Cardiac 2-Adrenergic receptor phosphorylation at Ser355/356 Regulates receptor internalization and functional resensitization

Abstract

Previous studies have demonstrated that 2-adrenergic receptors (2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting 2AR internalization and desensitization. However, the exact physiological function of 2ARs in cardiomyocytes is unknown. In this study, we showed that neonatal mouse cardiomyocytes had different contraction and internalization responses to sustained or repeated, transient agonist stimulation. Specifically, short-time stimulation (10 min) with epinephrine or norepinephrine increased the cardiomyocyte contraction rate, reaching a maximum at 5 min, followed by a slow decline. When the agonist was re-added after a 60-min wash-out period, the increase in the cardiomyocyte contraction rate was similar to the initial response. In contrast, when cardiomyocytes were exposed continuously to epinephrine or norepinephrine for 60 min, the second agonist stimulation did not increase the contraction response. These results indicated that continuous 2AR stimulation caused functional desensitization. Phosphorylation of 2ARs at serine (Ser)355/356 GRK phosphorylation sites, but not at Ser345/346 PKA phosphorylation sites increased with continuous epinephrine stimulation for 60 min. Accordingly, 2AR internalization increased. Interestingly, 2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of 2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized 2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of -arrestin with internalized 2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of 2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.