Malapert M, Guizouarn H, Fievet B, Jahns R, Garcia-Romeu F, Motais R, Borgese F
Laboratoire Jean Maetz, URA 1855, Département de Biologie Cellulaire et Moléculaire CEA, Villefranche-sur-Mer, France.
J Exp Biol. 1997 Jan;200(Pt 2):353-60. doi: 10.1242/jeb.200.2.353.
The trout red blood cell Na+/H+ antiporter (beta NHE) plays two interesting properties: it is the only NHE own to be activated by cyclic AMP, and the activation process is followed by a desensitisation of the transport system itself. Cloning and expression of beta NHE have provided inificant information about Na+/H+ activation, in particular that activation by cyclic AMP is directly dependent upon the presence of two protein kinase A consensus sites in the cytoplasmic tail of the antiporter. Expression of beta NHE in fibroblasts demonstrates that the protein kinase A (PKA) and protein kinase C (PKC) activation pathways are independent and do not converge a common kinase. Moreover, the hydrophilic C-terminal fragment is essential to the mediation of the various hormonal responses. NHE1 (the human ubiquitous isoform) is not activated by cyclic AMP, but a "NHE1 transmembrane domain/beta NHE cytoplasmic domain' chimera is fully activated by cyclic AMP. In red cells, activation of beta NHE is the result of phosphorylation by PKA of at least two independent sites. Desensitisation, inhibited by the phosphatase inhibitor okadaic acid, may consist of the dephosphorylation of one of these two sites. Furthermore, Calyculin A (CIA), another specific protein phosphatase inhibitor, induces in unstimulated cells a Na+/H+ exchange activity whose exchange properties are very different from those of the adrenergically stimulated antiporter. It is suggested that CIA may be able to revive "sequestered' antiporters. We propose that the molecular events underlying beta NHE desensitisation could be similar to those involved in rhodopsin desensitisation. Antibodies were generated against trout red cell arrestin in order to analyse the binding of arrestin to the activated exchanger. Recombinant trout arrestin was produced in a protease-deficient strain of Escherichia coli and its functionality tested in a reconstituted rhodopsin assay.
鳟鱼红细胞钠/氢反向转运体（β-NHE）具有两个有趣的特性：它是唯一一种由环磷酸腺苷（cAMP）激活的NHE，并且在激活过程之后，转运系统本身会出现脱敏现象。β-NHE的克隆和表达为钠/氢激活提供了重要信息，特别是cAMP激活直接依赖于反向转运体胞质尾中两个蛋白激酶A共有位点的存在。β-NHE在成纤维细胞中的表达表明，蛋白激酶A（PKA）和蛋白激酶C（PKC）激活途径是独立的，不会汇聚到一个共同的激酶。此外，亲水性C末端片段对于介导各种激素反应至关重要。NHE1（人类普遍存在的异构体）不会被cAMP激活，但“NHE1跨膜结构域/β-NHE胞质结构域”嵌合体可被cAMP完全激活。在红细胞中，β-NHE的激活是PKA对至少两个独立位点进行磷酸化的结果。脱敏现象可被磷酸酶抑制剂冈田酸抑制，可能是这两个位点之一的去磷酸化所致。此外，另一种特异性蛋白磷酸酶抑制剂Calyculin A（CIA）在未受刺激的细胞中诱导出一种钠/氢交换活性，其交换特性与肾上腺素刺激的反向转运体非常不同。有人认为CIA可能能够使“被隔离”的反向转运体重现活性。我们提出，β-NHE脱敏背后的分子事件可能与视紫红质脱敏所涉及的事件相似。为了分析抑制蛋白与激活的交换体的结合，制备了针对鳟鱼红细胞抑制蛋白的抗体。重组鳟鱼抑制蛋白在蛋白酶缺陷型大肠杆菌菌株中产生，并在重组视紫红质测定中测试其功能。
