Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.
Mass spectrometric identification of phosphorylation sites in guanylyl cyclase A and B.
复制标题
鸟苷酸环化酶 A 和 B 磷酸化位点的质谱鉴定。
DOI：
10.1021/bi101700e
复制
发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Potter,LincolnR
中科院分区：
文献类型：
--
作者：
Yoder,AndreaR;Stone,MatthewD;Griffin,TimothyJ;Potter,LincolnR
观嘌呤环化酶A和B （GC-A和GC-B）是介导利钠肽生理效应的跨膜观嘌呤环化酶受体。大鼠GC-A和GC-B的一些磷酸化位点是已知的，但没有关于人类同源物的磷酸化位点信息。在这里，我们使用质谱法鉴定了两个物种的GC-A和GC-B的磷酸化位点。用nLC-MS-MS对HEK293细胞中纯化的受体进行分离和分析。在大鼠GC-A中鉴定出7个磷酸化位点（S497、T500、S502、S506、S510、T513和S487），除S510和T513外，其余位点均在人GC-A中发现。在大鼠GC-B中鉴定出6个磷酸化位点（S513、T516、S518、S523、S526和T529），在人GC-B中也鉴定出6个磷酸化位点。GC-A和GC-B有5个位点相同。GC-A中的S487和GC-B中的T529是新的、未被鉴定的位点。丙氨酸取代S487不影响初始配体依赖的GC-A活性，但谷氨酸取代使活性降低20%。在野生型、S487A和S487E型GC-A中观察到相似水平的anp依赖性脱敏。谷氨酸或丙氨酸取代T529分别增加或降低了GC-B的配体依赖性环化酶活性，T529E也增加了含有谷氨酸的GC-B突变体中所有五个先前确定的位点的环化酶活性。总之，我们在GC-A和GC-B中发现并表征了新的磷酸化位点，并首次提供了在人类冠酰环化酶中磷酸化位点的证据。
Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologues. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487), and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529), and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild-type, S487A, and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.