Role of beta ARK in long-term agonist-promoted desensitisation of the beta 2-adrenergic receptor.
Phosphorylation of the beta 2-adrenergic receptor (beta 2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term beta 2AR 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 2AR and found that receptor phosphorylation in cells treated with 1 microM isoproterenol for 24 h was approximately 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 2AR lacking the determinants of phosphorylation by the beta AR kinase (beta ARK), PKA or both. In CHW cells expressing the WT beta 2AR, pretreatment with 1 microM 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 after 24 h. The lower level of PKA activity after 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 2AR 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 2AR as well.
McGraw
D W
DW
Department of Medicine (Pulmonary), University of Cincinnati College of Medicine, Ohio, USA.
Donnelly
E T
ET
Eason
M G
MG
Green
S A
SA
Liggett
S B
SB
D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D015536:Down-Regulation; D005347:Fibroblasts; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D051552:beta-Adrenergic Receptor Kinases
D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D000262:Adenylyl Cyclases; D007545:Isoproterenol