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Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.

Publication ,  Journal Article
Ferguson, SS; Ménard, L; Barak, LS; Koch, WJ; Colapietro, AM; Caron, MG
Published in: J Biol Chem
October 20, 1995

The beta 2-adrenergic receptor (beta 2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta 2AR mutant (beta 2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta 2AR. However, this mutant receptor not only desensitized in response to agonist stimulation but down-regulated normally. In an attempt to understand the basis for the properties of this mutant, we have examined the ability of this regulation-defective mutant to undergo agonist-mediated phosphorylation. When expressed in 293 cells, the maximal response for phosphorylation of the beta 2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta 2AR-Y326A mutants, demonstrated that the beta 2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the beta-adrenergic receptor kinase 1 (beta ARK1). However, overexpression of beta ARK1 led to the agonist-dependent phosphorylation of the beta 2AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta 2AR-Y326A sequestration could be prevented by mutating putative beta ARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type beta 2AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C20 beta ARK1-K220M). These findings implicate a role for beta ARK1-mediated phosphorylation in facilitating wild-type beta 2AR sequestration.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

October 20, 1995

Volume

270

Issue

42

Start / End Page

24782 / 24789

Location

United States

Related Subject Headings

  • beta-Adrenergic Receptor Kinases
  • Receptors, Adrenergic, beta-2
  • Phosphorylation
  • Mutation
  • Cyclic AMP-Dependent Protein Kinases
  • Cricetinae
  • CHO Cells
  • Biochemistry & Molecular Biology
  • Animals
  • Adrenergic beta-Agonists
 

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Ferguson, S. S., Ménard, L., Barak, L. S., Koch, W. J., Colapietro, A. M., & Caron, M. G. (1995). Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. J Biol Chem, 270(42), 24782–24789. https://doi.org/10.1074/jbc.270.42.24782
Ferguson, S. S., L. Ménard, L. S. Barak, W. J. Koch, A. M. Colapietro, and M. G. Caron. “Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.J Biol Chem 270, no. 42 (October 20, 1995): 24782–89. https://doi.org/10.1074/jbc.270.42.24782.
Ferguson SS, Ménard L, Barak LS, Koch WJ, Colapietro AM, Caron MG. Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. J Biol Chem. 1995 Oct 20;270(42):24782–9.
Ferguson, S. S., et al. “Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1.J Biol Chem, vol. 270, no. 42, Oct. 1995, pp. 24782–89. Pubmed, doi:10.1074/jbc.270.42.24782.
Ferguson SS, Ménard L, Barak LS, Koch WJ, Colapietro AM, Caron MG. Role of phosphorylation in agonist-promoted beta 2-adrenergic receptor sequestration. Rescue of a sequestration-defective mutant receptor by beta ARK1. J Biol Chem. 1995 Oct 20;270(42):24782–24789.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

October 20, 1995

Volume

270

Issue

42

Start / End Page

24782 / 24789

Location

United States

Related Subject Headings

  • beta-Adrenergic Receptor Kinases
  • Receptors, Adrenergic, beta-2
  • Phosphorylation
  • Mutation
  • Cyclic AMP-Dependent Protein Kinases
  • Cricetinae
  • CHO Cells
  • Biochemistry & Molecular Biology
  • Animals
  • Adrenergic beta-Agonists