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

Abstract

The Na/H exchanger regulatory factor (NHE-RF) was first identified as a co-factor for cAMP dependent protein kinase regulation of the rabbit epithelial Na/H exchanger. Subsequently, this protein which contains two PDZ motifs, was shown to interact with multiple cellular targets. To understand more fully the function of NHE-RF and its regulation, we have cloned the full-length cDNA for mouse NHE-RF and a portion of the mouse gene containing the promoter elements. NHE-RF cDNA, isolated from a mouse kidney cDNA library, predicted a polypeptide of 356 amino acids that shares striking sequence conservation within the two PDZ domains and in-vitro phosphorylation sites with the human and rat homologs. The nucleotide sequence 5′ of the transcription start site, identified by primer extension analysis, was highly ‘GC’ rich and lacked canonical TATA or CAAT sequences. Using a luciferase reporter construct, deletion analyses localized the critical segment for gene expression in mouse medullary thick ascending limb cells to 114 bp 5′ of the transcription start site. Although NHE-RF has been recently identified as an estrogen-inducible gene, the lack of an estrogen-response element in the mouse NHE-RF 5′-non-coding-sequence and the inability to demonstrate estrogen stimulation of reporter gene expression in MCF-7 cells suggests a non-conventional or indirect mechanism for NHE-RF regulation by estrogen.