DESENSITIZATION OF THE ISOLATED BETA-2-ADRENERGIC RECEPTOR BY BETA-ADRENERGIC-RECEPTOR KINASE, CAMP-DEPENDENT PROTEIN-KINASE, AND PROTEIN-KINASE-C OCCURS VIA DISTINCT MOLECULAR MECHANISMS

Exposure of beta-2-adrenergic receptors (beta-2ARs) to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase r esponse. Phosphorylation of the beta-2AR by several distinct kinases p lays an important role in this desensitization phenomenon. In this stu dy, we have utilized purified hamster lung beta-2AR and stimulatory gu anine nucleotide binding regulatory protein (G(s)), reconstituted in p hospholipid vesicles, to investigate the molecular properties of this desensitization response. Purified hamster beta-2AR was phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), or be ta-AR kinase (beta-ARK), and receptor function was determined by measu ring the beta-2AR-agonist-promoted G(s)-associated GTPase activity. At physiological concentrations of Mg+ (< 1 mM), receptor phosphorylatio n inhibited coupling to G(s) by 60% (PKA), 40% (PKC), and 30% (beta-AR K). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ suffic ient to promote receptor-independent activation of G(s) (> 5 mM). Addi tion of retinal arrestin, the light transduction component involved in the attenuation of rhodopsin function, did not enhance the uncoupling effect of beta-ARK phosphorylation of beta-2AR when assayed in the pr esence of 0.3 mM free Mg2+. At concentrations of Mg2+ ranging between 0.5 and 5.0 mM, however, significant potentiation of beta-ARK-mediated desensitization was observed upon arrestin addition. At a free Mg2+ c oncentration of 5 mM, arrestin did not potentiate the inhibition of re ceptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the recept or phosphorylated either by PKA or PKC or alternatively by beta-ARK.