Effect of Different G Protein-coupled Receptor Kinases on Phosphorylation and Desensitization of the α1B-Adrenergic Receptor

The α1B-adrenergic receptor (α1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of β-adrenergic receptor kinase (βARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type α1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative βARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the α1BAR. Increasing coexpression of βARK1 or βARK2 resulted in the progressive attenuation of the α1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of βARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated α1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type α1BAR, but not for its truncated mutant, when the receptor was coexpressed with β-arrestin 1 or β-arrestin 2. Despite their pronounced effect on phosphorylation of the α1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that βARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the α1BAR. They also identify the α1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the α1BAR remain to be elucidated. The α1B-adrenergic receptor (α1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of β-adrenergic receptor kinase (βARK) 1 (GRK2) or 2 (GRK3) could increase epinephrine-induced phosphorylation of the wild type α1BAR above basal as compared to that of the receptor expressed alone. On the other hand, overexpression of the dominant negative βARK (K220R) mutant impaired agonist-induced phosphorylation of the receptor. Overexpression of GRK6 could also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhanced basal but not agonist-induced phosphorylation of the α1BAR. Increasing coexpression of βARK1 or βARK2 resulted in the progressive attenuation of the α1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of βARK1 or 2 at low levels did not significantly impair the PI response mediated by the truncated α1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response was also observed for the wild type α1BAR, but not for its truncated mutant, when the receptor was coexpressed with β-arrestin 1 or β-arrestin 2. Despite their pronounced effect on phosphorylation of the α1BAR, overexpression of GRK5 or GRK6 did not affect the receptor-mediated response. In conclusion, our results provide the first evidence that βARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the α1BAR. They also identify the α1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the α1BAR remain to be elucidated.