ROLE OF PHOSPHORYLATION IN AGONIST-PROMOTED BETA(2)-ADRENERGIC RECEPTOR SEQUESTRATION - RESCUE OF A SEQUESTRATION-DEFECTIVE MUTANT RECEPTOR BY BETA-ARK1

The beta(2)-adrenergic receptor (beta(2)AR) belongs to the large family of G protein coupled receptors. Mutation of tyrosine residue 326 to an alanine resulted in a beta(2)AR mutant (beta(2)AR-Y326A) that was defective in its ability to sequester and was less well coupled to adenylyl cyclase than the wild-type beta(2)AR. 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(2)AR-Y326A mutant was impaired by 75%. Further characterization of this phosphorylation, using either forskolin stimulation or phosphorylation site-deficient beta(2)AR-Y326A mutants, demonstrated that the beta(2)AR-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(2)AR-Y326A mutant and rescue of its sequestration. beta ARK1-mediated rescue of beta(2)AR-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(2)AR could be attenuated by overexpressing a dominant-negative mutant of beta ARK1 (C-20 beta ARK1-K220M). These findings implicate a role for beta ARK1 mediated phosphorylation in facilitating wild-type beta(2)AR sequestration.