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J Biol Chem, Vol. 273, Issue 44, 28568-28575, October 30, 1998
From the Department of Clinical Nutrition, School of Medicine,
Tokushima University, Tokushima 770, Japan
We have isolated three unique NaPi-2-related
protein cDNAs (NaPi-2 A functional analysis demonstrated that NaPi-2 Renal phosphate (Pi) reabsorption is an essential
aspect of the maintenance of plasma Pi homeostasis (1).
Several mammalian renal Na+-dependent
Pi cotransporters have recently been isolated and well characterized (2-7). 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 (8-12). The rat (NaPi-2), human (NaPi-3), and murine
(NaPi-7) type II Na+/Pi cotransporter showed
common characteristics in electrophysiological studies (13, 14). The
apparent Km 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 (15, 16).
In contrast, there are several conflicting results regarding the
molecular structure and regulation of type II Na/Pi
cotransporter (8, 17-19). 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 (19).
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 (3). 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 (3).
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
(19). 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 (20).
To address the molecular structure of renal
Na+-dependent Pi cotransporter, we
have cloned NaPi-2 cDNA Library Construction and Screening--
A cDNA
library in vector
Positive clones of four types (NaPi-2, NaPi-2 RNA Isolation and Northern Blot 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 (12). Total RNA samples were
separated by electrophoresis on 1.2% agarose gels containing 2.2 M 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-2
Hybridization with the 32P-labeled NaPi-2-, NaPi-2 Cloning Cosmid and Phage Clones--
To isolate genomic DNA
encoding NaPi-2, we synthesized oligonucleotide primers specific for
the NaPi-2 cDNA sequence described by Magagnin et al.
(3). 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 [
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 (21). 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 (22).
In Vitro cRNA Synthesis--
Plasmids encoding NaPi-2,
NaPi-2 In Vitro Translation of cRNA--
Rat NaPi-2, NaPi-2 Western Blot Analysis--
Brush-border membrane vesicles
(BBMVs) were prepared from the rat kidney by the Ca2+
preparation method previously described (25). 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 (3). An N-terminal cysteine
residue was introduced for conjugation with keyhole limpet hemocyanine
(Sigma) using
m-maleimidobenzoyl-N-hydroxysuccinimide ester.
For the Western blot analysis, membrane proteins were separated by
SDS-PAGE and electrotransferred onto nitrocellulose sheets (16). 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.
Oocyte Injections and Transport Assay
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 (20) were used for the measurement
of Na+-independent uptake, with incubation as above but
substituting 100 mM choline for NaCl.
Isolation of NaPi-2
The NaPi-2
NaPi-2 Identification of the NaPi-2 Gene--
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
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).
Mapping of the Sequences of NaPi-2
The detailed analysis of the gene mapping showed that NaPi-2 Northern Blot Analysis of NaPi-2 In Vitro Synthesis of NaPi-2 Detection of NaPi-2 Functional Analysis of NaPi-2 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 In addition, in the in vitro translation analysis,
NaPi-2 In a previous study, we isolated the human NaPi-3 gene and
characterized its structure (21). The structure of the rat NaPi-2 gene
was highly similar to those of the human NaPi-3 and mouse NaPi-7 genes
(21, 26). The 13 exons were mapped in the two Xiao et al. (27) 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
(29). Kohl et al. (30) tested the functional consequences of
an interrupted protein backbone in the type II Na/Pi
cotransporter as proposed by Xiao et al. (27). 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 vitro
translation 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 (30).
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 (30).
In vitro transcribed RNA of these isoforms was injected into
X. 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-2 In our analysis of NaPi-2 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 (31).
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 (32). 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 (33). 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-2 Finally, we investigated the molecular structure and regulation of the
type II Na/Pi cotransporter. NaPi-2 We are grateful to Drs. H. Murer and J. Biber
for providing rat NaPi-2 cDNA clone.
*
This work were supported in part by grants-in-aid from the
Ministry of Education, Science, Sports and Culture of Japan, and Grants-in-Aid from Setsuro Fujii Memorial Foundation, and Salt Science
Research Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB013453, AB013454, AB013455.
The abbreviations used are:
PCR, polymerase
chain reaction; BBMV, brush-border membrane vesicle; Pi, inorganic phosphate; bp, base pair(s); kb, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; HHRH, hereditary hypophosphatemic
rickets with hypercalciuria.
Identification of Three Isoforms for the
Na+-dependent Phosphate Cotransporter (NaPi-2)
in Rat Kidney*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, NaPi-2
, and NaPi-2
) from a rat
kidney library. NaPi-2 cDNA encodes 337 amino acids which have
high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2 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-2 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 , , and were found to be
36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2 and
NaPi-2 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.
and -2 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.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, , and which have partially conserved
NaPi-2 cDNA. The present results suggest that NaPi-2, , and
are splicing variants of the NaPi-2 gene and could modulate the
function of NaPi-2 in renal proximal tubules.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
gt10 (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 (11). The 32P-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) (3).
, NaPi-2, and
NaPi-2) 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 (21). 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 (3). 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.
, NaPi-2, and NaPi-2 cDNA
sequences. These sequences of the upstream and downstream primers were:
NaPi-2, 5'-GTTCAGAGCCAGGTAAGACGATAC-3'(nucleotide positions +941 to
+964 relative to the translation start site of NaPi-2 cDNA) and
5'-CAGCTCTTTGAAAGCCACTGGGCC-3' (nucleotide positions +1139 to +1162
relative to the translation start site of NaPi-2 cDNA);
NaPi-2, 5'-GCAACCTCCTCTTCTGGCTTTGG-3'(nucleotide positions +647 to
+669 relative to the translation start site of NaPi-2) and
5'-GGTGCGCC GGTGCGCCCAGGCCAAACAGTGGG-3'(nucleotide positions +912 to
+935 relative to the translation start site of NaPi-2); NaPi-2,
5'-GGCCATATTCCTGAGGTATTTCG-3'(nucleotide positions 
600 to 578
relative to the translation start site of NaPi-2) and
5'-CACAGCCTGGGGGCGGAGCTAAG-3'(nucleotide positions 469 to 447
relative to the start site of NaPi-2).
-,
NaPi-2-, and NaPi-2-specific cDNA probes was performed in a
buffer containing 50% formamide, 5 × SSPE (0.15 M
NaCl/10 mM sodium phosphate (pH 7.4), 1 mM
EDTA), 2 × Denhardt's solution and 1% (w/v) SDS, after which
the membranes were analyzed with a Fuji (Tokyo) BAS-2000 image analysis
system.
-32P]dCTP (110 TBq/mmol)(ICN) by the Megaprime DNA labeling system (Amersham).
, NaPi-2, and NaPi-2 cDNA were linearized using
NotI and used for the in vitro synthesis of cRNA,
using T7 RNA polymerase (11).
,
NaPi-2, and NaPi-2 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 (23,
24).
--
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, mM
MgCl2, 10 mM Hepes/Tris, pH 7.5) in order to
remove the follicular membrane (11).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, , and cDNAs from Rat
Kidney cDNA Library--
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-2 encoded the
N-terminal region of NaPi-2, showing the amino acid sequence in Fig.
1A. NaPi-2 cDNA was
2389 bp in length, including 1011 bp of the open reading frame which encodes 337 amino acids. NaPi-2 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-2 had a quite different. The putative structure
showed that this clone has three trans-membrane domains and one
glycosylation site (Figs. 2 and
3).
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Fig. 1.
Nucleotide and amino acid sequences of
NaPi-2-related cDNA clones. A, nucleotide and amino
acid sequences of NaPi-2 cDNA. B, nucleotide and
amino acid sequences of NaPi-2 cDNA. C, nucleotide
and amino acid sequences of NaPi-2 cDNA.
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Fig. 2.
Comparison of amino acid sequences between
NaPi-2 and NaPi-2 , , and . A, comparison of
amino acid sequence of NaPi-2 and NaPi-2. B, comparison
of amino acid sequence of NaPi-2 and NaPi-2. C,
comparison of amino acid sequence of NaPi-2 and NaPi-2.
Dots in the NaPi-2 sequence indicate amino acids identical
to those of NaPi-2. Boxes indicate a novel sequence
in NaPi-2 or .
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Fig. 3.
Membrane models of NaPi-2 , , and
. 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-2 is reversed to NaPi-2. NaPi-2 has three
transmembrane domains and one phosphorylation site. NaPi-2 has three
transmembrane domains and a leucine-zipper motif in the C-terminal
region. NaPi-2 has four transmembrane domains and one glycosylation
site. B, comparison of leucine zipper motifs of NaPi-2 and
NaPi-2. NaPi-2 is a novel leucine zipper motif in the fourth
transmembrane domain.
cDNA was 1166 bp. The open reading frame was 981 bp
and encoded 327 amino acids (Fig. 1B). The 174-amino acid in
the N-terminal region of NaPi-2 were identical to those of NaPi-2,
while the 153-amino acid region in the C-terminal region of NaPi-2
were quite different from those of NaPi-2. However, the putative
structure of NaPi-2 showed four transmembrane domains and a leucine
zipper motif in the third transmembrane domain (Figs. 2 and 3).
cDNA was 1961 bp. The open reading frame was 804 bp
encoding 268 amino acids (Fig. 1C). The hydropathy plot
analysis shows that NaPi-2 has four transmembrane protein and one
glycosylation site, and one protein kinase C phosphorylation site in
the intracellular domain. NaPi-2 was completely identical to the
NaPi-2 C-terminal region of 268 amino acids (Figs. 2 and 3).
NP-1, NP-2, and NP-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. NP-1
was identical to NP-3. A partial sequence analysis and Southern blot
hybridization revealed that both NP-1 and NP-2 encompass the
entire coding region of the NaPi-2 cDNA. Clone NP-1 also
contained an extensive 5'-flanking region, whereas NP-2 contained
only exon 9 to exon 13 of the NaPi-2 gene (Fig.
4).
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Fig. 4.
Gene localization of NaPi-2 and NaPi-2 ,
, and transcripts. A, organizational map of the
complete NaPi-2 gene. A human placenta cosmid and phage library were
screened with a NaPi-2 cDNA probe to obtain two clones (NP-1 and
NP-2) that spanned the entire rat NaPi-2 gene. Restriction mapping
and Southern blot analysis revealed the overlapping nature of the
clones as well as the relative position of each exon within the clone
as indicated. B, mapping of NaPi-2, , and transcripts. The NaPi-2, , and -specific DNA probes are
described under "Experimental Procedures." The novel sequence
region which is not NaPi-2 cDNA is shown by hatched
boxes.
, , and cDNA--
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 NP-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).
uses
1400 bp of intron 8 as the exon and that this region also encodes the
3'-noncoding region. NaPi-2 also uses exon 1 to exon 5, and skips
exon 6 to exon 13. The mapping of NaPi-2 in the cosmid clone
indicated that the new exon of the 3'-region of NaPi-2 is used ~10
kb downstream of exon 13 of the NaPi-2 gene. The gene structure of
NaPi-2 is more unique. The 5'-untranslated sequence is mapped at
intron 8. The putative capping site of NaPi-2 cDNA is present
278 bp upstream of the exon/intron junction of exon 9. NaPi-2 uses
intron 9 of the NaPi-2 gene as the 5'-untranslational region. The
3'-nontranslational region of NaPi-2 used exon 13 of the NaPi-2
gene.
, , and --
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-2 hybridized at 9.5 and 4.6 kb.
The NaPi-2 probe was hybridized at 1.2 kb. In contrast, NaPi-2
was hybridized at 9.5 and 2.6 kb.
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Fig. 5.
Northern blot analysis of NaPi-2 , , and
transcripts in rat kidney cortex. Kidneys were obtained from
male Wistar rats fed a low-Pi diet. Total RNA from the
kidneys was prepared as described previously (23). Twenty µg of total
RNA was denatured, fractionated by electrophoresis on a 1.2% agarose
gel containing formaldehyde, transferred to a nylon membrane, and
subjected to hybridization with randomly primed,
32P-labeled NaPi-2, , and -specific cDNA
probes.
, , and --
As shown in
Fig. 6, we performed in vitro
translation for NaPi-2, , and cRNAs. In rabbit reticulocyte
lysate, NaPi-2 was 36 kDa, NaPi-2 was 36 kDa, and NaPi-2 was
29 kDa in SDS-PAGE. In the presence of microsome membrane, NaPi-2
and NaPi-2 were glycosylated and migrated to the following molecular
masses: NaPi-2 migrated to 45 kDa and NaPi-2 migrated to 35 kDa,
but NaPi-2 was not glycosylated.
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Fig. 6.
In vitro translation of NaPi-2 , ,
and rat NaPi-2, NaPi-2, NaPi-2 and NaPi-2 cDNA clones
were subjected to in vitro translation in the presence or
absence of pancreatic microsomes using a rabbit reticulocyte lysate
translation system. The samples were heated at 100 °C for 3 min
and subjected to a 10% SDS-PAGE. For autoradiography, the gels were
dried and exposed to x-ray film overnight at room temperature.
A, NaPi-2: lane 1, added microsomal membrane;
lane 2, no microsomal membrane fraction. B,
NaPi-2: lane 1, no microsomal membrane; lane
2, added microsomal membrane. C, NaPi-2: lane
1, no microsomal membrane; lane 2, added microsomal
membrane. D, NaPi-2: lane 1, no microsomal
membrane; lane 2, added microsomal membrane.
, , and in Isolated Brush-border
Membrane Vesicles from Rat Kidney Cortex--
To detect the presence
of the NaPi-2, , and 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/Pi
cotransporter RNaPi-1, we could not detect the dissociation of the
protein band (66 kDa) regardless of the presence or absence of
reducing regent.
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Fig. 7.
Western blot analysis of the
brush-border membrane vesicles from rat kidney proximal tubule.
The proteins were incubated with polyclonal antibodies raised against
synthetic C- and N-terminal NaPi-2 peptides (C-peptide and N-peptide).
For peptide protection, the peptides were included as indicated.
Labeled antigens were revealed by autoradiography with ECL Western
blotting solution. Comparable data were obtained in three independent
experiments performed in duplicate. DTT,
dithiothreitol.
, , and in Xenopus
Oocytes--
To elucidate the functional roles of NaPi-2, , and
, we analyzed Na+/Pi transport activity in
Xenopus oocytes (Fig. 8).
In vitro transcribed RNA of these isoforms was injected into
X. laevis oocytes either separately or combined in equimolar
proportions. When 5 ng of NaPi-2 cRNA was microinjected into
Xenopus 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-2, , or cRNA
alone. The co-injection of NaPi-2 and NaPi-2 , or of NaPi-2
and NaPi-2, had no effect on the Na+/Pi
cotransport activity. In addition, the Pi transport
activity in Xenopus oocytes co-expressing NaPi-2 and
NaPi-2 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-2 was
completely suppressed to that in oocytes microinjected with NaPi-2
alone.
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Fig. 8.
Functional assay of NaPi-2 , , and in Xenopus oocytes. Plasmid encoding NaPi-2, and
NaPi-2, NaPi-2, and NaPi-2 cDNA were linearized using
NotI and used for the in vitro synthesis of cRNA.
Five nanograms of each cRNA were microinjected into healthy oocytes.
After 3 days, 20 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. , absence; +, presence. Data are the mean ± S.E.
(n = 10) *p < 0.01, **p < 0.001 compared with NaPi-2 cRNA injection. 
, sodium dependent; 
,
sodium independent.
DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References
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.
, , and 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-2, , and 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 (20). Under reducing conditions, additional proteins of 45-49 (p45) and 40 kDa (p40) were detected with N-terminal and C-terminal antibodies, respectively (19, 27). Beliveau and co-workers
(19, 27) demonstrated that p40 and p45 have been shown to be
glycosylated and up-regulated by a low Pi diet. The amounts
of NaPi-2, 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-2 and NaPi-2 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 (27). 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-2 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-2. To
clarify the presence of NaPi-2, 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-2. 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-2 protein in the BBMV, in addition to the characterization of the 40-kDa
protein.
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 (21, 26). Kohl et al. (28) 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 (3). Obviously, the membrane spanning regions 1 to 3 and 4 to 8 are separated by a large hydrophilic loop into two distinct domains
(27, 28). This division is reflected on the genomic level regarding the exon/intron organization. Kohl et al. (28) suggest the
presence of the duplicated gene products in renal proximal tubular
cells. Indeed, NaPi-2 is splicing products from exon 1 to exon 8, and NaPi-2 is from 9 to 13 of the NaPi-2 gene.
/ and NaPi-2
(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-2 into Xenopus oocytes completely inhibited the
Na+-dependent Pi uptake. NaPi-2
also partially inhibited the Pi uptake. However, NaPi-2
did not affect NaPi-2 function. These results suggest that NaPi-2
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 (20). The finding of the dominant negative isoforms of
Na/Pi type II transporters may have important
physiopathological implications in X-linked hypophosphatemia.
, , and , NaPi-2 was found to have
three transmembrane domains and involve Asn-298, but not Asn-328. However, NaPi-2 did not encode the region including Asn-328. On the
basis of the NaPi-2 putative membrane structure, the extracellular domains of NaPi-2 has no glycosylation site. However, the
glycosylation experiment demonstrated that NaPi-2 is glycosylated by
microsomal membrane, suggesting that the structure of NaPi-2 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.
, , and may be helpful to resolve the molecular basis
of HHRH.
, , and 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.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Clinical
Nutrition, School of Medicine, Tokushima University, Kuramoto-Cho 3, Tokushima City, Tokushima 770, Japan. Tel.: 81-886-31-7095; Fax:
81-886-33-7094; E-mail:miyamoto{at}nutr.med.tokushima-u.ac.jp.
REFERENCES
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Abstract
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References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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