Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF)
Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF)
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DOI：
10.1172/jci204
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发表时间：
1998-05-15
影响因子：
15.9
通讯作者：
Shenolikar, S
中科院分区：
文献类型：
--
作者：
Weinman, EJ;Steplock, D;Shenolikar, S
cAMP依赖性蛋白激酶PKA对肾刷状缘膜Na/H交换的抑制需要最近克隆的调节辅因子Na/H交换调节因子（NHE-RF）的参与。NHE-RF蛋白的氨基酸11-101和氨基酸150-241共有74%的总体同源性，表明这些含PDZ的结构域的重复，第289位和第340位的丝氨酸残基被认为是PKA介导的磷酸化的最可能位点。为了研究NHE-RF和PKA介导的抑制兔BBM Na/H交换的结构-功能关系，代表全长NHE-RF以及截短和突变形式的NHE-RF的重组蛋白的作用用重建测定法测定。重建测定采用了兔BBM蛋白的一部分，其含有在ATP和Mg存在下不受PKA调节的Na/H交换活性，NHE-RF但不受PKA调节，以浓度依赖性抑制Na/H交换活性。在PKA存在下，剂量-反应关系显著左移，10（-12）M NHE-RF抑制Na/H交换转运30%在PKA存在但不存在的情况下，代表氨基酸1-151的重组多肽（结构域I）在PKA（代表氨基酸149-358的多肽）存在或不存在的情况下不影响Na/H交换转运（结构域II）在ATP和Mg的存在下，但不PKA，抑制Na/H交换活性的浓度依赖性的方式。在PKA的存在下，有一个左移的剂量-反应关系。10（-12）M结构域II多肽在PKA存在下抑制转运18%，但在PKA不存在下不抑制转运。丝氨酸残基287、289和290突变为丙氨酸在PKA不存在下不影响抑制作用，但消除PKA引起的剂量-反应关系的左移，丝氨酸残基339和340突变为丙氨酸对PKA依赖的Na/H交换转运调节没有影响。这些研究表明，NHE-RF抑制基础兔肾BBM Na/H交换活性-PKA增强的效果。仅含有NHE-RF的NH 2-末端PDZ结构域的多肽中的氨基酸序列不具有作为抑制剂的固有活性，但似乎是全长NHE-RF表达其对BBM Na/H交换剂的完全抑制作用所需的。在位置287、289和/或290处的一个或多个丝氨酸残基代表NHE-RF蛋白上介导cAMP对肾BBM Na/H交换剂的生理作用的关键PKA磷酸化位点。
Inhibition of the renal brush border membrane (BRM) 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 mariner, 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.