Role of βARK in long-term agonist-promoted desensitisation of the β2-adrenergic receptor

Phosphorylation of the beta(2)-adrenergic receptor (beta(2)AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term beta(2)AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type beta(2)AR and found that receptor phosphorylation in cells treated with 1 mu M isoproterenol for 24 h was similar to 4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated beta(2)AR lacking the determinants of phosphorylation by the beta AR kinase (beta ARK), PKA or both. In CHW cells expressing the WT beta(2)AR, pretreatment with 1 mu M isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 +/- 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 +/- 6%, P = NS compared to WT). In contrast, desensitisation was only 49 +/- 4% (P < 0.001 compared to WT) when the beta ARK sites were similarly substituted. Removal of both the beta ARK and PKA sites impaired desensitisation to the same extent as the beta ARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular beta ARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels alter 24 h. The lower level of PKA activity alter long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with beta(2)AR lacking beta ARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, beta ARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the beta(2)AR as well. (C) 1998 Elsevier Science Inc.