Borgese F, Malapert M, Fievet B, Pouyssegur J, Motais R
Laboratoire Jean Maetz, Départment de Biologie Cellulaire et Moléculaire du Commissariat à l'Energie Atomique, Villefranche-sur-Mer, France.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5431-5. doi: 10.1073/pnas.91.12.5431.
Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) are difficult to interpret as variable results have been reported for the different isoforms when expressed in various cell types. We took advantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the molecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate the critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced by the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, is essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decreased by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP-mediated activation by 72%. The residual activation required a cytoplasmic fragment (aa 559-661) that contains four sequences considered likely as putative PKA consensus sites. The results obtained with the chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exchange so induced can be modulated by the nature of the interaction between the N- and C-terminal domains.
关于环磷酸腺苷（cAMP）对克隆的钠氢交换体（NHEs）作用的研究难以解释，因为当在各种细胞类型中表达时，不同亚型的结果各不相同。我们利用了这样一个事实，即人类NHE1和鳟鱼红细胞β-NHE在同一细胞系PS120中表达时，对cAMP的反应不同（NHE1不敏感，β-NHE被激活），以分析cAMP激活的分子机制。我们构建了NHE1和β-NHE之间的嵌合体以及一组β-NHE突变体，以确定参与激活过程的分子关键部分。当NHE1的胞质结构域被β-NHE的胞质结构域取代时，NHE1变得对cAMP有反应；因此，β-NHE的胞质C末端包含两个cAMP依赖的共有序列，对于赋予cAMP依赖性至关重要。仅对两个蛋白激酶A（PKA）共有位点之一进行丝氨酸到甘氨酸的替换，使cAMP激活钠氢交换的能力降低了60%。同时对两个PKA共有位点进行丝氨酸到甘氨酸的替换，使cAMP介导的激活降低了72%。残余的激活需要一个胞质片段（氨基酸559 - 661），该片段包含四个被认为可能是假定的PKA共有位点的序列。用嵌合NHE获得的结果还表明，如果胞质C末端在激素激活中起关键作用，那么如此诱导的钠氢交换速率可由N末端和C末端结构域之间相互作用的性质来调节。
