title: Cardiac β2-Adrenergic Receptor Phosphorylation at Ser355/356 Regulates Receptor Internalization and Functional Resensitization.
abstract: Previous studies have demonstrated that β2-adrenergic receptors (β2ARs) can be phosphorylated by G protein-coupled receptor kinases (GRKs) and protein kinase A (PKA), affecting β2AR internalization and desensitization. However, the exact physiological function of β2ARs 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 β2AR stimulation caused functional desensitization. Phosphorylation of β2ARs 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, β2AR internalization increased. Interestingly, β2AR internalization was blocked by mutations at the GRK phosphorylation sites, but not by mutations at the PKA phosphorylation sites. Furthermore, inhibition of β2AR dephosphorylation by okadaic acid, a phosphatase 2A inhibitor, impaired the recovery of internalized β2ARs and reduced the cardiomyocyte contraction rate in response to epinephrine. Finally, epinephrine treatment induced the physical interaction of β-arrestin with internalized β2ARs in cardiomyocytes. Together, these data revealed the essential role of the Ser355/356 phosphorylation status of β2ARs in regulating receptor internalization and physiological resensitization in neonatal cardiomyocytes to contraction functions.
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Fan
authors.affiliation: Department of Endocrine and Metabolic Diseases, 1st Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Xuejiang
authors.initials: X
authors.lastname: Gu
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Ru
authors.initials: R
authors.lastname: Zhao
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Qingqing
authors.initials: Q
authors.lastname: Zheng
authors.affiliation: Department of Pathology, Shantou University Medical College, Shantou, Guangdong, P.R. China.
authors.forename: Lan
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Pathology, Shantou University Medical College, Shantou, Guangdong, P.R. China.
authors.forename: Wenbing
authors.initials: W
authors.lastname: Yang
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Lu
authors.initials: L
authors.lastname: Ding
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Feng
authors.initials: F
authors.lastname: Xue
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Junming
authors.initials: J
authors.lastname: Fan
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Gong
authors.affiliation: Institute of Hypoxia Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Yongyu
authors.identifier: http://orcid.org/0000-0002-8804-3472
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004837:Epinephrine / Q000494:pharmacology; D005260:Female; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D018342:Receptors, Adrenergic, beta-1; D018343:Receptors, Adrenergic, beta-2; D012694:Serine; D009638:Norepinephrine; D004837:Epinephrine