Mukhin Y V, Vlasova T, Jaffa A A, Collinsworth G, Bell J L, Tholanikunnel B G, Pettus T, Fitzgibbon W, Ploth D W, Raymond J R, Garnovskaya M N
Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center, and Department of Medicine of the Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2001 May 18;276(20):17339-46. doi: 10.1074/jbc.M010834200. Epub 2001 Feb 16.
We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein-coupled receptor, the bradykinin B2 receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein fluorescence measuring the accelerated rate of pH(i) recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B2 receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kinase C and phosphatidylinositol 3'-kinase. Immunoprecipitation studies showed that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. CaM appears to be a direct substrate for phosphorylation by Jak2 as measured by an in vitro kinase assay. We propose that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of CaM and most likely by increasing the binding of CaM to NHE-1.
我们使用了一种内髓集合管的培养鼠细胞模型（mIMCD - 3细胞），以研究典型的G蛋白偶联受体缓激肽B2受体对普遍存在的钠 - 质子交换器，即钠/氢交换体1型（NHE - 1）的调节作用。通过对静止细胞进行质子微生理学测定以及通过测量从施加的酸负荷中pH(i)恢复的加速速率的2'-7'-双[2 - 羧甲基]-5(6)-羧基荧光素荧光测定法评估，缓激肽以浓度依赖性方式快速激活NHE - 1。缓激肽B2受体、磷脂酶C、Ca2+/钙调蛋白（CaM）和Janus激酶2（Jak2）的抑制剂可阻断NHE - 1的激活，但百日咳毒素以及蛋白激酶C和磷脂酰肌醇3'-激酶的抑制剂则不能。免疫沉淀研究表明，缓激肽刺激了一种信号转导复合物 的组装，该复合物包括CaM、Jak2和NHE - 1。通过体外激酶测定法测量，CaM似乎是Jak2磷酸化的直接底物。我们提出，Jak2是NHE - 1活性的一种新的间接调节因子它通过增加CaM的酪氨酸磷酸化，很可能还通过增加CaM与NHE - 1的结合来调节NHE - 1的活性。
