Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland.
Mol Pharmacol. 2013 Jan;83(1):129-41. doi: 10.1124/mol.112.080440. Epub 2012 Oct 11.
The β(1)-adrenergic receptor (β(1)AR) is the predominant βAR in the heart and is the main target for β-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) β(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in β(1)AR regulation, focusing on the effects of long-term treatment with β-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hβ(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the β(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of β-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of β(1)AR levels. This phenomenon may contribute to the exceptional resistance of β(1)ARs to downregulation and tendency toward upregulation following long-term ligand treatments.
β(1)-肾上腺素能受体（β(1)AR）是心脏中的主要βAR，也是β-肾上腺素能拮抗剂的主要靶点，广泛用于治疗心血管疾病。此前，我们已经表明，人（h）β(1)AR 在其 N 端被金属蛋白酶切割，无论是组成型的还是受体激活依赖性的。在这项研究中，我们研究了 β(1)AR 调节中涉及的具体事件，重点关注长期使用β-肾上腺素能配体对稳定转染的人胚肾 293(i)细胞中受体加工的影响。关键发现使用瞬时转染的 hβ(1)AR 和新生大鼠心肌细胞中内源性表达的受体进行了验证。通过使用流式细胞术和 Western blot 分析，我们证明异丙肾上腺素、S-普萘洛尔、CGP-12177[4-[3-[(1,1-二甲基乙基)氨基]2-羟基丙氧基]-1,3-二氢-2H-苯并咪唑-2-酮]、普萘洛尔和噻吗洛尔，这些配体在腺苷酸环化酶或丝裂原激活蛋白激酶信号通路中对β(1)AR 具有激动作用，诱导成熟细胞表面受体的切割。相比之下，美托洛尔、比索洛尔和 CGP-20712[1-[2-((3-氨甲酰基-4-羟基)苯氧基)乙基氨基]-3-[4-(1-甲基-4-三氟甲基-2-咪唑基)苯氧基]-2-丙醇]，这些配体没有激动活性，只有很小的或没有作用。重要的是，激动剂还稳定了细胞内受体前体，可能通过其药理学伴侣作用，并在体外稳定了受体。因此，两种受体形式的相反作用导致质膜上切割的受体片段数量增加。这些结果强调了β-肾上腺素能配体的多维度性，并将这种特性从受体激活和信号传递扩展到β(1)AR 水平的调节。这种现象可能有助于β(1)AR 对下调的异常抵抗力，并在长期配体治疗后倾向于上调。
