ROLE OF BETA-ARK IN LONG-TERM AGONIST-PROMOTED DESENSITIZATION OF THEBETA(2)-ADRENERGIC RECEPTOR

Phosphorylation of the beta(2)-adrenergic receptor (beta(2)AR) is the initial event that underlies rapid agonist-promoted desensitisation. H owever, the role of phosphorylation in mediating long-term beta(2)AR d esensitisation is not known. To investigate this possibility, we perfo rmed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type beta(2)AR and found that recept or phosphorylation in cells treated with 1 mu M isoproterenol for 24 h was similar to 4-fold over the basal state. This finding suggested th at persistent phosphorylation of the receptor might contribute to func tional long-term desensitisation which we further explored with mutate d 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 isopr oterenol-stimulated cAMP response by 82 +/- 5%. Substitution of the PK A sites with alanines had no effect on the extent of desensitisation ( 77 +/- 6%, P = NS compared to WT). In contrast, desensitisation was on ly 49 +/- 4% (P < 0.001 compared to WT) when the beta ARK sites were s imilarly substituted. Removal of both the beta ARK and PKA sites impai red desensitisation to the same extent as the beta ARK mutant. The ext ent of receptor loss (downregulation) was the same among all of the ce ll lines used and therefore could not account for the observed differe nces in desensitisation. Cellular beta ARK activity, assessed by a rho dopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamical ly regulated, increasing 4-fold over basal levels after 15 min of isop roterenol and returning to near basal levels alter 24 h. The lower lev el of PKA activity alter long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Non etheless, long-term desensitisation was clearly attenuated with beta(2 )AR lacking beta ARK phosphorylation sites. These findings show that i n 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 Scie nce Inc.