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Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

ABSTRACT: Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, respectively. Both genes are approximately 16 kb and are comprised of 13 exons and 12 introns, the junctions of which conform to donor and acceptor site consensus sequences. Putative CAAT and TATA boxes are located, respectively, at positions -147 and -40 of the Npt2 gene and -143 and -51 of the NPT2 gene, relative to nucleotide 1 of the corresponding cDNAs. The translation initiation site is within exon 2 of both genes. The first 220 bp of the mouse and human promoter regions exhibit 72% identity. Two transcription start sites (at positions -9 and - 10 with respect to nucleotide 1 of NaPi-7 cDNA) and two polyadenylylation signals were identified in the Npt2 gene by primer extension, 5' and 3' rapid amplification of cDNA ends (RACE). A 484-bp 5' flanking region of the Npt2 gene, comprising the CAAT box, TATA box, and exon 1, was cloned upstream of a luciferase reporter gene; this construct significantly stimulated luciferase gene expression, relative to controls, when transiently transfected into OK cells, a renal cell line expressing type II Na+ -Pi cotransporter activity. The present data provide a basis for detailed analysis of cis and trans elements involved in the regulation of Npt2/NPT2 gene transcription and facilitate screening for mutations in the NPT2 gene in patients with autosomally inherited disorders of renal Pi reabsorption.

SUBMITTER: Hartmann CM

PROVIDER: S-EPMC38998 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

Hartmann C M CM Hewson A S AS Kos C H CH Hilfiker H H Soumounou Y Y Murer H H Tenenhouse H S HS

Proceedings of the National Academy of Sciences of the United States of America 19960701 14

Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, res ...[more]

PMID: 8693007

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Project description:Phosphate plays essential biological roles and its plasma level in humans requires tight control to avoid bone loss (insufficiency) or vascular calcification (excess). Intestinal absorption and renal reabsorption of phosphate are mediated by members of the SLC34 family of sodium-coupled transporters (NaPi-IIa,b,c) whose membrane expression is regulated by various hormones, circulating proteins, and phosphate itself. Consequently, NaPi-II proteins are also potentially important pharmaceutical targets for controlling phosphate levels. Their crucial role in Pi homeostasis is underscored by pathologies resulting from naturally occurring SLC34 mutations and SLC34 knockout animals. SLC34 isoforms have been extensively studied with respect to transport mechanism and structure-function relationships; however, the three-dimensional structure is unknown. All SLC34 transporters share a duplicated motif comprising a glutamine followed by a stretch of threonine or serine residues, suggesting the presence of structural repeats as found in other transporter families. Nevertheless, standard bioinformatic approaches fail to clearly identify a suitable template for molecular modeling. Here, we used hydrophobicity profiles and hidden Markov models to define a structural repeat common to all SLC34 isoforms. Similar approaches identify a relationship with the core regions in a crystal structure of Vibrio cholerae Na(+)-dicarboxylate transporter VcINDY, from which we generated a homology model of human NaPi-IIa. The aforementioned SLC34 motifs in each repeat localize to the center of the model, and were predicted to form Na(+) and Pi coordination sites. Functional relevance of key amino acids was confirmed by biochemical and electrophysiological analysis of expressed, mutated transporters. Moreover, the validity of the predicted architecture is corroborated by extensive published structure-function studies. The model provides key information for elucidating the transport mechanism and predicts candidate substrate binding sites.

Dataset Information

Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

Publications

Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

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