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

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