK2) interractome unveils new physiopathological targets. Br J Pharmacol 160:821–832CrossRefPubMedPubMedCentralGoogle Scholar
  22. Peregrin S, Jurado-Pueyo M, Campos PM, Sanz-Moreno V, Ruiz-Gómez A, Crespo P, Mayor F Jr, Murga C (2006) Phosphorylation of p38 by GRK2 at the docking groove unveils a novel mechanism for inactivating p38MAPK. Curr Biol 16:2042–2047CrossRefPubMedGoogle Scholar
  23. Pronin AN, Morris AJ, Surguchov A, Benovie JL (2000) Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J Biol Chem 275:26515–26522CrossRefPubMedGoogle Scholar
  24. Reiter E, Lefkowitz RJ (2006) GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol Metab 17:159–165CrossRefPubMedGoogle Scholar
  25. Ruiz-Gómez A, Humrich J, Murga C, Quitterer U, Lohse MJ, Mayor F Jr (2000) Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2. J Biol Chem 275:29724–29730CrossRefPubMedGoogle Scholar
  26. Ruiz-Gómez A, Mellström B, Tornero D, Morato E, Savignac M, Holguín H, Aurrekoetxea K, González P, González-García C, Ceña V, Mayor F Jr, Naranjo JR (2007) G protein-coupled receptor kinase 2-mediated phosphorylation of downstream regulatory element antagonist modulator regulates membrane trafficking of Kv4.2 potassium channel. J Biol Chem 282:1205–1215CrossRefPubMedGoogle Scholar
  27. Sanchez-Perez A, Kumar S, Cook DI (2007) GRK2 interacts with and phosphorylates Nedd4 and Nedd4-2. Biochem Biophys Res Commun 359:611–615CrossRefPubMedGoogle Scholar
  28. Wan KF, Sambi BS, Frame M, Tate R, Pyne NJ (2001) The inhibitory γ subunit of the type 6 retinal cyclic guanosine monophosphate phosphodiesterase is a novel intermediate regulating p42/p44 mitogen-activated protein kinase signaling in human embryonic kidney 293 cells. J Biol Chem 276:37802–37808PubMedGoogle Scholar
  29. Woodall MC, Ciccarelli M, Woodall BP, Koch WJ (2014) G protein-coupled receptor kinase 2: a link between myocardial contractile function and cardiac metabolism. Cir Res 114:1661–1670CrossRefGoogle Scholar
  30. Yoshida N, Haga K, Haga T (2003) Identification of sites of phosphorylation by G-protein-coupled receptor kinase 2 in β-tubulin. Eur J Biochem 270:1154–1163CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  1. 1.Department of MicrobiologySt. Marianna University School of MedicineKawasakiJapan
  2. 2.Department of Advanced Medical Initiatives, Faculty of Medical SciencesKyushu UniversityFukuokaJapan
  3. 3.Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)IkedaJapan
  4. 4.Division of Biopharmaceutics and PharmacokineticsNational Cerebral and Cardiovascular Center Research InstituteSuitaJapan

Personalised recommendations