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 response. Phosphorylation of the beta 2AR by several distinct kinases plays an important role in this desensitization phenomenon. In this study, we have utilized purified hamster lung beta 2AR and stimulatory guanine nucleotide binding regulatory protein (Gs), reconstituted in phospholipid 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 beta AR kinase (beta ARK), and receptor function was determined by measuring the beta 2AR-agonist-promoted Gs-associated GTPase activity. At physiological concentrations of Mg2+ (less than 1 mM), receptor phosphorylation inhibited coupling to Gs by 60% (PKA), 40% (PKC), and 30% (beta ARK). The desensitizing effect of phosphorylation was, however, greatly diminished when assays were performed at concentrations of Mg2+ sufficient to promote receptor-independent activation of Gs (greater than 5 mM). Addition 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 presence 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+ concentration of 5 mM, arrestin did not potentiate the inhibition of receptor function observed on PKA or PKC phosphorylation. These results suggest that distinct pathways of desensitization exist for the receptor phosphorylated either by PKA or PKC or alternatively by beta ARK.