Weinman E J, Steplock D, Tate K, Hall R A, Spurney R F, Shenolikar S
Department of Medicine, West Virginia University School of Medicine, Morgantown, West Virginia 26506, USA.
J Clin Invest. 1998 May 15;101(10):2199-206. doi: 10.1172/JCI204.
Inhibition of the renal brush border membrane (BBM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recently cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-RF). As deduced from the cDNA of this 358-amino acid protein, amino acids 11-101 and amino acids 150-241 of the NHE-RF protein share 74% overall homology suggesting duplication of these PDZ containing domains. The serine residues at amino acid position 289 and 340 are considered to be the most likely sites for PKA mediated phosphorylation. To study the structure- function relation between NHE-RF and PKA mediated inhibition of the rabbit BBM Na/H exchanger, the effect of recombinant proteins representing full-length NHE-RF as well as truncated and mutant forms of NHE-RF were determined using a reconstitution assay. The reconstitution assay employed a fraction of rabbit BBM proteins that contains Na/H exchanger activity that is not regulated by PKA. NHE-RF in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a significant left shift in the dose-response relation such that 10(-12) M NHE-RF inhibited Na/H exchange transport by 30% in the presence but not in the absence of PKA. A recombinant polypeptide representing amino acids 1-151 (Domain I) did not affect Na/H exchange transport in the presence or absence of PKA. A polypeptide representing amino acids 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhibited Na/H exchange activity in a concentration-dependent manner. In the presence of PKA, there was a left shift in the dose-response relation. 10(-12) M of Domain II polypeptide inhibited transport by 18% in the presence but not in the absence of PKA. Mutation of serine residues 287, 289, and 290 to alanine did not affect the inhibitory effect in the absence of PKA but abolished the left shift in the dose-response relation elicited by PKA. Mutation of serine residues 339 and 340 to alanine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit renal BBM Na/H exchange activity-an effect which is augmented by PKA. The amino acid sequences in the polypeptide containing only the NH2-terminal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor but appears to be required for the full-length NHE-RF to express its full inhibitory effect on the BBM Na/H exchanger. One or more of the serine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the physiologic effect of cAMP on the renal BBM Na/H exchanger.
环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）对肾刷状缘膜（BBM）钠/氢交换体的抑制作用需要一种最近克隆的调节辅助因子——钠/氢交换体调节因子（NHE-RF）的参与。从这种358个氨基酸的蛋白质的cDNA推导可知，NHE-RF蛋白的第11至101位氨基酸和第150至241位氨基酸总体上有74%的同源性，这表明这些含PDZ结构域发生了重复。第289位和340位氨基酸处的丝氨酸残基被认为是PKA介导的磷酸化最可能的位点。为了研究NHE-RF与PKA介导的兔BBM钠/氢交换体抑制作用之间的结构-功能关系，使用重组实验确定了代表全长NHE-RF以及NHE-RF的截短和突变形式的重组蛋白的作用。重组实验采用了兔BBM蛋白的一部分，该部分含有不受PKA调节的钠/氢交换体活性。在ATP和镁存在但PKA不存在的情况下，NHE-RF以浓度依赖性方式抑制钠/氢交换活性。在PKA存在的情况下，剂量-反应关系有显著的左移，以至于在有PKA但无PKA时，10^(-12) M的NHE-RF抑制钠/氢交换转运达30%。代表第1至151位氨基酸（结构域I）的重组多肽在有或无PKA时均不影响钠/氢交换转运。代表第149至358位氨基酸（结构域II）的多肽在ATP和镁存在但PKA不存在的情况下，以浓度依赖性方式抑制钠/氢交换活性。在PKA存在的情况下，剂量-反应关系有左移。10^(-12) M的结构域II多肽在有PKA但无PKA时抑制转运达18%。将第287、289和290位丝氨酸残基突变为丙氨酸在无PKA时不影响抑制作用，但消除了PKA引起的剂量-反应关系的左移。将第339和340位丝氨酸残基突变为丙氨酸对PKA依赖性的钠/氢交换转运调节没有影响。这些研究表明，NHE-RF抑制兔肾基础BBM钠/氢交换活性——PKA可增强这种作用。仅包含NHE-RF氨基末端PDZ结构域的多肽中的氨基酸序列作为抑制剂没有内在活性，但似乎是全长NHE-RF对BBM钠/氢交换体发挥其完全抑制作用所必需的。第287、289和/或290位的一个或多个丝氨酸残基代表NHE-RF蛋白上关键的PKA磷酸化位点，其介导cAMP对肾BBM钠/氢交换体的生理作用。
