INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

CALCIUM INFLUX HAS an important role in the regulation of many cellular processes in both excitable and nonexcitable cells (1, 2, 4, 8, 21). In excitable cells, Ca²⁺ enters the cytosol through voltage-dependent channels that have been characterized in great detail (2, 8). In nonexcitable cells, stimulation of plasma membrane receptors by a variety of hormones, growth factors, and other agonists induces a G protein-dependent activation of phosphatidylinositol 4,5,-bisphosphate-specific phospholipase C (PLC) to produce D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] (1, 3, 8, 20, 21). Ins(1,4,5)P₃ is a critical intracellular second messenger, since it diffuses into the cytosol and binds to the Ins(1,4,5)P₃ receptor in the endoplasmic reticulum (ER), resulting in the release of Ca²⁺ from an internal store. The depletion of Ca²⁺ from the ER store activates a Ca²⁺ influx pathway in the plasma membrane, whereas refill of the stores inactivates Ca²⁺ influx (20). This phenomenon has been described as store-operated or capacitive Ca²⁺ entry. The molecular nature and mechanism of the Ca²⁺ influx is not yet known. Recent studies have suggested that this Ca²⁺ influx is mediated by a channel that has been referred to as Ca²⁺ release-activated Ca²⁺ channel or store-operated Ca²⁺ channel (SOC). Inward currents due to Ca²⁺ influx via SOCs have been measured in a number of different cell types (8, 16). A critical and as yet unanswered question concerns the mechanism that relays the status of the internal Ca²⁺ store to the plasma membrane to either activate or inactivate Ca²⁺ influx.

Searching for genes encoding the SOC has initiated the cloning of mammalian homologues of the Drosophila transient receptor potential (Trp) gene (5, 13, 14). The visual signal transduction cascade in the Drosophila eye is coupled to the activation of PLC and has been suggested to involve Ins(1,4,5)P₃-induced Ca²⁺ mobilization (13). Trp and Trp-like (Trpl) genes have been cloned from Drosophila, and these have been proposed to have a role in store-operated Ca²⁺ influx. (13, 14). Expression of the Drosophila Trp (dTrp) cDNA in insect Sf9 cells and Xenopus oocytes was associated with the appearance of a novel Ca²⁺-selective channel that was activated when internal Ca²⁺ stores were depleted by thapsigargin treatment, i.e., via inhibition of ER Ca²⁺-ATPases (11, 17, 24). Mammalian homologues of the Trp genes have been reported in several mammalian species and tissues, including human brain and embryonic kidney cells (hTrp) (26, 28, 32), mouse brain and mouse pancreatic B cells (mTrp) (5, 22), rat brain (rTrp) (10), and bovine adrenal and endothelial cells (bTrp) (9). These genes appear to be part of a gene family and presently include six genes: Trp1, Trp2, Trp3, Trp4, Trp5, and Trp6. Full-length cDNAs of hTrp1, mTrp1, bTrp3, hTrp3, bTrp4, rTrp4, mTrp5, and mTrp6 have been reported. In addition, splice variants of hTrp1, bTrp1, and mTrp1, have been identified, which primarily differ in the amino acid sequence of the amino-terminal region of the protein (7, 15, 18, 22, 26, 28, 32). The long form of this gene, referred to as Trp1, has an additional 34-amino acid sequence (amino acids 126-159) that is lacking in the short, Trp1, isoform. Recently a shorter Trp gene, a splice variant of bTrp4 encoding a protein of 495 amino acids, has been reported (9).

hTrp1, hTrp3, bTrp4, mTrp5, and mTrp6 genes have been expressed in cells such as HEK-293, CHO, and COS, and some functional studies have been carried out, including intracellular Ca²⁺ mobilization and Ca²⁺ current measurements (5, 6, 15, 17, 18, 29-32). A number of these studies have revealed that the functional characteristics of the Ca²⁺ influx in cells expressing the Trp gene product(s) are distinct from those of the endogenous store-operated Ca²⁺ influx in the nontransfected cells. Furthermore, expression of hTrp3 and mTrp6 resulted in increased levels of Ca²⁺ influx activity that was associated with PLC activation but not with internal Ca²⁺ store depletion (29, 31). However, the expression of TRPC1A, which is equivalent to the -isoform of Trp1 gene, and bTrp4 (bovine CCE1) resulted in the appearance of a nonselective cation channel in response to internal Ca²⁺ store depletion (32). The most convincing evidence relating Trp to store-operated Ca²⁺ entry was reported by Zhu et al. (30), who showed that expression of a mixture of RNA in the antisense direction (encoding all six mTrp isoforms, mTrp1 through mTrp6) attenuated store-operated Ca²⁺ influx in murine Ltk cells. Thus there is a strong possibility that one of the Trp isoforms that has already been cloned, or an as yet unidentified Trp gene, might encode the SOC. Although the use of tag sequences has been useful for the detection of the expressed gene product (25, 29), the presence of endogenous Trp protein(s) has not yet been demonstrated, and neither has its function been clearly determined. It has been proposed that the expressed Trp protein might form homo- or heterodimers or multimers via interactions between the expressed Trp isoforms or between the expressed and endogenous Trp proteins (27). However, to test this hypothesis, it is first necessary to develop tools for determining the presence and function of the endogenous Trp protein(s) in various tissues.

Toward this goal, we isolated a full-length Trp1 cDNA from rat brain and identified the presence of both the Trp1 and Trp1 variants in rat brain. Furthermore, on the basis of the deduced amino acid sequence of the carboxy-terminal region of this gene, we have synthesized a Trp1-specific peptide, which was used to generate polyclonal antiserum. This antibody was used to demonstrate, for the first time, the presence of the endogenous Trp1 protein in human and rat brain and to localize the protein in the plasma membrane of HEK-293 cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

All reagents used in the study were of the highest grade available. hTrp3-expressing, hTrp1-expressing, or control (vasopressin receptor-expressing) HEK-293 cells were cultured as described (29, 30). Rat tissues were excised from 4-wk-old male Wistar rats (Harlan Sprague-Dawley) and immediately frozen in liquid nitrogen. Human (cadaver) brain samples were obtained from the National Institute on Aging.

RNA isolation, synthesis of first-strand cDNA, and RT-PCR analysis. Total RNA was extracted from the frozen tissues using TRIzol reagent (GIBCO BRL). mRNA was isolated using the Oligotex mRNA minikit (Qiagen). Total RNA was treated with DNase I (amplification grade, GIBCO BRL) at a concentration of 1 U/µg RNA in the reaction buffer, containing (in mM) 20 Tris · HCl (pH 8.4), 2 MgCl₂, and 50 KCl, for 15 min at room temperature. The reaction was terminated by adding EDTA at a final concentration of 2.5 mM and heated at 65°C for 10 min.

RACE library preparation and RACE-PCR. The cDNA libraries for rapid amplification of cDNA ends (RACE) were prepared using the Marathon cDNA amplification kit (Clontech), with some modifications. Briefly, the first strand was synthesized in 10 µl of reaction solution containing 1 µg of RNA, 1 µl of cDNA synthesis primer (10 µM), 2 µl of 5× first-strand buffer, 1 µl of dNTP mix (10 mM), and 1 µl of MMLV RT (100 U/µl), which was incubated at 42°C for 1 h. The second strand was then synthesized in a reaction tube containing 10 µl of the first-strand reaction solution, 48.4 µl of water, 16 µl of 5× second-strand buffer, 1.6 µl of dNTP (10 mM), and 4 µl of 20× second-strand enzyme cocktail, and the tube was incubated at 16°C for 1.5 h. After incubation, 2 µl of T4 DNA polymerase were added to the solution, which was further incubated at 16°C for 45 min. The reaction was stopped by adding 4 µl of the EDTA-glycogen mixture. Double-strand cDNA (ds cDNA) was purified using the Qiagen nucleotide purification kit. Finally, the adaptors were ligated to the ds cDNA in 10 µl of reaction medium containing 5 µl of ds cDNA, 2 µl of Marathon cDNA adaptor (10 µM), 2 µl of 5× DNA ligation buffer, and 1 µl of T4 DNA ligase (1 U/µl). This reaction mixture was incubated at 16°C overnight and then was stopped by heating at 70°C for 5 min. RACE-PCR was performed in 50 µl of reaction mixture containing 37 µl of water, 5 µl of 10× KlenTaq PCR reaction buffer, 5 µl of dNTP (10 mM), 1 µl of advantage KlenTaq polymerase mix (50×), 1 µl of adaptor primer (AP1; 10 µM), and 1 µl of Trp genespecific primer (10 µM). The PCR conditions were initial denaturation at 94°C for 1 min, followed by three different temperature cycles as follows: 1) 5 cycles of denaturation at 94°C for 30 s and annealing and extension at 72°C for 4 min, 2) 5 cycles of denaturation at 94°C for 30 s and annealing and extension at 70°C for 4 min, and 3) 25 cycles of denaturation at 94°C for 30 min and annealing and extension at 68°C for 4 min. After these cycles, the reaction mixture was incubated for an additional 7 min at 68°C, followed by soaking at 4°C before termination of the reaction.

Gene cloning and ligation. The RACE-PCR product was cloned into a T/A cloning-based vector, pT-Adv (Clontech), which contains lacZ fragment for -complementation in Escherichia coli and ampicillin resistance and kanamycin resistance genes for selection. The ligation reaction was performed at 14°C overnight in 10 µl of mixture that contained 1 µl of 10× ligation buffer (Clontech), 2 µl of the vector (25 ng/µl), 1-6 µl of PCR product, and 1 µl of T4 DNA ligase (10 U/µl). Ligated plasmids were transformed to TOP10F' E. coli competent cells and plated on LB plates with X-gal/isopropyl -D-thiogalactopyranoside and 50 µg/ml ampicillin. The plates were incubated at 37°C for 18-20 h and put into 4°C for color development. Single white clones were selected for plasmid DNA isolation, restriction enzyme analysis, and DNA sequencing.

DNA sequencing and DNA/protein sequence analysis. DNA sequencing was performed by automatic sequencing, using an ABI 377 sequencer in the National Institute of Dental and Craniofacial Research core facility. The DNA and the deduced protein sequences were analyzed by several DNA sequence analysis software packages: Mac Vector, MacLing (Molecular), GCG (Genetics Computer Group), and the Blast and ClustalW online service of the National Center for Biotechnology Information.

Northern blot analysis. The probes were labeled with [-³²P]dCTP (Amersham Life Sciences) by using the Primer-it room temperature random primer labeling kit (Stratagene) and purified by passage through ProbeQuant G-50 microcolumns (Pharmacia Biotech) to remove unincorporated ³²P-labeled nucleotides. The multiple tissue Northern blots (Clontech) were prehybridized in ExpressHyb solution (Clontech) at 68°C for 1-2 h and were then hybridized in the same solution with the probes at 68°C overnight. The blots were rinsed in a solution of 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and 0.05% SDS three to four times and then washed twice in this solution at room temperature (15-30 min each). The blots were then washed twice (20-40 min each time) in a solution containing 0.1× SSC and 0.1% SDS at 50°C. After the washing, the blots were exposed to the X-OMAT films (Kodak) for detection of the hybridization signals.

Generation of anti-Trp1 antiserum. Polyclonal antibody was generated against the sequence Gln-Leu-Tyr-Asp-Lys-Gly-Tyr-Thr-Ser-Lys-Glu-Gln-Lys-Asp (see Fig. 3). The synthetic purified peptide was conjugated and used to immunize one rabbit. Peptide synthesis and antibody generation were carried out by Lofstrand Labs (Gaithersburg, MD). The antiserum was affinity purified using the peptide and Immunopure Ag/Ab immobilization kit no. 2 (Sulfolink gel, Pierce). The antibody was effective in Western blotting, immunoprecipitation, and immunolocalization studies.

Membrane preparation and Western blot analysis. Crude membrane protein was prepared from frozen human and rat brain tissues and HEK-293 cells in buffer containing 50 mM Tris · HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF; Calbiochem), and 19 µl/ml aprotinin (Sigma). The tissues or cells were homogenized using a Polytron homogenizer, and the homogenate was centrifuged at 3,000 rpm (1,090 g) at 4°C for 10 min. The supernatants were filtered and centrifuged at 19,000 rpm (43,700 g) for 30 min. The pellets were suspended in the above buffer, aliquoted, frozen, and stored at 70°C until use.

Immunolocalization of Trp proteins in HEK-293 cells. hTrp3-expressing, hTrp1-expressing, or control (vasopressin receptor-expressing) HEK-293 cells were cultured on glass coverslips coated with poly-L-lysine (25 or 50 µg/ml) for 2 days. These cells were rinsed once with PBS (pH 7.5), fixed with 3% formaldehyde-1× PBS for 30 min, treated with 100 mM glycine-PBS for 30 min, permeabilized with methanol at 70°C on dry ice for 5 min, and then washed three times with PBS. After the washes, the samples were blocked with 5% donkey serum for 1 h, then incubated with the primary antibodies, mouse monoclonal anti-HA (Boehringer-Mannheim) or rabbit polyclonal anti-Trp1 at 1:150 or 1:100 dilution, and then washed three times. The samples were then incubated with FITC-conjugated anti-rabbit IgG or anti-mouse IgG at 1:150 dilution for 1 h, washed with PBS three times, and mounted on glass slides. Slides were examined on a Leica TCS 4D CLSM confocal microscope equipped with an argon-krypton laser; 488-nm light was used for excitation of the FITC-labeled antibodies, and images were collected using a 100× oil immersion objective (Leica Lasertechnik, Heidelberg, Germany). Differential interference contrast images of the same fields of cells were also collected.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cloning of a full-length cDNA of Trp1 isoform from rat brain. Alignment of the sequences of the reported Trp genes demonstrated conserved DNA sequences in the amino acid regions Glu-Trp-Lys-Phe-Ala-Arg-(Ser) and (Phe)-Gly-Pro-Leu-Gln-Ser, as was previously reported (30). Based on these conserved regions, the primer pairs AW2A/AW2B and AW3A/AW3B were synthesized (see Table 1 for the sequences) and used to amplify Trp-homologous sequences, by RT-PCR, from total RNA isolated from the rat submandibular gland, a nonexcitable tissue. The results of the RT-PCR reaction (Fig. 1A) demonstrated a single product (~400 bp). This sequence (Fig. 1B), encoding the conserved domain present in Trp genes, showed 94% homology to mTrp1 and 88% homology to hTrp1.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   RT-PCR amplification of a Trp1-homologous sequence from rat submandibular gland RNA. A: arrow indicates RT-PCR product amplified from rat submandibular gland RNA (lane 1) by using primer pair AW3A/AW3B (based on conserved Trp domains). Lane labeled M shows DNA size markers (100 bp). B: DNA sequence of RT-PCR products from rat submandibular glands. Boxed sequences were used for design of primers for rapid amplification of cDNA ends (RACE)-PCR amplification.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Cloning of Trp1-homologous cDNA from rat brain by RACE-PCR. A: amplification of 3' end of Trp1 cDNA from rat brain (lane 2). RACE-PCR product size is ~2.4 kb. Lanes 1 and 3 are molecular size markers. B: amplification of 5' end of Trp1 cDNA from rat brain (results from 2 experiments are shown in lanes 2 and 3). RACE-PCR product size is ~1.6 kb. Lanes 1 and 4 are molecular markers as in A. C: PCR amplification of overlapping region of 5' and 3' clones shown in A and B. Size of product is ~700 bp. Lanes 1 and 4 are molecular markers as in A; lanes 2 and 3 show results of two experiments.

View larger version (48K): [in this window] [in a new window]   Fig. 3.   A: alignment of rat Trp1 (rTrp1) with Trp1 genes from different species. Deduced amino acid sequences of reported Trp1 genes. MTrp1, mouse Trp1 isoform; RTrp1, rat Trp1 -isoform (present report); HTrp1, alternatively spliced short form (D34) of human Trp1; BTrp1, long () form of bovine Trp1. Amino acid differences between species are indicated in bold. Major difference is additional 34-amino acid sequence (amino acids 126-159) in amino-terminal region of -isoform that is missing in truncated -isoforms (indicated by dashes). Underlined sequence was used to synthesize a peptide for generation of polyclonal anti-Trp1 antiserum. B: hydrophilicity plot of rTrp1 protein. Kyte-Doolittle analysis of hydropathy was done with a window of 21 amino acids. * Seven putative transmembrane domains located between amino acids 320 and 620.

Expression of Trp1 transcripts in rat and mouse tissues. To determine whether both - and -isoforms of rTrp1 are present in rat brain, we performed PCR amplification of the region (nucleotides 316-449) by using the primers AW29A and AW29B (see Table 1). Two products of the expected molecular masses were amplified from rat brain RNA (Fig. 4, lane 2) and represent the two isoforms of rTrp1, rTrp1 (long form, with 34 extra amino acids) and rTrp1 (short form, without the 34-amino acid sequence). Furthermore, using the same set of primers, only the smaller product was amplified from the pT-rb-5' clone, confirming that it is the Trp1 isoform (Fig. 4, lane 1).

View larger version (50K): [in this window] [in a new window]   Fig. 4.   Detection of both - and -isoforms of rTrp1 in rat brain. RT-PCR analysis was performed using specific primer pairs (AW29A and AW29B) to amplify - and -isoforms. Both isoforms are present in rat brain (lane 2). Lane 1 is amplification of pT-rb-5' clone by using primers AW29A and AW29B. Lanes marked M represent nucleic acid size standards of 100-bp DNA ladder.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Expression of rTrp1 in rat and mouse tissues. Rat and mouse multiple-tissue mRNA blots (Clontech) were hybridized with rTrp1 cDNA probe (Kpn I and SexA I fragment; 1.7 kb) under high-stringency conditions (see MATERIALS AND METHODS). Lanes 1-8 represent mRNA from tissues of heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis, respectively. A: hybridization results of rTrp1 probe with rat blot (12-h exposure). B: results with mouse blot (60-h exposure). C: hybridization of -actin probe with rat blot (12-h exposure). Mouse and human RNA blots gave similar results with -actin probe (not shown).

Detection of the endogenous Trp1 protein in rat and human brain. The hTrp3 and mTrp6 genes have been expressed and the ³⁵S-labeled Trp proteins have been detected by using an antibody against a carboxy-terminal HA tag sequence (5, 25). However, the presence of endogenous Trp protein in any tissue or cell type has not yet been demonstrated. Toward this goal, we synthesized a peptide corresponding to the deduced amino acid sequence of rTrp1 (amino acids 523-536, with sequence Gln-Leu-Tyr-Asp-Lys-Gly-Tyr-Thr-Ser-Lys-Glu-Gln-Lys-Asp) and used it to generate polyclonal antisera in rabbits. The antigenicity of this sequence was determined by the BLAST program. Furthermore, it should be noted that this region appears to be present only in Trp1 and not in other reported Trp genes. The antiserum was affinity purified using the peptide and was initially used for immunoprecipitation of the expressed hTrp1 gene product. ³⁵S-labeled hTrp1 (Fig. 6A, lanes 2-4), but not mTrp4 (Fig. 6A, lane 1) or hTrp3 (not shown), was immunoprecipitated by this antibody from lysates of COSM6 cells transfected with the respective Trp cDNAs. Similar results were obtained on Western blots. Figure 6B shows the proteins from membranes isolated from HEK-293 cells expressing either hTrp3 protein (lanes 2 and 4) or the control vector (vasopressin receptor; lanes 1 and 3) reacting with anti-Trp1 (lanes 1-4) and anti-HA tag (lane 5) antibodies. With the anti-Trp1 antibody (Fig. 6B), reactivity was detected at similar levels with a protein of ~92 kDa (lanes 1 and 2) in both control and hTrp3-expressing cells, which could be blocked by preincubation of the antibody with the peptide (lanes 3 and 4). Proteins of ~148, 120, and 100 kDa displayed reactivity toward the anti-HA tag antibody (Fig. 6B, lane 5; note that in this case the reactivity was only seen in membranes of cells transfected with the hTrp3 cDNA, data not shown). The multiple bands seen in the case of hTrp3 are consistent with the glycosylation states of this protein (5, 25, 29). Importantly, proteins in this molecular mass range were not detected by the Trp1 antibody in the same blot. In aggregate, the data demonstrate 1) the specificity of the anti-Trp1 antibody for the Trp1 protein and 2) the presence of endogenous Trp1 protein in HEK-293 cells.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Detection of endogenous Trp1 protein. A: 35S-labeled hTrp1 (lanes 2-4, 1:30, 1:100, and 1:300 dilution of anti-Trp1, respectively; arrow), but not mTrp4 (lane 1, 1:100 dilution) was immunoprecipitated from COSM6 cell lysates. Cells were transfected with respective cDNAs and selected by G418 (30). B: Western blot analysis of plasma membrane proteins from HEK-293 expressing hTrp3 cDNA (lanes 2, 4, and 5) or control (vasopressin-receptor-expressing) vector (lanes 1 and 3). Anti-Trp1 (lanes 1-4) or anti-HA tag (lane 5) antibodies were used. Endogenous Trp1 protein was detected (lanes 1 and 2); arrow at left indicates reactivity with a protein of ~92 kDa. This reactivity was blocked by preincubation of antibody with peptide (lanes 3 and 4). Proteins of ~148, 120, and 100 kDa displayed reactivity toward the anti-HA tag antibody (lane 5; arrows at right). C: Western blot analysis of proteins in a crude membrane fraction of human (lane 1) and rat (lane 2) brain. Reactivity to anti-Trp1 was detected with 92- and 56-kDa proteins (arrows). Reactivity was competed against by preincubation of antibody with peptide (not shown).

Immunolocalization of endogenous Trp1 protein in HEK-293 cells. Because HEK-293 cell membranes showed high levels of reactivity with the anti-Trp1 antibody, we used anti-Trp1 antibody in combination with an FITC-labeled secondary antibody to detect the endogenous protein in these cells by confocal microscopy. Figure 7A shows the localization of the endogenous Trp1 protein in HEK-293 cells transfected with the hTrp3 cDNA. A strong reaction to the antibody was observed in the cells. Localization of the Trp1 protein was clearly seen in the plasma membrane of these cells, although some reactivity was seen inside the cell, in a nonnuclear region. The image shown is a z-series section through approximately the middle of the cell. Notably, the reactivity was much reduced and not detected in the plasma membrane in the absence of the primary antibody (image was similar to that shown in Fig. 8E). When anti-Trp1 was first incubated with the peptide, the reactivity in the plasma membrane was attenuated significantly (Fig. 7B), although the reactivity inside the cells was not completely competed out. Similar results were obtained with control HEK-293 cells (i.e., transfected with the vasopressin receptor cDNA; data not shown). Thus HEK-293 cells appear to contain the Trp1 protein at levels high enough to be detected by immunofluorescence. The localization of the immunofluorescence (e.g., at the cell boundary) was further confirmed by superimposing the FITC image on a light-field image (not shown). To our knowledge, this is the first evidence for the presence and localization of an endogenous Trp protein in any cell type.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   Immunolocalization of endogenous hTrp1 protein in HEK-293 cells by confocal microscopy. HEK-293 cells were plated onto glass coverslips and cultured overnight. Cells were fixed and permeabilized as described in MATERIALS AND METHODS and then treated with anti-Trp1 antibody (A) or anti-Trp1 antibody-peptide (B) followed by a secondary FITC-linked anti-rabbit IgG. Image in A is a stacked image of ~15 z-series sections in middle region of cells. Plasma membrane areas are indicated by arrows.

View larger version (52K): [in this window] [in a new window]   Fig. 8.   Immunolocalization of hTrp1 and hTrp3 proteins. After culture, HEK-293 cells, expressing either HA-tagged hTrp1 protein (A, B, and D) or HA-tagged hTrp3 protein (C), were fixed and permeabilized with methanol and then treated either with anti-Trp1 antibody (A and D) or with anti-HA antibody (B and C) and then with required FITC-labeled secondary antibody. D: anti-Trp1 was first incubated with synthetic Trp1 peptide. E: cells were treated only with anti-mouse secondary antibody (treatment with anti-rabbit IgG alone gave similar results). Unlabeled arrows indicate plasma membrane region; arrows labeled 2 indicate intracellular fluorescence.

Immunolocalization of hTrp1 and hTrp3 proteins in HEK-293 cells. To confirm the localization of Trp1 protein, we used HEK-293 cells transfected with the HA-tagged hTrp1 cDNA. Immunolocalization of Trp1 in these cells was examined by confocal microscopy using either anti-Trp1 or anti-HA and the respective FITC-labeled secondary antibody. A strong reactivity was observed in the plasma membrane region of these cells with either antibody. Figure 8, A and B, shows the images obtained with anti-Trp1 and anti-HA, respectively, in hTrp1-expressing HEK-293 cells. In ~20% of the cells, a prominent cytosolic localization was also seen (Fig. 8, A and B). Figure 9 shows a ×4 zoom image of such a cell using the anti-HA (a similar pattern was seen in cells treated with anti-Trp1). Immunofluorescence was associated with the plasma membrane and a reticular structure inside the cell, which appeared to exclude the nuclear region. We have not yet identified this intracellular organelle. When anti-HA was first incubated with the HA peptide (Fig. 8D) or in the absence of the primary antibody (Fig. 8E shows the reactivity detected with anti-mouse IgG, which was similar to that seen with anti-rabbit IgG), the signal was considerably dampened and the plasma membrane or ER localizations were not seen. Thus the expressed hTrp1 protein, like the endogenous Trp1 protein, was localized primarily in the plasma membrane of the cell (compare Fig. 8, A and B). The reason for the heterogeneity in the Trp1 localization and the significance of its intracellular localization in some cells is not presently clear. One possible explanation is that, due to the high levels of expression, some of the protein might not be routed correctly. This needs to be examined in greater detail. Notably, such heterogeneity was not very prominent in localization of the hTrp3 protein in HEK-293 cells transfected with the HA-tagged hTrp3 cDNA. In these cells, the protein was clearly localized in the plasma membrane (see Fig. 8C; note that the same primary and secondary antibodies were used in Fig. 8, B and C). Intracellular reactivity was also observed in these cells. However, this was localized (like a "hot spot") to a specific region that was seen in almost every cell. Although we have not yet identified this subcellular location of the protein, given that the hTrp3 is glycosylated (5), this region is likely to be the Golgi apparatus. In a recent study, Vannier et al. (25), using the same cells and primary antibody, suggested that Trp3 is localized throughout the cell.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   Intracellular localization of hTrp1 in HEK-293 cells transfected with Trp1 cDNA. Approximately 20% of Trp1-expressing cells showed intracellular localization of protein (e.g., see cell labeled 2 in Fig. 8, A and B). A 4× zoom image of such a cell is shown. Immunofluorescence is present in a reticular intracellular structure. Image was obtained using anti-HA antibody. Similar pattern was seen with anti-Trp1 antibody. Arrow labeled 1 indicates plasma membrane region, and arrow labeled 2 indicates intracellular fluorescence.

Presence of endogenous hTrp1 and HA-tagged expressed hTrp3 proteins in plasma membrane fraction of transfected HEK-293 cells. The data in Figs. 7 and 8 show that although the Trp1 and Trp3 proteins are localized in the plasma membrane they are also present in intracellular locations in HEK-293 cells. To further demonstrate that these two proteins are localized in the plasma membrane of the cell, we have fractionated Trp3-expressing HEK-293 cells and isolated the plasma membrane by density gradient centrifugation using Percoll. The presence of endogenous Trp1 (Fig. 10A) and the expressed HA-tagged Trp3 (Fig. 10B) proteins in the various fractions was examined by Western blotting using the anti-Trp1 antibody and the anti-HA antibody, respectively. The fractions examined for Fig. 10 were unbroken cells (lane 1), 4,500-rpm supernatant (lane 2; starting fraction for plasma membrane isolation), 13,500-rpm supernatant (lane 3; fraction remaining after plasma membranes were pelleted, which contains most of the ER component of the cell), and purified plasma membrane fraction (lane 4). Both proteins were similarly enriched in the purified plasma membrane fraction (compare lanes 2 and 4 in Fig. 10, A and B). It should be noted that each lane contained 5 µg of protein. In the case of hTrp3 the ECL reaction was stopped after 1 min, whereas in the case of hTrp1 the reaction was carried out for >1 h to detect a signal. This difference can be explained on the basis of the expected expression levels of the two proteins, since hTrp3 is overexpressed in these cells compared with the endogenous hTrp1. The lower band seen with the anti-HA antibody (Fig. 10B) is not the Trp3 protein, since it is also detected by this antibody in the control, vasopressin receptor-expressing cells (data not shown). Note that this band does not appear to be enriched in any fraction.

View larger version (25K): [in this window] [in a new window]   Fig. 10.   Enrichment of hTrp1 and hTrp3 proteins in HEK-293 cell plasma membrane fraction. hTrp3-expressing HEK-293 cells were grown to confluence and harvested. Plasma membranes were purified as described in MATERIALS AND METHODS; 5 µg of same protein samples were loaded as follows in both gels (A and B). Lane 1, unbroken cells; lane 2, 13,5000-rpm pellet, crude plasma membrane fraction; lane 3, 13,500 rpm supernatant, contains bulk of endoplasmic reticulum; lane 4, Percoll density gradient-purified plasma membrane fraction. Blots of gels were incubated with either anti-Trp1 (A) or anti-HA (B) and respective secondary antibodies. Enhanced chemiluminescence reaction was performed for 30 s for B and 2 h for A. Endogenous hTrp1 protein (A) and expressed HA-tagged hTrp3 (B) are indicated by arrows. In both cases, protein is relatively enriched in purified plasma membrane fraction (compare intensities of bands in lanes 2 and 4 in both gels).