Journal of Biological Chemistry
Volume 270, Issue 42, 20 October 1995, Pages 24782-24789
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Cell Biology and Metabolism
Role of Phosphorylation in Agonist-promoted β2-Adrenergic Receptor Sequestration: RESCUE OF A SEQUESTRATION-DEFECTIVE MUTANT RECEPTOR BY βARK1 (∗)

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The β2-adrenergic receptor (β2AR) belongs to the large family of G protein-coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a β2AR mutant (β2AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type β2AR. 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 β2AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient β2AR-Y326A mutants, demonstrated that the β2AR-Y326A mutant can be phosphorylated by cAMP-dependent protein kinase (PKA) but does not serve as a substrate for the β-adrenergic receptor kinase 1 (βARK1). However, overexpression of βARK1 led to the agonist-dependent phosphorylation of the β2AR-Y326A mutant and rescue of its sequestration. βARK1-mediated rescue of β2AR-Y326A sequestration could be prevented by mutating putative βARK phosphorylation sites, but not PKA phosphorylation sites. In addition, both sequestration and phosphorylation of the wild-type β2AR could be attenuated by overexpressing a dominant-negative mutant of βARK1 (C20βARK1-K220M). These findings implicate a role for βARK1-mediated phosphorylation in facilitating wild-type β2AR sequestration.

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This work was supported by National Institutes of Health Grant NS 19576 (to M. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

These authors contributed equally to this work.

Fellow of the Medical Research Council of Canada.

∗∗

Recipient of a Howard Hughes postdoctoral fellowship.