The human Na(+)/H(+) exchanger NHE2 gene: genomic organization and promoter characterization.

The Na(+)/H(+) exchanger (NHE) 2 belongs to a family of plasma membrane transporters involved in intracellular pH and cell volume regulation. We recently reported cloning of human NHE2 (hNHE2) from a colonic cDNA library. Northern blot analysis has identified NHE2 mRNA only in small intestine, prostate, kidney, colon, and skeletal muscle. In this study, we describe the structure and 5'-regulatory region of the hNHE2 gene. The hNHE2 gene spans >90 kb and is organized in 12 exons intervened by 11 introns. All introns contain the conserved GT and AG dinucleotides at the donor and acceptor sites, respectively. The hNHE2 gene was mapped to chromosome 2q11.2. Primer extension analysis revealed a single transcription initiation site in human colonic adenocarcinoma cell lines. Analysis of the DNA nucleotide sequences of a 1.4-kb fragment of the 5'-flanking region shows no canonical TATA or CAAT boxes. However, the promoter region contains several potential cis-regulatory elements such as Sp1, early growth response-1, activator protein-2, MyoD, p300, nuclear factor-kappaB, myeloid zinc finger protein-1, caudal-related homeobox (Cdx) gene A, and Cdx protein-2 binding sites. In transient transfection studies, a reporter construct containing the 1.4-kb promoter region exhibited low luciferase activity levels. However, after deletion upstream of -664, its activity increased approximately threefold. Thus our data suggest that an inhibitory element may exist in the NHE2 promoter 5'-upstream region.

Molecular cloning, tissue distribution, and functional expression of the human Na(+)/H(+) exchanger NHE2.

In the present report, we describe the cloning of a human colonic cDNA that describes the full-length Na(+)/H(+) exchanger (NHE) 2 coding region. The human NHE2 (hNHE2) cDNA encodes for a polypeptide of 812 amino acids with a 90% overall identity to both rabbit and rat NHE2 isoforms. In comparison with SLC9A2, recently reported as the human NHE2, the hNHE2 polypeptide is 115 amino acids longer in the NH(2)-terminal end and shows only an 84% DNA nucleotide sequence identity. Northern blot analysis revealed that hNHE2 message has an uneven tissue distribution, with high levels in the skeletal muscle, colon, and kidney and lower levels in the testis, prostate, ovary, and small intestine. Protein expression studies with hNHE2 clone showed that a 75-kDa protein was expressed. Stable expression of transfected full-length hNHE2 cDNA in Na(+)/H(+) exchange-deficient LAP1 cells exhibited Na(+)-dependent pH recovery after an acid prepulse that was inhibited by 0.1 mM amiloride. These data indicate that this cDNA is the true human NHE2 cDNA and that the encoded protein is capable of catalyzing Na(+)/H(+) exchange activity.

Intestinal distribution of human Na+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA.

The identity of Na+/H+ exchanger (NHE) isoforms in the human small intestine and colon and their role in vectorial Na+ absorption are not known. The present studies were undertaken to examine the regional and vertical axis distribution of NHE-1, NHE-2, and NHE-3 mRNA in the human intestine. Ribonuclease protection assays were used to quantitate the levels of mRNA of these isoforms in various regions of the human intestine. In situ hybridization technique was used to localize NHE-2 and NHE-3 mRNA in the colon. The NHE-1 isoform message was present uniformly throughout the length of the human intestine. In contrast, mRNA levels for human NHE-2 and NHE-3 isoforms demonstrated significant regional differences. The NHE-3 abundance was found in decreasing order: ileum > jejunum > proximal colon = distal colon. The NHE-2 message level in the distal colon was significantly higher than in the proximal colon but was evenly distributed in the small intestine. In addition, NHE-2 mRNA was present in surface epithelial cells as well as in cells of the crypt region, suggesting the presence of NHE-2 message throughout the vertical axis of the colonic crypts. In contrast, NHE-3 mRNA was localized to surface colonocytes in the proximal colon. On the basis of this tissue-specific localization of NHE-2 and NHE-3 mRNA, it can be speculated that the relative contribution of NHE-2 and NHE-3 isoforms in Na+ absorption in the human intestine may be region specific, and these putative apical isoforms may be differentially regulated.

Specific sequences in the signal anchor of the beta-galactoside alpha-2,6-sialyltransferase are not essential for Golgi localization. Membrane flanking sequences may specify Golgi retention.

The beta-galactoside alpha-2,6-sialyltransferase is a trans Golgi/trans Golgi network glycosyltransferase which adds sialic acid residues to Asn-linked oligosaccharides of glycoproteins. Previous results suggested that the sialyltransferase stem and signal anchor including flanking sequences may be two independent Golgi retention regions. However, other experiments demonstrated that the sequence of the signal anchor itself was not important. To investigate whether the sialyltransferase signal anchor was necessary and sufficient for Golgi retention, several mutant and chimeric proteins were expressed and localized in Cos-1 and Chinese hamster ovary cells. We found that the signal anchor and flanking sequences were able to retain the sialyltransferase catalytic domain in the Golgi. However, efficient Golgi retention was still observed when the signal anchor was altered or entirely replaced in either the presence or absence of most of the luminal stem region. Chimeric proteins consisting of the sialyltransferase cytoplasmic tail and signal anchor fused to the extracellular domains of two different cell surface proteins demonstrated poor Golgi retention. A significant increase in the Golgi retention of one of these chimeras was observed when two lysines were placed next to the signal anchor on the luminal side. Taken together these results suggest that the sialyltransferase signal anchor is not necessary or sufficient for Golgi retention, rather, appropriately spaced cytoplasmic and luminal flanking sequences are the important elements of the sialyltransferase Golgi retention region.