STRUCTURE-FUNCTION OF RECOMBINANT NA H EXCHANGER REGULATORY FACTOR (NHE-RF)/

Inhibition of the renal brush border membrane (BRM) Na/H exchanger by cAMP-dependent protein kinase, PKA, requires participation of a recent ly cloned regulatory cofactor, Na/H exchanger-regulatory factor (NHE-R F), As deduced from the cDNA of this 358-amino acid protein, amino aci ds 11-101 and amino acids 150-241 of the NHE-RF protein share 74% over all homology suggesting duplication of these PDZ containing domains, T he serine residues at amino acid position 289 and 340 are considered t o be the most likely sites for PKA mediated phosphorylation, To study the structure-function relation between NHE-RF and PKA mediated inhibi tion of the rabbit BBM Na/H exchanger, the effect of recombinant prote ins representing full-length NHE-RF as well as truncated and mutant fo rms of NHE-RF were determined using a reconstitution assay. The recons titution assay employed a fraction of rabbit BBM proteins that contain s 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 i n 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 acid s 149-358 (Domain II) in the presence of ATP and Mg but not PKA, inhib ited Na/H exchange activity in a concentration-dependent manner. In th e presence of PKA, there was a left shift in the dose-response relatio n. 10(-12) M of Domain II polypeptide inhibited transport by 18% in th e 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 t he absence of PKA but abolished the left shift in the dose-response re lation elicited by PKA, Mutation of serine residues 339 and 340 to ala nine were without effect on PKA dependent regulation of Na/H exchange transport. These studies indicate that NHE-RF inhibits basal rabbit re nal BBM Na/H exchange activity-an effect which is augmented by PKA. Th e amino acid sequences in the polypeptide containing only the NH2-term inal PDZ domain of NHE-RF have no intrinsic activity as an inhibitor b ut appears to be required for the full-length NHE-RF to express its fu ll inhibitory effect on the BBM Na/H exchanger. One or more of the ser ine residues at positions 287, 289, and/or 290 represent the critical PKA phosphorylation site(s) on the NHE-RF protein that mediates the ph ysiologic effect of cAMP on the renal BBM Na/H exchanger.