Institute of Physiology, University of Würzburg, Würzburg, Germany.
FEBS J. 2010 Jun;277(11):2440-53. doi: 10.1111/j.1742-4658.2010.07658.x. Epub 2010 Apr 26.
Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3',5'-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications.
心钠肽（ANP）通过其鸟苷酸环化酶 A（GC-A）受体和细胞内鸟苷 3'，5'-环单磷酸的产生，在调节血压方面起着至关重要的作用。在慢性心力衰竭患者中，ANP 的血浆水平升高，但心血管作用严重减弱，表明存在受体或受体后缺陷。对代谢标记的 GC-A 过表达细胞的研究表明，GC-A 广泛磷酸化，并且 ANP 诱导的 GC-A 同源脱敏与受体去磷酸化相关，这一机制可能导致体内功能丧失。在这项研究中，在人胚肾细胞系 HEK293 中表达的 GC-A 受体的串联质谱分析明确表明，受体的细胞内结构域在多个残基处磷酸化：Ser487、Ser497、Thr500、Ser502、Ser506、Ser510 和 Thr513。基于多重反应监测的 MS 定量分析表明，ANP 引起的脱敏伴随着受体磷酸化和去磷酸化的复杂模式。完全磷酸化的 GC-A 群体减少。然而，有趣的是，暴露于 ANP 后，GC-A 的 Ser487 磷酸化选择性增强。通过定点突变分析研究了这一观察结果的功能相关性。用谷氨酸（模拟磷酸化）取代 Ser487 削弱了 ANP 对 GC-A 受体的激活，但阻止了进一步的脱敏。我们的数据证实了先前的研究，表明 GC-A 对 ANP 的反应性受磷酸化调节。然而，除了先前假定的位点（Ser497、Thr500、Ser502、Ser506、Ser510）去磷酸化之外，同源脱敏似乎还涉及 GC-A 上 Ser487 的磷酸化，这是一个新发现的磷酸化位点。鉴定和进一步表征涉及 GC-A 对 ANP 反应性下调的特定机制可能具有重要的病理生理学意义。
