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Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Publication ,  Journal Article
Oppermann, M; Diversé-Pierluissi, M; Drazner, MH; Dyer, SL; Freedman, NJ; Peppel, KC; Lefkowitz, RJ
Published in: Proc Natl Acad Sci U S A
July 23, 1996

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

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Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

July 23, 1996

Volume

93

Issue

15

Start / End Page

7649 / 7654

Location

United States

Related Subject Headings

  • beta-Adrenergic Receptor Kinases
  • Transfection
  • Receptors, G-Protein-Coupled
  • Receptors, Cell Surface
  • Receptor Protein-Tyrosine Kinases
  • Rabbits
  • Protein Serine-Threonine Kinases
  • Phosphorylation
  • Neurons, Afferent
  • Myocardium
 

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Oppermann, M., Diversé-Pierluissi, M., Drazner, M. H., Dyer, S. L., Freedman, N. J., Peppel, K. C., & Lefkowitz, R. J. (1996). Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases. Proc Natl Acad Sci U S A, 93(15), 7649–7654. https://doi.org/10.1073/pnas.93.15.7649
Oppermann, M., M. Diversé-Pierluissi, M. H. Drazner, S. L. Dyer, N. J. Freedman, K. C. Peppel, and R. J. Lefkowitz. “Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.Proc Natl Acad Sci U S A 93, no. 15 (July 23, 1996): 7649–54. https://doi.org/10.1073/pnas.93.15.7649.
Oppermann M, Diversé-Pierluissi M, Drazner MH, Dyer SL, Freedman NJ, Peppel KC, et al. Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7649–54.
Oppermann, M., et al. “Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.Proc Natl Acad Sci U S A, vol. 93, no. 15, July 1996, pp. 7649–54. Pubmed, doi:10.1073/pnas.93.15.7649.
Oppermann M, Diversé-Pierluissi M, Drazner MH, Dyer SL, Freedman NJ, Peppel KC, Lefkowitz RJ. Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7649–7654.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

July 23, 1996

Volume

93

Issue

15

Start / End Page

7649 / 7654

Location

United States

Related Subject Headings

  • beta-Adrenergic Receptor Kinases
  • Transfection
  • Receptors, G-Protein-Coupled
  • Receptors, Cell Surface
  • Receptor Protein-Tyrosine Kinases
  • Rabbits
  • Protein Serine-Threonine Kinases
  • Phosphorylation
  • Neurons, Afferent
  • Myocardium