INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

Cell infection with amphotropic murine leukemia virus (A-MLV) is mediated by the binding of viral envelope glycoproteins to a cell surface receptor called PiT-2 (27, 52). PiT-2 is expressed in all tissues of all mammalian species, conferring susceptibility to A-MLV infection on all known mammalian cells, with the exception of certain hamster cells, like the CHO cell line, which produces soluble-factor-impairing envelope-receptor interactions (29, 30, 54). Gibbon ape leukemia virus (GaLV) and feline leukemia virus subgroup B (FeLV-B) use a receptor called PiT-1 (19, 32, 49), which is highly homologous to PiT-2 (27). Although PiT-1 is also widely expressed in mammalian tissues (51), receptors are functional for GaLV and FeLV infection in certain species only. The 10A1 murine leukemia virus is a natural variant of A-MLV that recognizes both PiT-2 and PiT-1 in human, feline, and simian cells (28, 57). The ability of PiT-1 and PiT-2 proteins to function as retrovirus receptors has been attributed to amino acid residues that differ between the receptors of the various species (6, 8, 15, 17, 20, 24, 34, 35, 46-48).

Independent of their capacity to mediate retrovirus binding and entry, PiT-1 and PiT-2 proteins serve as sodium-dependent phosphate (NaP_i) transporters (21, 33, 55). This finding led to the description of mammalian type III NaP_i transporters. In contrast with type I and type II transporters, which are expressed in specialized structures, like the brush border membranes of kidney and intestinal cells, and which contribute to phosphate homeostasis (4), type III transporters likely represent a general phosphate exchange system between the cells and the extracellular medium. PiT-1 and PiT-2 do not share homology with the members of the other NaP_i transporter families.

PiT-1 and PiT-2 activities are modulated in response to variations in extracellular inorganic phosphate concentrations ([P_i]). Phosphate starvation increases PiT-1 and PiT-2 gene expression (10, 11) and induces posttranslational modifications of PiT-2 that activate both phosphate transport and retrovirus entry (36). It was previously shown that only a fraction of PiT-2 molecules that are present at the cell surface are capable of processing virus entry, suggesting that the receptor may exist in an activated or inactivated form (2). It was found that PiT-2 is associated with the actin cytoskeleton network and forms high-molecular-weight complexes (36). As both interaction with actin and high-molecular-weight complex formation appeared inversely related to [P_i] (C. Salaün, unpublished data), and thus to receptor activity, an attractive hypothesis is that they participate in posttranslational changes important for function. Investigations that are necessary to better understand these molecular interactions require an unambiguous model of the membrane organization of PiT-2.

A topological organization of PiT-1 and PiT-2 has been proposed based on hydropathy plots. The model supposed 10 transmembranes (TMs), internal N- and C-terminal segments, and a large central intracytoplasmic domain (27, 34, 52). With the exception of the two extracellular domains that are involved in virus-envelope binding (reviewed in reference 44) and of the large intracellular loop that is recognized by specific antibodies (10), the predicted topology of PiT-1 and PiT-2 has not been confirmed experimentally. We examined this issue by a broad analysis of PiT-2-related sequences available in databanks and by a comprehensive experimental approach combining a study of protein glycosylation, the fusion of tagging epitopes, and the in vitro translation of truncation mutants. We propose a model of PiT-2 topology in the plasma membrane which accounts for the experimental findings and for the organization of the protein in several homology domains. This model differs from the predictions based on hydropathy plots and points out regions potentially important for PiT-2 functions.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Computational analysis. Multiple-sequence alignment was obtained based on the domain database collected by Corpet et al. (12), which is accessible at the University of Toulouse (http://protein.toulouse.inra.fr/cgi-bin/ReqProdomII.pl). Aligned sequences included the following (numbers in parentheses correspond to amino acid numbers in the aligned sequences): Y630 METJA, Methanococcus jannaschii unknown protein (17 to 131, 168 to 285); O29467 ARCFU, Archaeoglobus fulgidus permease (13 to 134, 174 to 296); O58858 PYRHO, Pyrococcus horikoshii transporter (13 to 124, 176 to 262); O54523 AAAAA, Halobacterium halobium permease (18 to 145); Q50684 MYCTU, Mycobacterium tuberculosis transporter (79 to 135, 405 to 533); YG04 HAEIN, Haemophilus influenzae permease HI1604 (21 to 155, 268 to 400); Q17454 CAEEL, Caenorhabditis elegans phosphate permease (44 to 118, 352 to 476); Q17455 CAEEL, C. elegans P4 phosphate permease (44 to 118, 342 to 477); P91840 CAEEL, C. elegans W05H5 protein (55 to 184); Q17404 CAEEL, C. elegans F09G2.3 protein (40 to 172, 348 to 483); O18697 CAEEL, C. elegans C48A7.2 protein (19 to 164, 348 to 483); O45220 CAEEL, C. elegans BO331.2 protein (29 to 92, 348 to 483); Q60421 CRIGR, Cricetulus griseus (Chinese hamster) PiT-2 (20 to 153, 503 to 632); Q63488 RAT, Rattus norvegicus PiT-1 (20 to 153, 501 to 633); Q08357 HUMAN, Homo sapiens PiT-2 (20 to 153, 500 to 632); Q61609 MOUSE, Mus musculus PiT-1 (39 to 172, 531 to 662); Q60422 CRIGR, C. griseus PiT-1 (35 to 168, 528 to 660); Q08344 HUMAN, H. sapiens PiT-1 (35 to 168, 528 to 660); O97596 FELCA, Felis silvestris catus PiT-1 (39 to 172, 532 to 664); YB8I YEAST, Saccharomyces cerevisiae permease YBR29C/PHO89 (21 to 155, 414 to 521); PHO4 NEUCR, Neurospora crassa PHO-4 (21 to 157, 436 to 568); O58374 PYRHO, P. horikoshii permease PHO 640 (18 to 114, 255 to 390); O28476 ARCFU, A. fulgidus permease (14 to 150, 190 to 317); PITB ECOLI, Escherichia coli phosphate transporter (26 to 124); PITA ECOLI, E. coli phosphate transporter (26 to 124); Q50173 MYCLE, Mycobacterium leprae transporter (23 to 114); O06411 MYCTU, Mycobacterium tuberculosis transporter (23 to 114); O34436 BACSU, Bacillus subtilis transporter (23 to 109, 231 to 284); O30499 BBBBB, Rhizobium meliloti permease (23 to 112, 228 to 315); Q38954 ARATH, Arabidopsis thaliana permease (167 to 301, 430 to 567); P93264 MESCR, Mesembryanthemum crystallinum permease (161 to 295, 428 to 529); Q9Z7M4 BBBBB, Chlamydia pneumoniae permease (14 to 148, 276 to 406); O84698 CHLTR, Chlamydia trachomatis permease (14 to 148, 276 to 406); O34734 BACSU, B. subtilis YLNA protein (11 to 102, 175 to 300); O37913 METTH, Methanobacterium thermautotrophicum sodium-dependent phosphate transporter (14 to 140, 186 to 307); Q9ZJC8 BBBBB, Helicobacter pylori permease (59 to 192, 301 to 405); and O26024 HELPY, H. pylori HP 1491 permease (59 to 192, 301 to 405).

Cell lines, materials, reagents, and transfections. Restriction enzymes and antiprotease Complete tablets were purchased from Boehringer Mannheim. The TNT quick-coupled transcription-translation system and canine pancreatic microsomes were from Promega. N-glycosidase F was from New England Biolabs. Pro-Mix ³⁵S labeling mix, horseradish peroxidase-coupled and cy3-coupled secondary antibodies, and the ECL+ kit were from Amersham Pharmacia Biotech. The 9E10-cy3 monoclonal antibody (MAb) was from Sigma. The 3F10 and 12CA5 MAbs were from Boehringer Mannheim. pCDNA3 and pCDNA3.1 Myc-His plasmids were from Invitrogen. The anti-PiT-2 rabbit serum was a generous gift from S. Kuhmann and D. Kabat (Oregon Health Sciences University, Portland).

Immunofluorescence on living cells. Cells were seeded on slides and cultured overnight. Slides were washed with cold phosphate-buffered saline (PBS) and placed on a 25-µl drop containing 12CA5 (1:500 dilution in culture medium containing 20 mM HEPES) or 9E10-cy3 (1:250 dilution) for 1 h at 4°C. After three washes with cold PBS, cells were fixed in 3% formaldehyde for 20 min at 4°C and quenched with 50 mM NH₄Cl in PBS for 10 min. Slides were placed on a 25-µl drop containing the cy3-coupled secondary antibody for 1 h at room temperature. Images were acquired using a Zeiss confocal fluorescence microscope and contrast enhanced using Adobe Photoshop 4.0 software.

Immunoblotting. After electrotransfer of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gels to nitrocellulose membranes using a Bio-Rad apparatus; membranes were blocked for 1 h in a solution containing 5% lowfat milk, 0.1% Tween 20, and PBS (PBS-milk); and the first antibody (anti-PiT-2, 1:2,000 dilution) was added for 1 h in PBS-milk at room temperature before the membranes were washed in 0.1% Tween-PBS. Horseradish peroxidase-coupled secondary antibodies were added for 1 h at room temperature. After washes in PBS-Tween 20, peroxidase activity was revealed using the ECL+ reagent and each membrane was exposed.

Plasmid construction. The PiT-2V plasmid contains a tagged version of human PiT-2 in which a peptide from the vesicular stomatitis virus G protein has been fused to the C terminus of the receptor (36). PiT-2-M was constructed by inserting human PiT-2 cDNA in the pCDNA3.1 Myc-His B plasmid vector, leading to in-frame ligation of the 3' extremity with sequences encoding a Myc-polyhistidine tag. This construct was then inserted downstream of a sequence encoding an influenza virus hemagglutinin (HA) epitope (MYPYDVPDYA) in the pCEP4-HA plasmid (a gift from S. Michelson), giving rise to the HA-PiT-2M plasmid.

Cell-free transcription-translation, immunoprecipitation, and posttranslational analysis. Cell-free transcription-translations were performed using the TNT quick-coupled system (Promega). Reactions were carried out for 90 min at 30°C in a final volume of 25 µl containing 20 µl of rabbit reticulocyte lysate, 1 µg of plasmid DNA, and 10 µCi of Pro-Mix ³⁵S label, with or without 2 µl of canine pancreatic microsomes. For glycosylation studies, vesicles were ultracentrifuged in a TL-100 rotor for 15 min, washed once, and resuspended in 25 µl of PBS before analysis by SDS-polyacrylamide gel electrophoresis. For immunoprecipitations, translation products were diluted in 150 µl PBS or PBS-0.5% Triton X-100 and incubated for 2 h with 0.5 µg of the 9E10 MAb. Magnetic beads coated with anti-mouse immunoglobulins (Igs) were then added and carefully washed with PBS using a magnetic tube holder. Recovered material was solubilized in loading buffer supplemented with 2% -mercaptoethanol or in a solution containing 1% Triton X-100, 0.5% SDS, 1% -mercaptoethanol, 1% NP-40, and 50 mM sodium phosphate (pH 7.5), and the material was incubated for 1 h at 37°C with 1,000 U of protein N-glycosidase F (PNGase F). N-glycosidase F treatment was similarly performed on cell lysates and on in vitro translation products.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Homology domains and predicted TM segments in human PiT-2. The sequences of human (32, 52), rat (27, 50), mouse (19, 42, 56), and hamster (9, 57) PiT-1 and PiT-2, as well as that of feline PiT-1 (39), have been determined. A search of the ProDom database (12) identified several domains within these proteins (Fig. 1A). They include domains specific for the mammalian PiT-1 and PiT-2 proteins, such as the N- and C-terminal extremities and a large central region, and domains for which homologies exist with nonmammalian proteins. These homology domains are referred to as PD1131 and PD7717 in the ProDom database.

View larger version (58K): [in this window] [in a new window]   FIG. 1.   Human PiT-2 homology domains and predicted membrane organization. (A) Various domains of human PiT-2. The amino- and carboxy-terminal copies of homology domain PD1131, which can be recognized in the 37 members of the PiT family, are shown as blue boxes. The homology domain 7717, which is not found in bacteria or plants, is shown in red. Green boxes indicate domains that are found in mammalian PiT-1 and PiT-2 sequences only. They include the amino- and carboxy-terminal extremities and the large hydrophilic central domain. (B) Alignment of human PiT-2 N- and C-terminal PD1131 homology domains. Identical residues found at the same position are indicated by a bar. Residues shown in green boxes correspond to amino acids conserved at these positions in more than 85% of aligned PD1131 sequences (n = 51 to 68, depending on the location). Conserved residues define four conserved blocks, indicated in green circles. Regions predicted as potential TM domains are shown in bold blue letters. Numbering is that of the human PiT-2 sequence. (C) Alignment of sequences of 11 PD7717 homology domains. Sequence identifiers (SEQ ID), organisms of origin (org.), the start (st.) and the end of the aligned sequences, and the weight (w.) of the sequence in the alignment are indicated. Sequences are as specified in Materials and Methods. Hydrophobic amino acids are boxed in yellow. Blue lines indicate TM regions that are predicted in all shown sequences. They correspond to predicted TM-5, TM-6, and TM-7 in human PiT-2. (D) Location of human PiT-2 regions (shown as open boxes with roman numerals I to XI) which are predicted to form TM domains when the PredictProtein algorithm is run on the entire human PiT-2 sequence. Arabic numerals between boxes refer to loops (L-1 to L-10). Numbers in green boxes within PD1131 homology domains refer to highly conserved amino acid blocks.

Experimental investigations of human PiT-2 TM organization. (i) Glycosylation of asparagine 81. The currently used 10-TM model of PiT-2 topology predicts that all potential N-linked glycosylation sites (residues 81, 328, and 383) would be located in intracellular domains (27, 34, 52). Thus, according to this prediction, PiT-2 should not bear N-linked oligosaccharide chains. We examined this issue in CHO cells by expressing human PiT-2. Cell extracts were analyzed by Western blotting using a rabbit antiserum directed against the intracellular loop L-7 (Fig. 2A). A signal at 70-kDa was visible in naive CHO cells as well as in CHO-PiT-2 cells. Although the size of this signal is consistent with hamster PiT-2, we presume that it is nonspecific background, as, in contrast with PiT-2 signals, migration was not affected by heat (data not shown) (10). A 73-kDa human PiT-2-specific signal was detected in CHO-PiT-2 cells (Fig. 2A, PiT-2.gly+). The digestion of cell extracts with PNGase F, which removes N-linked oligosaccharide chains, induced a shift of this signal to 68-kDa species (Fig. 2A, PiT-2.gly). This shift indicated that PiT-2 carries N-linked oligosaccharide chains. Similar observations were made with the tagged versions of human PiT-2 that are described below (data not shown). Since asparagines 328 and 383 are located in the intracellular L-7 segment, asparagine 81 appeared to be the only potential candidate for N-linked glycosylation. Site-directed mutagenesis was performed on this residue, such that asparagine was changed to valine (PiT-2.N81V). Expression of PiT-2.N81V in CHO cells conferred susceptibility to A-MLV infection (data not shown). Western blotting performed on CHO cells expressing PiT-2.N81V showed a signal at 68 kDa, the migration of which was not modified by treating cell extracts with PNGase F (Fig. 2A). Wild-type human PiT-2 and PiT-2.N81V proteins were also synthesized in vitro using reticulocyte lysates (Fig. 2B). The synthesis of wild-type human PiT-2 in the absence of microsomal vesicles produced a nonglycosylated protein migrating at 68 kDa (Fig. 2B, PiT-2.gly). The addition of microsomes allowed for glycosylation and produced a 73-kDa species (Fig. 2B, PiT-2.gly+) that was susceptible to PNGase F digestion. Glycosylation was not observed for the PiT-2.N81V mutant. We concluded from these experiments that PiT-2 is a glycoprotein that carries an N-linked oligosaccharide on asparagine 81. Since this amino acid is located in L-2, we concluded that L-2 is located at the cell surface.

View larger version (56K): [in this window] [in a new window]   FIG. 2.   Glycosylation of human PiT-2 on asparagine 81. (A) Western blot analysis of cell extracts using rabbit serum directed against the central intracytoplasmic domain (L-7) of human PiT-2 (hPiT-2). Extracts were prepared from naive CHO cells (), CHO-PiT-2 cells (wild type), and CHO cells expressing a mutant human PiT-2 in which asparagine 81 was changed to valine (N81V) and treated (+) or not treated () with an enzyme removing N-linked oligosaccharide (PNGase F). (B) Analysis of radiolabeled in vitro translation products. In vitro translation products of human PiT-2 (wild type) and the glycosylation mutant (N81V) were synthesized in either the presence (+) or absence () of microsomal vesicles and treated (+) or not treated () with PNGase F. Signals corresponding to glycosylated (PiT-2.gly+) and deglycosylated (PiT-2.gly) species are indicated. Molecular mass markers are in kilodaltons.

(ii) Tagging of the N- and C-terminal extremities of human PiT-2. It was previously reported that an antigenic epitope fused at the C-terminal extremity of PiT-2 could be detected at the cell surface (36). This observation was made using a tag derived from the vesicular stomatitis virus G envelope glycoprotein. In order to confirm this result with a different tag and to extend the observation to the N-terminal extremity, human PiT-2 was fused to an HA tag at the N-terminal extremity and to a c-Myc tag at the C-terminal extremity (HA-PiT-2M). The construction was expressed in CHO cells, conferring A-MLV envelope binding and susceptibility to A-MLV infection (data not shown). Living cells were incubated at 4°C either with the anti-HA MAb 12CA5 or with the anti-Myc MAb 9E10-cy3, then fixed, and stained with fluorescent anti-IgG. Incubation at 4°C prevented receptor internalization. Both the HA and the c-Myc signals were detected at the cell margin (Fig. 3). As a control, cells transfected with an expression vector for an HA-tagged version of the intracellular Rho protein were not stained unless the plasma membrane was permeabilized (data not shown). These results indicate that the N- and C-terminal extremities of human PiT-2 are accessible to antibodies at the cell surface. Thus, both the N- and C-terminal extremities of epitope-tagged human PiT-2 fusion protein are extracellular.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Detection of N- and C-terminal tags at the surface of living cells. Naive CHO cells or CHO cells expressing human PiT-2 bearing an N-terminal fused influenza virus HA tag and a C-terminal fused c-Myc tag (CHO-HA-PiT-2M) were grown on glass slides and incubated at 4°C for 1 h with either a MAb against the HA tag (anti-HA 12CA5) or a MAb against the Myc tag (anti-Myc 9E10-cy3). Cells were then washed at 4°C and fixed before incubation with a fluorescent anti-mouse IgG secondary antibody.

(iii) In vitro translation of human PiT-2 C-terminal-truncation mutants. C-terminal-truncation mutants of PiT-2 were constructed with a c-Myc tag fused at the C-terminal extremity. Proteins were synthesized in vitro using rabbit reticulocyte lysates. The addition of microsomal vesicles allowed for processing, glycosylation, and incorporation into membranes. The presence of the C-terminal tag of truncation mutants at the surface of microsomal vesicles was examined with an aim to determine their orientation in the membrane. Tags were detected by incubating in vitro translation products with the anti-Myc 9E10 MAb. Vesicles were disrupted either before the addition of the antibody, giving access to both intra- and extravesicular epitopes, or after the addition of the antibody, which could bind to extravesicular tags only. Immune complexes were precipitated and analyzed for the presence of glycosylated products. As only a fraction of the translation products was incorporated into vesicles, analysis of glycosylated material allowed a focus on processed products that had been actually incorporated into vesicles. Thus, immunoprecipitation of glycosylated species indicated that the mutant protein was oriented with its C-terminal extremity at the surface of the vesicles, whereas failure to immunoprecipitate glycoslylated species indicated that the mutant protein was oriented with its C-terminal extremity inside the vesicles.

View larger version (41K): [in this window] [in a new window]   FIG. 4.   Immunoprecipitation of in vitro translation products of human PiT-2 C-terminal-truncation mutants. Plasmids encoding C-terminally truncated forms of human PiT-2 and bearing a C-terminal Myc-His tag were transcribed in vitro, and transcription products were translated in a rabbit reticulocyte lysate assay in the presence of 35S-labeled methionine and cysteine and of microsomal vesicles. Vesicles were then either disrupted by a detergent (Triton X-100) (+) or kept intact (). A MAb directed against the Myc tag (9E10) was added for 4 h at 4°C, and immune complexes were precipitated by binding to antimouse magnetic beads. After elution from beads, translation products were treated (+) or not treated () with PNGase F, an enzyme removing N-linked oligosaccharides. C-terminal truncations were performed at the C-terminal extremities of the predicted L-2, L-3, L-4, L-5, L-6, L-7, L-8, L-9, and L-10 loops (see Materials and Methods and Fig. 1D). A control reaction was performed with wild-type human PiT-2 (wild type). Signals corresponding to glycosylated (gly+) and deglycosylated (gly-) species are indicated. The deduced orientation of C-terminal-truncation mutants in vesicles is indicated. Molecular mass markers are in kilodaltons.

Proposed topology of human PiT-2. Two regions have been recognized as important for the interaction of PiT-1 or PiT-2 with their cognate retroviral envelopes. Amino acid 522 (8, 17, 20) and a surrounding region of 13 amino acids in L-8 (15, 35), often referred to as region A, together determine recognition by GaLV, A-MLV, or FeLV-B envelopes. This region is a presumed binding site for the VRA receptor-binding domain of the envelopes (46, 47). The other important region for envelope recognition is located in L-4, between amino acids 132 and 142 (34). This region is presumably a binding site for the VRB receptor-binding domain of the envelopes (46, 47). These published data unambiguously indicate that L-4 and L-8 are extracellular.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Proposed model of human PiT-2 topology. The various domains of human PiT-2 and the predicted TMs and loops are shown in the top panel. The expected extracellular (E) or intracellular (I) localization of the N- and C-terminal extremities and of each predicted loop is indicated in the middle panel, according to the various investigation methods. The bottom panel shows the proposed topology of human PiT-2. Blue segments are the N-PD1131 and C-PD1131 homology domains, in which numbers in green circles indicate the conserved amino acid blocks. The red segment is PD7717. As the orientation of loop 6 is still unrevealed, this segment appears as a striped line. Black lines and open boxes correspond to segments for which homologous sequences were found in mammals only.