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Identification of Three Isoforms for the Na+-dependent Phosphate Cotransporter (NaPi-2) in Rat Kidney

1998; Elsevier BV; Volume: 273; Issue: 44 Linguagem: Inglês

10.1074/jbc.273.44.28568

1083-351X

Sawako Tatsumi, Ken–ichi Miyamoto, Tomoko Kouda, Keiko Motonaga, Kanako Katai, Ichiro Ohkido, Kyoko Morita, Hiroko Segawa, Yoshiko Tani, Hironori Yamamoto, Yutaka Taketani, Eiji Takeda,

Medical Imaging and Pathology Studies

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2α, NaPi-2β, and NaPi-2γ) from a rat kidney library. NaPi-2α cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2β encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2γ encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 α, β, and γ were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2α and NaPi-2γ were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms.A functional analysis demonstrated that NaPi-2γ and -2α markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter. We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2α, NaPi-2β, and NaPi-2γ) from a rat kidney library. NaPi-2α cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2β encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2γ encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 α, β, and γ were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2α and NaPi-2γ were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2γ and -2α markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter. polymerase chain reaction brush-border membrane vesicle inorganic phosphate base pair(s) kilobase pair(s) polyacrylamide gel electrophoresis hereditary hypophosphatemic rickets with hypercalciuria. Renal phosphate (Pi) reabsorption is an essential aspect of the maintenance of plasma Pi homeostasis (1Murrer H. Biber J. Seldin D.W. Giebisch G. The Kidney: Pathophysiology. 2nd Ed. Raven Press, New York1992: 2481-2509Google Scholar). Several mammalian renal Na+-dependent Pi cotransporters have recently been isolated and well characterized (2Werner A. Moore M.L. Mantei N. Biber J. Semenza G. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9608-9612Crossref PubMed Scopus (236) Google Scholar, 3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar, 4Sorribas V. Markovich D. Hayes G. Stange G. Forgo J. Biber J. Murer H. J. Biol. Chem. 1994; 269: 6615-6621Abstract Full Text PDF PubMed Google Scholar, 5Verri T. Markovich D. Perego C. Norbis F. Stange G. Sorribas V. Biber J. Murer H. Am. J. Physiol. 1995; 268: F626-F633PubMed Google Scholar, 6Collins J.F. Ghishan F.K. FASEB J. 1994; 8: 862-868Crossref PubMed Scopus (89) Google Scholar, 7Michael P.K. Kabat D Kidney Int. 1996; 49: 959-963Abstract Full Text PDF PubMed Scopus (160) Google Scholar). The cDNA of these transporters can be divided into at least three types (types I-III) in the kidney cortex. It has been demonstrated that the type II transporter is a major functional Na+-dependent phosphate cotransporter in the proximal tubules (8Biber J. Custer M. Magagnin S. Hayes G. Werner A. Lotscher M. Kaissling B. Murer H. Kidney Int. 1996; 49: 981-985Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 9Murer H. Biber J. Annu. Rev. Physiol. 1996; 58: 607-618Crossref PubMed Scopus (95) Google Scholar, 10Murer H. Biber J. Pflugers Arch. 1997; 433: 379-389Crossref PubMed Scopus (90) Google Scholar, 11Miyamoto K. Tatsumi S. Sonoda T. Yamamoto H. Minami H. Taketani Y. Takeda E. Biochem. J. 1995; 305: 81-85Crossref PubMed Scopus (66) Google Scholar, 12Miyamoto K. Segawa H. Morita K. Nii T. Tatsumi S. Taketani Y. Takeda E. Biochem. J. 1997; 327: 735-739Crossref PubMed Scopus (25) Google Scholar). The rat (NaPi-2), human (NaPi-3), and murine (NaPi-7) type II Na+/Pi cotransporter showed common characteristics in electrophysiological studies (13Busch A. Waldegger S. Herzer T. Biber J. Markovich D. Hayes G. Murer H. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8205-8208Crossref PubMed Scopus (74) Google Scholar, 14Hartmann C.M. Wagner C.A. Busch A.E. Markovich D. Biber J. Lang F. Murer H. Pflugers Arch. 1995; 430: 830-836Crossref PubMed Scopus (73) Google Scholar). The apparent K m for Pi and Na+in the type II Na/Pi transporter are in good agreement with previous Pi uptake studies in vesicles of renal brush-border membranes (15Loghman Adham M. Motock G.T. Wilson P. Levi M. Am. J. Physiol. 1995; 269: F93-F102PubMed Google Scholar, 16Katai K. Segawa H. Haga H. Morita K. Arai H. Tatsumi S. Taketani Y. Miyamoto K. Hisano S. Fukui Y. Takeda E. J. Biochem. (Tokyo). 1997; 121: 50-55Crossref PubMed Scopus (49) Google Scholar). In contrast, there are several conflicting results regarding the molecular structure and regulation of type II Na/Picotransporter (8Biber J. Custer M. Magagnin S. Hayes G. Werner A. Lotscher M. Kaissling B. Murer H. Kidney Int. 1996; 49: 981-985Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 17Werner A. Kempson S.A. Biber J. Murer H. J. Biol. Chem. 1994; 269: 6637-6639Abstract Full Text PDF PubMed Google Scholar, 18Levi M. Lotscher M. Sorribas V. Custer M. Arar M. Kaissling B. Murer H. Biber J. Am. J. Physiol. 1994; 267: F900-F908PubMed Google Scholar, 19Boyer C.J. Xiao Y. Dugre A. Vincent E. Delisle M.C. Beliveau R. Biochim. Biophys. Acta. 1996; 1281: 117-123Crossref PubMed Scopus (15) Google Scholar). Concerning the molecular structure of NaPi-2, its related proteins have been identified and partly characterized from the kidney proximal tubules of rat and mouse (19Boyer C.J. Xiao Y. Dugre A. Vincent E. Delisle M.C. Beliveau R. Biochim. Biophys. Acta. 1996; 1281: 117-123Crossref PubMed Scopus (15) Google Scholar). These proteins were analyzed with the Western blot technique using polyclonal antibodies raised against the C- and N-terminal proteins of the rat NaPi-2 as deduced from the nucleotide sequence of its cloned cDNA (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). A novel protein of 40 kDa (p40) was detected and appears to be derived from a protein of 75 kDa (p70), which is closer to the predicted molecular mass of 68.7 kDa deduced from NaPi-2 cDNA (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). The p40 and p70 proteins possessed similar physicochemical properties and p40 was regulated in the same fashion as p70 in rats given a low-Pi diet, suggesting that p40 may play an important role in the regulation of the renal Na/Pi cotransport system (19Boyer C.J. Xiao Y. Dugre A. Vincent E. Delisle M.C. Beliveau R. Biochim. Biophys. Acta. 1996; 1281: 117-123Crossref PubMed Scopus (15) Google Scholar). Similar results using polyclonal antibodies directed against the deduced N- and C-terminal amino acid sequences of NaPi-2 have been obtained by other workers (20Collins J.F. Bulus N. Ghishan F.K. Am. J. Physiol. 1995; 268: G917-G924PubMed Google Scholar). To address the molecular structure of renal Na+-dependent Pi cotransporter, we have cloned NaPi-2α, β, and γ which have partially conserved NaPi-2 cDNA. The present results suggest that NaPi-2α, β, and γ are splicing variants of the NaPi-2 gene and could modulate the function of NaPi-2 in renal proximal tubules. A cDNA library in vector λgt10 (5 × 105 independent recombinants) was constructed from rat kidney poly(A)+ RNA by oligo(dT)-primed cDNA synthesis (Life Technologies, Inc., Gaithersburg, MD). Plaques were screened by hybridization under low-stringency conditions as described previously (11Miyamoto K. Tatsumi S. Sonoda T. Yamamoto H. Minami H. Taketani Y. Takeda E. Biochem. J. 1995; 305: 81-85Crossref PubMed Scopus (66) Google Scholar). The32P-labeled rat NaPi-2 cDNA probes were used as the following set primers: 1) SA-AA; 2) SB-AB; 3) SC-AC, primer SA, 5′-CTGGACAAGTCTGTGATTACCAGC-3′ (nucleotide positions +834 to +857 relative to the translation start site of NaPi-2 cDNA); primer AA, 5′-CTCCCTGGGGCTGGCCAGTGCTGC-3′(nucleotide positions +1359 to +1382); primer SB, 5′-ATGATGTCCTACAGCGAGAGATTGG-3′ (nucleotide positions +1 to +25); primer AB, 5′-CACGTCCAGGGAGCAGACAAAGAGG-3′ (nucleotide positions +336 to +360); SC, 5′-CAGGCTGGCAGGCTATGGTCGGCTTG-3′ (nucleotide positions +1601 to +1626); and AC, 5′-GAGCCGGGTGGCATTGTGGTGAGC-3′ (nucleotide positions +1888 to +1911) (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). Positive clones of four types (NaPi-2, NaPi-2α, NaPi-2β, and NaPi-2γ) were isolated and subcloned into the EcoRI site of pBluescript II SK(+) and characterized by restriction mapping with proper restriction enzymes. Both strands of the cDNA inserts were sequenced by using vector-derived primers, and synthetic oligonucleotides derived from the cDNA sequence (21Taketani Y. Miyamoto K. Tanaka K. Katai K. Chikamori M. Tatsumi S. Segawa H. Yamamoto H. Morita K. Takeda E. Biochem. J. 1997; 324: 927-934Crossref PubMed Scopus (45) Google Scholar). NaPi-2 cDNA clones (pNaPi-2-A and pNaPi-2-B) were isolated containing 2440 and 2527 base pairs of insert, respectively. Sequencing data indicated that the two clones have the same open reading frame, encoding a 637-amino acid protein, but have different polyadenylation signals (data not shown). The sequence of the pNaPi-2-A clone was completely identical to that of the previous report (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). pNaPi-2-B is approximately 87 base longer in 3′-untranslated sequence upstream of its poly(A) signal. pNaPi-2-B was used for functional analysis. Kidneys were obtained from male Wistar rats (body weight, 170 to 200 g). The animals were anesthetized with Nembutal (50 mg/kg of body weight, intraperitoneally) and killed by aortic puncture. Total RNA was isolated from rat kidney cortex by acid guanidine thiocyanate/phenol/chloroform extraction (12Miyamoto K. Segawa H. Morita K. Nii T. Tatsumi S. Taketani Y. Takeda E. Biochem. J. 1997; 327: 735-739Crossref PubMed Scopus (25) Google Scholar). Total RNA samples were separated by electrophoresis on 1.2% agarose gels containing 2.2m formaldehyde and transferred to Hybond-N membranes (Amersham, Buckinghamshire, United Kingdom) and covalently cross-linked by exposure to UV light. We synthesized oligonucleotide primers specific for the NaPi-2, NaPi-2α, NaPi-2β, and NaPi-2γ cDNA sequences. These sequences of the upstream and downstream primers were: NaPi-2α, 5′-GTTCAGAGCCAGGTAAGACGATAC-3′(nucleotide positions +941 to +964 relative to the translation start site of NaPi-2α cDNA) and 5′-CAGCTCTTTGAAAGCCACTGGGCC-3′ (nucleotide positions +1139 to +1162 relative to the translation start site of NaPi-2α cDNA); NaPi-2β, 5′-GCAACCTCCTCTTCTGGCTTTGG-3′(nucleotide positions +647 to +669 relative to the translation start site of NaPi-2β) and 5′-GGTGCGCC GGTGCGCCCAGGCCAAACAGTGGG-3′(nucleotide positions +912 to +935 relative to the translation start site of NaPi-2β); NaPi-2γ, 5′-GGCCATATTCCTGAGGTATTTCG-3′(nucleotide positions −600 to −578 relative to the translation start site of NaPi-2γ) and 5′-CACAGCCTGGGGGCGGAGCTAAG-3′(nucleotide positions −469 to −447 relative to the start site of NaPi-2γ). Hybridization with the 32P-labeled NaPi-2-, NaPi-2α-, NaPi-2β-, and NaPi-2γ-specific cDNA probes was performed in a buffer containing 50% formamide, 5 × SSPE (0.15 mNaCl/10 mm sodium phosphate (pH 7.4), 1 mmEDTA), 2 × Denhardt's solution and 1% (w/v) SDS, after which the membranes were analyzed with a Fuji (Tokyo) BAS-2000 image analysis system. To isolate genomic DNA encoding NaPi-2, we synthesized oligonucleotide primers specific for the NaPi-2 cDNA sequence described by Magagnin et al.(3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). The sequences of the upstream and downstream primers were 5′-CGTGGTGCTTGTTAACGTCCTGCAG-3′ (nucleotide positions +1656 to +1680 relative to the translation start site of NaPi-2 cDNA) and 5′-CTAGAGCCGGGTGGCATTGTG-3′ (nucleotide positions +1891 to +1914 relative to the translation start site), respectively. Rat genomic DNA was subjected to polymerase chain reaction (PCR)1 amplification with the two primers and Taq DNA polymerase (Takara, Kyoto, Japan) for 30 cycles of denaturation at 95 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. The PCR product was subcloned into pBluescript II SK(+)(Stratagene, La Jolla, CA) with the TA cloning system (Promega, Madison, WI). The plasmid was digested with PstI and XhoI, and the released DNA fragment was labeled with [α-32P]dCTP (110 TBq/mmol)(ICN) by the Megaprime DNA labeling system (Amersham). We screened a genomic DNA library (CLONTECH, Palo Alto, CA) constructed in EMBL3 from fragments of rat kidney DNA generated by Sau3AI digestion. Plaques (1 × 108) were transferred to a nitrocellulose membrane (Hybond-C extra; Amersham), and hybridization and washing were performed as described previously (21Taketani Y. Miyamoto K. Tanaka K. Katai K. Chikamori M. Tatsumi S. Segawa H. Yamamoto H. Morita K. Takeda E. Biochem. J. 1997; 324: 927-934Crossref PubMed Scopus (45) Google Scholar). Positive clones were purified and DNA was extracted with the use of large-scale liquid cultures. A cosmid library (pWE15, CLONTECH) was used for the screening of the NaPi-2 gene. Colonies (1.5 × 106) were transformed on nylon-based membranes (Colony/Plaque ScreenTM, NEN Research Products, Boston, MA). Positive clones were isolated as described previously (22Miyamoto K. Kesterson R.A. Tamamoto H. Taketani Y. Nishiwaki E. Tatsumi S. Inoue Y. Morita K. Takeda E. Pike J.W. Mol. Endocrinol. 1997; 11: 1165-1179Crossref PubMed Scopus (270) Google Scholar). Plasmids encoding NaPi-2, NaPi-2α, NaPi-2β, and NaPi-2γ cDNA were linearized usingNotI and used for the in vitro synthesis of cRNA, using T7 RNA polymerase (11Miyamoto K. Tatsumi S. Sonoda T. Yamamoto H. Minami H. Taketani Y. Takeda E. Biochem. J. 1995; 305: 81-85Crossref PubMed Scopus (66) Google Scholar). Rat NaPi-2, NaPi-2α, NaPi-2β, and NaPi-2γ cDNA clones were subjected to in vitro translation in the presence or absence of pancreatic microsomes using a rabbit reticulocyte lysate translation system. In the absence of microsomes, the reaction was set up as follows: 1 μg of cRNA, 17.5 μl of rabbit reticulocyte lysate, 0.5 μl of amino acid mixture minus methionine (1 mm), 2.0 μl of [35S]methionine (1200 Ci/mmol), 1 μl of RNasin ribonuclease inhibitor (40 units/μl), and nuclease-free water up to 25 μl. In the presence of microsomes, 0.5 μg of cRNA, 17.5 μl of rabbit reticulocyte lysate, 0.5 μl of amino acid mixture minus methionine (1 mm), 2.0 μl of [35S]methionine (1200 Ci/mmol), 1 μl of RNasin ribonuclease, 1.8 μl of canine pancreatic microsomes, and nuclease-free water up to 25 μl. Both reactions were incubated at 37 °C for 90 min and then placed on ice. The samples were heated at 100 °C for 3 min and subjected to a 10% SDS-polyacrylamide gel electrophoresis (PAGE). For autoradiography, the gels were dried and exposed to x-ray film overnight at room temperature (23Pelham R.B. Jakson R.J. Eur. J. Biochem. 1976; 67: 247-256Crossref PubMed Scopus (2433) Google Scholar,24Walter P. Blobel G. Methods Enzymol. 1983; 96: 84-111Crossref PubMed Scopus (475) Google Scholar). Brush-border membrane vesicles (BBMVs) were prepared from the rat kidney by the Ca2+preparation method previously described (25Minami H. Kim K.R. Tada K. Takahashi F. Miyamoto K. Nakabou Y. Sakai K. Hagihira H. Gastroenterology. 1993; 105: 692-697Abstract Full Text PDF PubMed Scopus (23) Google Scholar). The antibodies were raised against a peptide that represented an amino acid sequence (Leu-Ala-Leu-Pro-Ala-His-His-Asn-Ala-Thr-Arg-Leu, amino acids 626–637) in the C-terminal region of NaPi-2 or an amino acid sequence (Met-Met-Ser-Tyr-Ser-Glu-Arg-Leu-Gly-Gly Pro-Ala-Val-Ser, amino acids 1–15) in the N-terminal region of NaPi-2 (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). An N-terminal cysteine residue was introduced for conjugation with keyhole limpet hemocyanine (Sigma) usingm-maleimidobenzoyl-N-hydroxysuccinimide ester. For the Western blot analysis, membrane proteins were separated by SDS-PAGE and electrotransferred onto nitrocellulose sheets (16Katai K. Segawa H. Haga H. Morita K. Arai H. Tatsumi S. Taketani Y. Miyamoto K. Hisano S. Fukui Y. Takeda E. J. Biochem. (Tokyo). 1997; 121: 50-55Crossref PubMed Scopus (49) Google Scholar). The samples were mixed with sample buffer containing a final concentration of 20% (w/v) glycerol, 1% (w/v) SDS, 0.05% bromphenol blue, and 0.625 m Tris-HCl (pH 6.8), with or without 5 mm dithiothreitol. Xenopus laevis females were obtained from Hamamatsu Jikkenn (Shizuoka, Japan). Small clumps of oocytes were treated twice for 90 min with 2 mg/ml collagenase in a Ca+-free solution (ORII solution: 82.5 mm NaCl, 2 mm KCl, mmMgCl2, 10 mm Hepes/Tris, pH 7.5) in order to remove the follicular membrane (11Miyamoto K. Tatsumi S. Sonoda T. Yamamoto H. Minami H. Taketani Y. Takeda E. Biochem. J. 1995; 305: 81-85Crossref PubMed Scopus (66) Google Scholar). After extensive washing, first with ORII solution and then with modified Barth's solution overnight at 18 °C, healthy oocytes were injected with cRNA (dissolved in water at concentrations from 1 mg/ml) or water using a manual injector (Narishige, Tokyo, Japan). Twenty oocytes were washed in Na+ containing uptake solution (96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm Hepes/Tris, pH 7.5, 0.1 mM KH2PO4 (10 μCi/ml)) for 60 min at room temperature, and the other oocytes (20Collins J.F. Bulus N. Ghishan F.K. Am. J. Physiol. 1995; 268: G917-G924PubMed Google Scholar) were used for the measurement of Na+-independent uptake, with incubation as above but substituting 100 mm choline for NaCl. We screened a rat kidney cDNA library by using N- and C-terminal-specific cDNA fragments as described under “Experimental Procedures.” Eight cDNA clones were obtained and sequenced. Three clones, termed NaPi-2α encoded the N-terminal region of NaPi-2, showing the amino acid sequence in Fig. 1 A. NaPi-2α cDNA was 2389 bp in length, including 1011 bp of the open reading frame which encodes 337 amino acids. NaPi-2α was found to have high homology to the N-terminal region of NaPi-2, but 25 amino acids in the C-terminal region of NaPi-2α had a quite different. The putative structure showed that this clone has three trans-membrane domains and one glycosylation site (Figs. 2 and 3).Figure 1Nucleotide and amino acid sequences of NaPi-2-related cDNA clones. A, nucleotide and amino acid sequences of NaPi-2α cDNA. B, nucleotide and amino acid sequences of NaPi-2β cDNA. C, nucleotide and amino acid sequences of NaPi-2γ cDNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Comparison of amino acid sequences between NaPi-2 and NaPi-2α, β, and γ. A, comparison of amino acid sequence of NaPi-2 and NaPi-2α. B, comparison of amino acid sequence of NaPi-2 and NaPi-2β. C,comparison of amino acid sequence of NaPi-2 and NaPi-2γ.Dots in the NaPi-2 sequence indicate amino acids identical to those of NaPi-2αβγ. Boxes indicate a novel sequence in NaPi-2α or β.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Membrane models of NaPi-2α, β, and γ. A, putative membrane spanning regions (M1-M8) are depicted as cylinders. Putative N-glycosylation sites and protein kinase C sites are marked. The leucine zipper motif was marked as L-L-L. In the locations of the intra- and extracellular loops of M4-M8, NaPi-2γ is reversed to NaPi-2. NaPi-2α has three transmembrane domains and one phosphorylation site. NaPi-2β has three transmembrane domains and a leucine-zipper motif in the C-terminal region. NaPi-2γ has four transmembrane domains and one glycosylation site. B, comparison of leucine zipper motifs of NaPi-2 and NaPi-2β. NaPi-2β is a novel leucine zipper motif in the fourth transmembrane domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The NaPi-2β cDNA was 1166 bp. The open reading frame was 981 bp and encoded 327 amino acids (Fig. 1 B). The 174-amino acid in the N-terminal region of NaPi-2β were identical to those of NaPi-2, while the 153-amino acid region in the C-terminal region of NaPi-2β were quite different from those of NaPi-2. However, the putative structure of NaPi-2β showed four transmembrane domains and a leucine zipper motif in the third transmembrane domain (Figs. 2 and 3). NaPi-2γ cDNA was 1961 bp. The open reading frame was 804 bp encoding 268 amino acids (Fig. 1 C). The hydropathy plot analysis shows that NaPi-2γ has four transmembrane protein and one glycosylation site, and one protein kinase C phosphorylation site in the intracellular domain. NaPi-2γ was completely identical to the NaPi-2 C-terminal region of 268 amino acids (Figs. 2 and 3). We screened approximately 1.0 × 108 plaques of a human genomic DNA library and detected three positive clones. These clones were purified and are referred to as λNP-1, λNP-2, and λNP-3. DNA from the three clones was further analyzed by restriction enzyme mapping, and the insert lengths were estimated to be about 15 and 14 kb, respectively. λNP-1 was identical to λNP-3. A partial sequence analysis and Southern blot hybridization revealed that both λNP-1 and λNP-2 encompass the entire coding region of the NaPi-2 cDNA. Clone λNP-1 also contained an extensive 5′-flanking region, whereas λNP-2 contained only exon 9 to exon 13 of the NaPi-2 gene (Fig. 4). Similarly, we have isolated a cosmid clone (CNP-1) which has about 40 kb of the NaPi-2 gene. In this clone, we also mapped the phage clone and each exon (data not shown). The sequences of the intron-exon junctions between exons 1 and 13 are compatible with the consensus sequences (AG-GT) for splicing junctions. Sequence data were obtained by analyzing the DNA insert of clone λNP-1 and the PCR products of this clone obtained with primers based on the cDNA sequence. Intron sizes were estimated by comparison either with a DNA size marker on agarose gel electrophoresis after digestion with restriction enzymes or with PCR products (data not shown). The detailed analysis of the gene mapping showed that NaPi-2α uses 1400 bp of intron 8 as the exon and that this region also encodes the 3′-noncoding region. NaPi-2β also uses exon 1 to exon 5, and skips exon 6 to exon 13. The mapping of NaPi-2β in the cosmid clone indicated that the new exon of the 3′-region of NaPi-2β is used ∼10 kb downstream of exon 13 of the NaPi-2 gene. The gene structure of NaPi-2 γ is more unique. The 5′-untranslated sequence is mapped at intron 8. The putative capping site of NaPi-2γ cDNA is present 278 bp upstream of the exon/intron junction of exon 9. NaPi-2 γ uses intron 9 of the NaPi-2 gene as the 5′-untranslational region. The 3′-nontranslational region of NaPi-2γ used exon 13 of the NaPi-2 gene. To identify the transcripts of each isoform, the specific sequence was chosen in each clone. We determined the size of each transcript for the NaPi-2 isoforms (Fig. 5). The NaPi-2 cDNA full-length probe hybridized to four transcripts (9.5, 4.6, 2.6, and 1.2 kb). The specific probe for NaPi-2α hybridized at 9.5 and 4.6 kb. The NaPi-2β probe was hybridized at 1.2 kb. In contrast, NaPi-2γ was hybridized at 9.5 and 2.6 kb. As shown in Fig. 6, we performed in vitrotranslation for NaPi-2α, β, and γ cRNAs. In rabbit reticulocyte lysate, NaPi-2α was 36 kDa, NaPi-2β was 36 kDa, and NaPi-2γ was 29 kDa in SDS-PAGE. In the presence of microsome membrane, NaPi-2α and NaPi-2γ were glycosylated and migrated to the following molecular masses: NaPi-2α migrated to 45 kDa and NaPi-2γ migrated to 35 kDa, but NaPi-2β was not glycosylated. To detect the presence of the NaPi-2α, β, and γ isoforms in BBMV isolated from rat renal proximal tubular cells, a Western blot analysis was carried out using the N- and C-terminal specific antibodies as described under “Experimental Procedures” (Fig. 7). The C-terminal antibodies reacted with 180, 70–90, and 37 kDa proteins in the presence of a reducing regent (+ dithiothreitol). The 37-kDa protein band was most pronounced and disappeared in the absence of the reducing regent. In contrast, the N-terminal antibodies could react with 90-, 45-, and 40-kDa proteins in the presence of the reducing regent (+dithiothreitol). In the absence of the reducing regent, we did not detect the 45- and 40-kDa proteins, but a prominent 180-kDa protein appeared. In the case of the type I Na/Picotransporter RNaPi-1, we could not detect the dissociation of the protein band (66 kDa) regardless of the presence or absence of reducing regent. To elucidate the functional roles of NaPi-2α, β, and γ, we analyzed Na+/Pi transport activity in Xenopus oocytes (Fig. 8).In vitro transcribed RNA of these isoforms was injected intoX. laevis oocytes either separately or combined in equimolar proportions. When 5 ng of NaPi-2 cRNA was microinjected intoXenopus oocytes, the Na+-dependent Pi transport activity was stimulated to an approximate 40-fold increase compared with that in the water-injected controls. Na+/Pi cotransport activity was not observed in Xenopus oocytes expressing NaPi-2α, β, or γ cRNA alone. The co-injection of NaPi-2α and NaPi-2 γ, or of NaPi-2β and NaPi-2γ, had no effect on the Na+/Picotransport activity. In addition, the Pi transport activity in Xenopus oocytes co-expressing NaPi-2 and NaPi-2α was suppressed compared with that in oocytes microinjected with NaPi-2 alone. In contrast, the Pi transport activity in Xenopus oocytes co-injected with NaPi-2 and NaPi-2γ was completely suppressed to that in oocytes microinjected with NaPi-2 alone. In the present study, we isolated three types of NaPi-2-related cDNA clones. The mapping of each cDNA clone showed that the novel sequence is present in the isolated λ clone including the NaPi-2 gene. A Northern blot analysis showed that each transcript of the cDNAs is consistent with the four transcripts hybridized by NaPi-2 cDNA, suggesting that these three NaPi-2-related clones are splicing variants of the NaPi-2 gene. In addition, in the in vitro translation analysis, NaPi-2α, β, and γ were revealed at 36, 36, and 29 kDa, respectively. These protein sizes were increased when the reaction was added to the mixture of microsomal membrane; the protein sizes of glycosylated NaPi-2α, β, and γ were 45, 36, and 35 kDa, respectively. Indeed, the sizes of the in vitro translation products and glycosylated proteins were very similar to those in the BBMV proteins (45, 40, and 35 kDa) reacted with NaPi-2 N- or C-terminal antibodies. Previous studies with polyclonal antibodies have demonstrated that NaPi-2 is present as proteins of 80–90 kDa in the rat renal BBM (20Collins J.F. Bulus N. Ghishan F.K. Am. J. Physiol. 1995; 268: G917-G924PubMed Google Scholar). Under reducing conditions, additional proteins of 45–49 (p45) and 40 kDa (p40) were detected with N-terminal and C-terminal antibodies, respectively (19Boyer C.J. Xiao Y. Dugre A. Vincent E. Delisle M.C. Beliveau R. Biochim. Biophys. Acta. 1996; 1281: 117-123Crossref PubMed Scopus (15) Google Scholar, 27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar). Beliveau and co-workers (19Boyer C.J. Xiao Y. Dugre A. Vincent E. Delisle M.C. Beliveau R. Biochim. Biophys. Acta. 1996; 1281: 117-123Crossref PubMed Scopus (15) Google Scholar, 27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar) demonstrated that p40 and p45 have been shown to be glycosylated and up-regulated by a low Pi diet. The amounts of NaPi-2α,β mRNA were significantly increased in rats fed a low Pi diet compared with those in rats fed a normal Pi diet (data not shown). These results suggest that NaPi-2α and NaPi-2γ may correspond to p45 and p40, respectively. In the present study, the N-terminal antibodies were reacted with 40-kDa protein, in addition to 45-kDa protein described previously (27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar). The 40-kDa protein was not detected in the BBMV isolated from rats fed a high Pi diet (data not shown). The size of NaPi-2β in the BBMV (40 kDa) is larger than the predicted 36 kDa. It is possible that the protein detected in the BBMV is not a product of NaPi-2β. To clarify the presence of NaPi-2β, we performed Western blotting experiments with antibodies directed against the region of these isoforms that are not conserved. We evaluated the generation of the specific antibodie recognition of 3 different epitopes (C-terminal region) of NaPi-2β. The antibodies obtained from rabbit immunized with the peptides did not produce any positive results (data not shown). Further study is needed to clarify the presence of NaPi-2β protein in the BBMV, in addition to the characterization of the 40-kDa protein. In a previous study, we isolated the human NaPi-3 gene and characterized its structure (21Taketani Y. Miyamoto K. Tanaka K. Katai K. Chikamori M. Tatsumi S. Segawa H. Yamamoto H. Morita K. Takeda E. Biochem. J. 1997; 324: 927-934Crossref PubMed Scopus (45) Google Scholar). The structure of the rat NaPi-2 gene was highly similar to those of the human NaPi-3 and mouse NaPi-7 genes (21Taketani Y. Miyamoto K. Tanaka K. Katai K. Chikamori M. Tatsumi S. Segawa H. Yamamoto H. Morita K. Takeda E. Biochem. J. 1997; 324: 927-934Crossref PubMed Scopus (45) Google Scholar, 26Hartmann C.M. Hewson A.S. Kos C.H. Hilfiker H. Soumounou Y. Murer H. Tenenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7409-7414Crossref PubMed Scopus (50) Google Scholar). The 13 exons were mapped in the two λ phages and one cosmid clone. The gene structural feature showed that the large intron is present between exon 8 and exon 9 among three species: human, mouse, and rat (21Taketani Y. Miyamoto K. Tanaka K. Katai K. Chikamori M. Tatsumi S. Segawa H. Yamamoto H. Morita K. Takeda E. Biochem. J. 1997; 324: 927-934Crossref PubMed Scopus (45) Google Scholar, 26Hartmann C.M. Hewson A.S. Kos C.H. Hilfiker H. Soumounou Y. Murer H. Tenenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7409-7414Crossref PubMed Scopus (50) Google Scholar). Kohl et al. (28Kohl B. Hülseweh B. Strundk U. Werner A. FASEB J. 1996; 10: A90Google Scholar) recently reported that the flounder type II transporter gene was divided by two independent genes. The hydrophobic analysis of NaPi-2 predicted eight transmembrane regions (3Magagnin S. Werner A. Markovich D. Sorribas V. Stange G. Biber J. Murer H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5979-5983Crossref PubMed Scopus (332) Google Scholar). Obviously, the membrane spanning regions 1 to 3 and 4 to 8 are separated by a large hydrophilic loop into two distinct domains (27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar, 28Kohl B. Hülseweh B. Strundk U. Werner A. FASEB J. 1996; 10: A90Google Scholar). This division is reflected on the genomic level regarding the exon/intron organization. Kohl et al. (28Kohl B. Hülseweh B. Strundk U. Werner A. FASEB J. 1996; 10: A90Google Scholar) suggest the presence of the duplicated gene products in renal proximal tubular cells. Indeed, NaPi-2α is splicing products from exon 1 to exon 8, and NaPi-2γ is from 9 to 13 of the NaPi-2 gene. Xiao et al. (27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar) suggested that the lower weight proteins (40–45 kDa) resulted from a specific post-translational proteolytic cleavage of the NaPi-2 polypeptide and that the cleavage site could thus be located between Asn-298 and Asn-328, which have been shown to constitute the only two N-glycosylated residues in NaPi-2 (29Hayes G. Busch A. Lotscher M. Waldegger S. Lang F. Verrey F. Biber J. Murer H. J. Biol. Chem. 1994; 269: 24143-24149Abstract Full Text PDF PubMed Google Scholar). Kohl et al. (30Kohl B. Wagner C.A. Hülseweh B. Busch A.E. Werner A. J. Physiol. 1998; 508: 341-350Crossref PubMed Scopus (35) Google Scholar) tested the functional consequences of an interrupted protein backbone in the type II Na/Picotransporter as proposed by Xiao et al. (27Xiao Y. Boyer C.J. Vincent E. Dugre A. Vachon V. Potier M. Beliveau R. Biochem. J. 1997; 323: 401-408Crossref PubMed Scopus (22) Google Scholar). The fragments were denoted 1–3 plus 4–8, and 1–5 plus 6–8 referring to the putative membrane-spanning segments in the proposed topological model of type II Na/Pi cotransporter. The in vitrotranslation experiments prove the integrity of the different cRNAs resulting in correctly translated protein fragments. However, none of the truncated transporters of the 1–5 plus 6–8 combination was efficiently processed in Xenopus oocytes. The coexpression experiments revealed that the complementing fragments 1–3 plus 4–8 could stabilize each other resulting in proper membrane delivery (30Kohl B. Wagner C.A. Hülseweh B. Busch A.E. Werner A. J. Physiol. 1998; 508: 341-350Crossref PubMed Scopus (35) Google Scholar). This implies a direct interaction of the two cognate constructs and correct folding of the individual fragments. This assumption is supported by the functional integrity of the combined fragments 1–3 and 4–8 (30Kohl B. Wagner C.A. Hülseweh B. Busch A.E. Werner A. J. Physiol. 1998; 508: 341-350Crossref PubMed Scopus (35) Google Scholar). In vitro transcribed RNA of these isoforms was injected intoX. laevis oocytes either separately or combined in equimolar proportions and assayed for Pi transport. However, we failed to detect the enhancement of Pi uptake in Xenopus oocytes co-expressing NaPi-2α/β and NaPi-2γ (1–3 plus 5–8/1–4 plus 5–8 referring to the putative membrane-spanning segments). In contrast, the co-injection of NaPi-2 and NaPi-2γ into Xenopus oocytes completely inhibited the Na+-dependent Pi uptake. NaPi-2α also partially inhibited the Pi uptake. However, NaPi-2β did not affect NaPi-2 function. These results suggest that NaPi-2 αγ are dominant negative inhibitors of NaPi-2 rather than a functional complex from two independent isoforms. Indeed, in hypophosphatemic mice (Hyp), Pi deprivation caused an 8-fold increase in immunoreactive type II transporter protein at the BBMV, but Na/Pi cotransport activity was a 2-fold increase in the BBMV, suggesting that the majority of this BBM protein is inactive (20Collins J.F. Bulus N. Ghishan F.K. Am. J. Physiol. 1995; 268: G917-G924PubMed Google Scholar). The finding of the dominant negative isoforms of Na/Pi type II transporters may have important physiopathological implications in X-linked hypophosphatemia. In our analysis of NaPi-2α, β, and γ, NaPi-2α was found to have three transmembrane domains and involve Asn-298, but not Asn-328. However, NaPi-2γ did not encode the region including Asn-328. On the basis of the NaPi-2 putative membrane structure, the extracellular domains of NaPi-2γ has no glycosylation site. However, the glycosylation experiment demonstrated that NaPi-2γ is glycosylated by microsomal membrane, suggesting that the structure of NaPi-2γ may be in reverse orientation to that of NaPi-2. Using immunohistochemical approaches we are now determining whether epitopes are located in the intra- or extracellular compartments. In addition, X-linked hypophosphatemia and hereditary hypophosphatemic rickets with hypercalciuria (HHRH) are Mendelian disorders of Pi homeostasis characterized by rachitic bone disease, hypophosphatemia, and impaired renal Pi reabsorption (31Tenenhouse H. J. Bone Miner. Res. 1997; 12: 159-164Crossref PubMed Scopus (56) Google Scholar). The mutant gene in patients with X-linked hypophosphatemia has recently been identified by positional cloning and was designated PEX to signify a Pi-regulating gene with homology to endopeptidases that maps to the X chromosome (32The Hyp Consortium Nat. Genet. 1995; 11: 130-136Crossref PubMed Scopus (963) Google Scholar). In contrast, the molecular basis for the renal defect in Pi reabsorption in HHRH has not yet been addressed. However, a recent study suggested that HHRH arises from a primary defect in the Na/Pi transporter (33Beck L. Karaplis A.C. Amizuka N. Hewson A.S. Ozawa H. Tenenhouse H.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5372-5377Crossref PubMed Scopus (517) Google Scholar). We have analyzed the human NaPi-3 gene, but could not find any mutation in Japanese HHRH patients. The presence of regulatory proteins such as NaPi-2α, β, and γ may be helpful to resolve the molecular basis of HHRH. Finally, we investigated the molecular structure and regulation of the type II Na/Pi cotransporter. NaPi-2α, β, and γ have partially conserved NaPi-2 cDNA and are splicing variants of the NaPi-2 gene. These NaPi-2-related proteins may modulate the function of NaPi-2 in the renal proximal tubules. We are grateful to Drs. H. Murer and J. Biber for providing rat NaPi-2 cDNA clone.