Molecular cloning of the cDNA and promoter sequences for the mouse sodium-hydrogen exchanger regulatory factor

The Na/H exchanger regulatory factor (NHE-RF) was first identified as a co- factor for cAMP dependent protein kinase regulation of the rabbit epithelia l Na/H exchanger. Subsequently, this protein which contains two PDZ motifs, was shown to interact with multiple cellular targets. To understand more f ully the function of NHE-RF and its regulation, we have cloned the full-len gth cDNA for mouse NHE-RF and a portion of the mouse gene containing the pr omoter elements. NHE-RF cDNA, isolated from a mouse kidney cDNA library, pr edicted a polypeptide of 356 amino acids that shares striking sequence cons ervation within the two PDZ domains and in-vitro phosphorylation sites with the human and rat homologs. The nucleotide sequence 5' of the transcriptio n start site, identified by primer extension analysis, was highly 'GC' rich and lacked canonical TATA or CAAT sequences. Using a luciferase reporter c onstruct, deletion analyses localized the critical segment for gene express ion in mouse medullary thick ascending limb cells to 114 bp 5' of the trans cription start site. Although NHE-RF has been recently identified as an est rogen-inducible gene, the lack of an estrogen-response element in the mouse NHE-RF 5'-non-coding-sequence and the inability to demonstrate estrogen st imulation of reporter gene expression in MCF-7 cells suggests a non-convent ional or indirect mechanism for NHE-RF regulation by estrogen. (C) 1999 Els evier Science B.V. All rights reserved.