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J. Biol. Chem., Vol. 275, Issue 47, 36483-36486, November 24, 2000

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ACCELERATED PUBLICATION
Expression of Truncated Transient Receptor Potential protein 1 (Trp1)

EVIDENCE THAT THE Trp1 C TERMINUS MODULATES STORE-OPERATED Ca²⁺ ENTRY^*

Brij B. Singh, Xibao Liu, and Indu S. Ambudkar

From the Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 7, 2000, and in revised form, September 7, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Transient receptor potential protein 1 (Trp1) has been proposed as a component of the store-operated Ca²⁺ entry (SOCE) channel. However, the exact mechanism by which Trp1 is regulated by store depletion is not known. Here, we examined the role of the Trp1 C-terminal domain in SOCE by expressing hTrp1 lacking amino acids 664-793 (Trp1) or full-length hTrp1 in the HSG (human submandibular gland) cell line. Both carbachol (CCh) and thapsigargin (Tg) activated sustained Ca²⁺ influx in control (nontransfected), Trp1-, and Trp1-expressing cells. Sustained [Ca²⁺]_i, following stimulation with either Tg or CCh in Trp1-expressing cells, was about 1.5-2-fold higher than in Trp1-expressing cells and 4-fold higher than in control cells. Importantly, (i) basal Ca²⁺ influx and (ii) Tg- or CCh-stimulated internal Ca²⁺ release were similar in all the cells. A similar increase in Tg-stimulated Ca²⁺ influx was seen in cells expressing 2Trp1, lacking the C-terminal domain amino acid 649-793, which includes the EWKFAR sequence. Further, both inositol 1,4,5-trisphosphate receptor-3 and caveolin-1 were immunoprecipitated with Trp1 and Trp1. In aggregate, these data suggest that (i) the EWKFAR sequence does not contribute significantly to the Trp1-associated increase in SOCE, and (ii) the Trp1 C-terminal region, amino acids 664-793, is involved in the modulation of SOCE.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ influx in non-excitable cells occurs via the store-operated Ca²⁺ entry (SOCE)¹ mechanism, which is activated by the depletion of Ca²⁺ from the internal Ca²⁺ store (1-4). However, the nature of the signal that is transmitted from the internal Ca²⁺ store to the plasma membrane to trigger activation or inactivation of SOCE is not yet known. Three main models have been proposed for the activation of SOCE: (i) activation by second messengers such as cGMP or inositol 1,4,5-trisphosphate (IP₃) or mediators such as the calcium influx factor, which are either generated with, or in response to, the release of Ca²⁺ from the internal Ca²⁺ store; (ii) recruitment of channels into the plasma membrane by a process involving vesicle fusion; (iii) a physical interaction between the SOC channel in the plasma membrane and the IP₃ receptor (IP₃R) in the internal Ca²⁺ store membrane, i.e. the conformational-coupling hypothesis (1, 2, 5, 6). A major hurdle in establishing the mechanism of activation for SOCE has been the lack of information regarding the identity of the SOCE channel protein(s).

Recently, mammalian homologues of the Drosophila trp gene have been suggested to encode the SOCE channel protein (2, 3, 7). Seven different trps have been cloned. Expression of trp1 and trp4 was associated with increased SOCE, whereas expression of their antisense cDNAs resulted in a loss of SOCE (7-10). Expression of trp3 and trp6 induced increases in agonist- but not thapsigargin-stimulated Ca²⁺ influx (8, 11-14). Thus, although it is possible that some Trp proteins might be involved in SOCE, it remains to be established whether the Trps in fact form the SOCE channel. More importantly, studies with Trp proteins have provided data consistent with the conformational coupling hypothesis proposed for the regulation of SOCE. Two Trp proteins that have been shown to be localized in the plasma membrane, Trp3 and Trp1, appear to interact with the IP₃ receptor(s) (15-18).

We have recently reported that Trp1 is a strong candidate for the SOCE mechanism in the human submandibular gland cell line (HSG) and that it is associated with a caveolar lipid domain where it is assembled in a signaling complex with proteins such as IP₃Rs, G_q/11, and caveolin (8, 17). We proposed that protein-protein interactions coordinated within this domain are involved in the activation or inactivation of SOCE. To determine which region of the Trp1 molecule is involved in the activation of SOCE, we have now expressed truncated forms of Trp1 that lack (i) the C-terminal domain aa 664-793 (Trp1) or (ii) the C-terminal domain aa 649-793, which includes the highly conserved EWKFAR sequence (2Trp1), in HSG cells and measured SOCE. The data demonstrate that activation of SOCE was not altered by expression of truncated Trp1. Importantly, SOCE was increased in these cells compared with those expressing full-length Trp1, and IP₃R3 was immunoprecipitated with Trp1. These data are novel and suggest that the C-terminal domain of Trp1 is involved in the modulation of Ca²⁺ influx via the SOCE pathway.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

DNA Manipulation, HSG Cell Culture, and Transfection-- The 3'-untranslated 1.5-kilobase pair region of htrp1 cDNA used previously (8, 10) was deleted and full-length htrp1 was cloned into the pcDNA3 vector at the KpnI site. For the truncation, htrp1 was cleaved at the XhoI site (trp1, Fig. 1A), and religated. The plasmid was used to transform DH5 competent cells (Life Technologies, Inc.), and individual clones were selected. The truncated Trp1 protein (Trp1) has 9 amino acids (GGALFYSVT) carried over from the vector at the C-terminus, which is not homologous to any reported protein sequence. Another trp1 construct, trp1^v, lacking the vector sequence, was made by inserting a stop codon at the end of the Trp1 sequence by using a PCR-based strategy. In addition, 2trp1, generated by PCR, lacked the C-terminal domain (), including the EWKFAR sequence conserved in all Trp proteins. 2Trp1 also had a 12-amino acid sequence (EGGPYSIVSPKC) that was carried over from the vector, which was different from that in Trp1. All of the constructs were analyzed using restriction analysis and DNA sequencing.

HSG cells were cultured and stably transfected as described earlier (8, 19, 20). Cells were lysed and crude membranes were prepared as described previously (8). Protein concentration was determined by the Bio-Rad protein assay.

Immunoprecipitation-- Crude membranes were treated with 0.5% Nonidet P-40 or with 1.5 mM octylglucoside + 0.5 M KI, and centrifuged at 45,000 × g for 60 min. 200 µg of the pre-cleared supernatant was incubated with 10 µg of anti-HA antibody (Roche Molecular Biochemicals (17, 21). Immunocomplexes were pulled down with protein A, washed, and treated with SDS solubilization buffer. Proteins were detected by Western blotting as described previously (8, 21). Anti-caveolin-1 and anti-IP₃R3 (Roche Molecular Biochemicals) were used at 1:1000 dilution.

Confocal Microscopy-- Cells were fixed, permeabilized, and treated with anti-HA antibody (1:50) and rhodamine-linked secondary antibody as described previously (8). Images were collected by confocal microscopy (8). The entire series of images was then collected into a single focused image using Confocal Assistant software supplied by the manufacturer.

[Ca2+]_i Measurements-- Fura2 fluorescence in single cells was measured as described earlier (8, 17). Analog plots of the fluorescence ratio (340/380) in single cells are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of Truncated and Full-length Trp1 in HSG Cells-- The C terminus of hTrp1 was deleted between aa 664 and 793 (see Fig. 1A). Truncated hTrp1 (Trp1) and full-length (Trp1; both with N-terminal HA tag) were stably expressed in HSG cells. Molecular sizes of the inserts (Fig. 1B) were consistent with the predicted sizes, i.e. trp1 was 387 base pairs less than the full-length gene. These inserts were further characterized by digestion with restriction enzymes and sequencing (data not shown).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Expression of Trp1 and Trp1 in HSG cells. A, linear diagram of htrp1 and htrp1. The N-terminal HA-tag sequence is shown by the green box, the predicted transmembrane regions are shown by black boxes, and the EWKFAR region aa sequence is indicated above the boxed area (residues in red show the caveolin-binding consensus sequence). CMV, cytomegalovirus; bp, base pairs. B, htrp1 and htrp1 inserts obtained by digestion with KpnI or KpnI/XbaI, respectively (lower bands, indicated by arrows) and pcDNA3 vector (upper bands). C, detection of Trp1 and Trp1 by Western blotting using an anti-HA antibody (shown by arrows). D, immunolocalization of stably expressed Trp1 in HSG cells by confocal microscopy using anti-HA antibody and rhodamine-linked secondary antibody. Arrows indicate fluorescence in the plasma membrane region of the cell. As reported earlier (8, 21), fluorescence was also seen in the cytoplasmic region of some cells. Minimal fluorescence was detected in the absence of primary antibody (E). Similar results were obtained with incubation of the primary antibody with the HA-peptide (data not shown).

Fig. 1C shows Trp1 and Trp1 in crude membranes isolated from stably transfected cells. The lower band (78 kDa) corresponds to the expected molecular weight of Trp1. The proteins were also detected by the anti-Trp1 antibody. Notably, the relative level of endogenous Trp1 in Trp1-expressing cells was not higher than in Trp1-expressing cells (data not shown).

Fig. 1D shows the immunolocalization of Trp1 in HSG cells. Fluorescence was detected in the plasma membrane region of transfected cells, similar to the localization of stably expressed HA-tagged Trp1 in HSG cells (data not shown; see Ref. 8). Thus, deletion of the C terminus does not interfere with the localization of the Trp1 protein in HSG cells. Further, these data demonstrate that relatively similar levels of Trp1 and Trp1 are expressed in HSG cells stably transfected with the respective cDNAs.

Thapsigargin- and Carbachol-stimulated Ca²⁺ Influx in Trp1- and Trp1-expressing HSG Cells-- Fig. 2 shows thapsigargin (Tg)-stimulated [Ca²⁺]_i changes in control cells (nontransfected, labeled HSG) in Trp1- and Trp1-expressing HSG cells. Measurements were made in Ca²⁺-containing medium (Fig. 2, A and B) and in nominally Ca²⁺-free medium (Fig. 2, C and D). Average data are shown in the bar graphs (Fig. 2, B and D). In the presence of external Ca²⁺ (1.0 mM), Tg induced an initial, relatively fast increase in [Ca²⁺]_i in control cells, which returned to resting values in about 10 min. Part of the initial increase in [Ca²⁺]_i and the relatively sustained increase in [Ca²⁺]_i are caused by SOCE (8, 17). However, in cells stimulated with Tg in a Ca²⁺-free medium, peak [Ca²⁺]_i increases were lower, and the resting levels were reached within 4 min (Fig. 1C). The transient [Ca²⁺]_i increase is the result of internal Ca²⁺ release. With external Ca²⁺, Tg-stimulated initial peak increase was significantly higher (about 1.5-fold) in HSG cells expressing Trp1. Further, the sustained elevation in [Ca²⁺]_i, measured at 7.5 min after stimulation was also increased in these cells (by about 3-fold). These results are consistent with our previous studies (8). Importantly, cells expressing Trp1, also demonstrated an initial peak increase in [Ca²⁺]_i in response to Tg stimulation, which was similar to Trp1 cells but significantly higher than in control cells. More interestingly, the sustained elevation of [Ca²⁺]_i (at 7.5 min after stimulation) seen in these cells was about 1.5- and 4-fold higher than in Trp1-expressing and control cells, respectively. Fig. 2C shows that Tg-stimulated similar [Ca²⁺]_i changes when cells were stimulated in a Ca²⁺-free medium. Thus, the internal Ca²⁺ store status and Ca²⁺ release are similar in control, Trp1-, and Trp1-expressing cells and do not account for the increase in Ca²⁺ influx. Additionally, the resting Ca²⁺ permeabilities of the three sets of cells were similar. These were determined by the re-addition of 1.0 mM Ca²⁺ to cells in a Ca²⁺-free medium or by adding 5 or 10 mM Ca²⁺ to cells in a Ca²⁺-containing medium (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Thapsigargin-stimulated Ca2+ mobilization in Trp1- and truncated Trp1-expressing cells. [Ca2+]i changes, i.e. 340/380 nm fluorescence ratio, induced by 2 µM Tg in cells in Ca2+-containing medium (A) and Ca2+-free medium (C) are shown. Average data for the two conditions are shown in B and D, respectively. **, indicates values significantly different (p < 0.01) from the unmarked values in the set of data (i.e. at peak or 7.5 min); n, number of cells imaged. E, shows thapsigargin-stimulated fura2 fluorescence ratios in control cells, cells expressing Trp1, Trp1v (Trp1 minus vector sequence), and 2Trp1 (Trp1-minus aa 649-793, i.e. without EWKFAR sequence). * and **, indicate values significantly different (p < 0.05 and p < 0.01, respectively) from the unmarked values.

To exclude effects due to the vector sequence carried over in Trp1, another truncated Trp1 (Trp1^v) was expressed in HSG cells, which lacked the vector sequence. 2Trp1 was also expressed, which lacked the C-terminal domain aa 649-793. Note that a vector sequence different from that in Trp1 was carried over in 2Trp1. Significantly, SOCE in cells stably transfected with these cDNAs, was similar to that in cells transfected with trp1 (Fig. 2, D and E). In all three cases, SOCE was higher than in control cells and those expressing Trp1. Thus, the increased SOCE in cells expressing the truncated Trp1(s) is not due to contributions from the vector sequence that was carried over. Interestingly, the EWKFAR sequence does not appear to significantly affect Trp1 function (Fig. 2E). In aggregate, the data from Fig. 2 show that the activation of SOCE by Tg is not affected in HSG cells expressing Trp1 lacking the C-terminal domain. Importantly, these cells have a higher level of SOCE than cells expressing the full-length protein.

Carbachol (CCh)-stimulated Ca²⁺ mobilization was also measured in control and Trp1 and Trp1 cells in Ca²⁺-containing (Fig. 3, A and B) and Ca²⁺-free (Fig. 3, C and D) medium. As seen with Tg, sustained [Ca²⁺]_i increase (measured 5 min after stimulation was significantly higher in Trp1 cells than in Trp1 cells and control cells, by 1.8 and 4.4-fold, respectively. However, the initial peak increase was not significantly different in the three sets of cell. Fig. 3C shows that CCh-stimulated internal Ca²⁺ release is similar in all the cells. Thus, these data demonstrate that: (i) initial Ca²⁺ signaling events related to CCh stimulation of HSG cells are not altered by expression of Trp1 or Trp1; and (ii) the sustained Ca²⁺ increase seen in cells in Ca²⁺-containing medium, i.e. Ca²⁺ influx, is increased in cells expressing the Trp1 proteins. The Ca²⁺ entry component was also assessed by adding Ca²⁺ to cells treated with either CCh or Tg in Ca²⁺-free medium. These results also demonstrated significantly (2-fold) higher [Ca²⁺]_i elevation in Trp1 cells compared with Trp1 cells (data not shown).

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Carbachol-stimulated Ca2+ mobilization in Trp1- and Trp1-expressing cells. [Ca2+]i changes induced by 1 mM carbachol (CCh) in cells in Ca2+-containing medium (A) and Ca2+-free medium (C) are shown. Average data for the two conditions are shown in B and D, respectively. **, indicates values significantly different (p < 0.01) from the unmarked values in the data set (i.e. at peak or 5 min); n, number of cells imaged. Other details are given in the figure.

In aggregate, the data from Figs. 2 and 3 clearly demonstrate that SOCE is increased in HSG cells expressing the Trp1 proteins. More significantly, the data show that higher levels of Ca²⁺ influx are achieved when the truncated Trp1 proteins are expressed. Our data also rule out the possibility that this increase in SOCE is due to changes in the status of internal Ca²⁺ stores, an increase in basal Ca²⁺ influx, or differences in the level of protein expression.

Interaction of Trp1 with IP₃R3 and Caveolin 1-- To examine whether the C-terminal deletion alters the interaction of Trp1 with IP₃R, an anti-HA antibody was used to immunoprecipitate either Trp1 (Fig. 4, lane 2) or Trp1 (Fig. 4, lane 1). The immunoprecipitates were then analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using either anti-IP₃R3 or anti-caveolin1. The reactions shown in Fig. 4 clearly demonstrate that the relative level of IP₃R3 or caveolin1 co-immunoprecipitated with Trp1 is similar to that seen with Trp1.

View larger version (63K): [in this window] [in a new window]   Fig. 4.   Coimmunoprecipitation of Trp1 and Trp1 with IP3R3 and caveolin 1. Anti-HA antibody was used to immunoprecipitate the expressed Trp proteins. IP3R3 and caveolin1 were detected (shown by arrows) in immunoprecipitates of hTrp1 (lane 1) and hTrp1 (lane 2) by Western blotting.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have previously reported that (i) expression of hTrp1 in HSG cells induces as increase in SOCE, and (ii) Trp1 in HSG cells interacts with IP₃R and caveolin 1 (8, 17). The main findings of this study are that deletions in the C-terminal region of Trp1 do not appear to alter its ability to increase SOCE in HSG cells. Further, the ability of Trp1 to interact with IP₃R3 and caveolin 1 is also not altered, as shown by the coimmunoprecipitation of these proteins with Trp1. Importantly, the relative amounts of IP₃R3 coimmunoprecipitated with Trp1 or Trp1 were similar. Previous studies have shown that the IP₃R N-terminal peptide (aa 1-787) induced spontaneous Trp3 channel activity, although when the IP₃R N terminus was linked to the IP₃R-transmembrane regions, IP₃ was required for channel activation (15). Thus, it was proposed that Trp3 interactions with the IP₃R under "resting" conditions inhibits the channel. When Ca²⁺ is released from the store in the presence of IP₃, there was an alteration in the Trp3-IP₃R interaction, resulting in opening of the channel. Another study (16) demonstrated that a C-terminal domain of Trp3 (aa 777-797) is conserved among various Trps and is the putative site for interaction with IP₃R. Expression of the Trp3 domain aa 742-795 induced an inhibition of agonist- and thapsigargin-stimulated Ca²⁺ influx in cells expressing Trp3. An N-terminal domain in the IP₃R3 (aa 638-926) was identified as the region interacting with the C terminus of hTrp3. Two subdomains within this IP₃R region, aa 751-821 and aa 669-698, were identified as having stimulatory and inhibitory effects, respectively, on Trp3 activity. Also, some homology was identified in these domains among the various IP₃Rs.

Based on these previous reports and our studies showing that Trp1 expression increases SOCE in HSG cells and Trp1 interacts with IP₃R3 (8, 17), we predicted the following possible outcomes of Trp1 expression in HSG cells. (i) There would be no effect on SOCE if the Trp1 C terminus interaction with IP₃R was required for opening the channel. In this case, Trp1 would not interact with IP₃R because of lack of the C terminus. (ii) There would be no effect on SOCE if Trp1 interacted with IP₃R via another domain but gating was mediated via the C terminus. In this case, Trp1 and IP₃R would interact, but the channel would be silent. (iii) SOCE would be constitutively activated if IP₃R interaction with the Trp1 C terminus during gating relieved an inhibition of the gate by the Trp1 C terminus. In this case, removing the inhibitory domain would allow the gate to be opened and Trp1 would function as an open channel. (iv) Inhibition of SOCE would occur if the truncated channel could not be gated but it interacted with IP₃R and competed with the endogenous SOCE channel, i.e. a dominant negative effect. All four of these possibilities were excluded by the data discussed above. We have shown that expression of Trp1 induced no change in the ability of either carbachol or thapsigargin to induce SOCE in HSG cells, ruling out predictions i, ii, and iv. Further, no increase in basal Ca²⁺ permeability was noted in Trp1-expressing cells, ruling out possibility iii. Importantly, the level of SOCE seen in Trp1 cells was greater than that seen in Trp1-expressing cells. The data suggest that the difference in the SOC between Trp1 and Trp1 cells is primarily in the sustained level of [Ca²⁺]_i, indicating that there is relatively more Ca²⁺ entering the Trp1-expressing cells.

The data described above suggest that the C terminus of Trp1 likely acts as an inhibitory domain of the SOCE channel and restricts the amount of Ca²⁺ entering the cells. This suggestion is based on the observation that there is an increase in SOCE when this domain is removed. As proposed previously, Trp1 might form the SOCE channel either by itself or in association with other subunits. Thus, the marked effects of the C-terminal truncation on SOCE are consistent with this proposal. Interestingly, a similar increase in Ca²⁺ influx activity was reported following deletion of the C terminus of the -subunit of the cardiac L-type voltage-gated Ca²⁺ channels (22). However, electrophysiological measurements of single-channel events will be required to understand exactly how C-terminal deletion of Trp1 results in increased SOCE. It is possible that putative phosphorylation sites, or direct binding of lipids or proteins such as calmodulin, might be involved in the Trp1 C terminus-mediated feedback regulation of SOCE. Alternatively, as suggested by previous reports and analogous to Trp3 (15, 16, 18), direct interactions of Trp1 with the IP₃R could modulate Ca²⁺ influx. Because IP₃R coimmunoprecipitates with Trp1, which lacks the C-terminal domain, our data suggest that IP₃R might bind to other sites on Trp1. However, presently, we cannot rule out the possibility that Trp1 indirectly associates with IP₃R via interactions with endogenous Trp or other protein(s), which in turn is associated with IP₃R. Despite how Trp1 interacts with IP₃R, the present data strongly suggest that the C-terminal region of hTrp1 exerts an inhibitory effect on SOCE, which likely provides a feedback regulation of Ca²⁺ influx. Further, we report here for the first time that the EWKFAR region in Trp1 does not contribute significantly to its functional effects on SOCE.

In conclusion, we have shown that deletion of the C terminus of hTrp1 does not affect its ability to be activated via store-depletion and increase SOCE in HSG cells. Further, the levels of Ca²⁺ influx associated with the expression of the truncated Trp1 proteins are higher than that associated with the expression of the full-length Trp1. Thus, our data support the suggestion that the C-terminal of hTrp1 is involved in modulating SOCE, likely by exerting a feedback inhibitory effect.

	ACKNOWLEDGEMENTS

We thank Deborah Liao for technical assistance, Drs. Bruce Baum, Robert Wellner, Suresh Ambudkar, and Tim Lockwich for invaluable help, and Drs. Michael Zhu and Shmuel Muallem for useful discussions.

	FOOTNOTES

^* 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.

To whom correspondence should be addressed: Bldg. 10, Rm. 1N-113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5298; Fax: 301-402-1228; E-mail: ambudkar@yoda.nidcr.nih.gov.

Published, JBC Papers in Press, September 8, 2000, DOI 10.1074/jbc.C000529200

	ABBREVIATIONS

The abbreviations used are: SOCE, store-operated Ca²⁺ entry; IP₃, inositol 1,4,5-trisphosphate; IP₃R, IP₃ receptor; HSG, human submandibular gland; aa, amino acid(s); Tg, thapsigargin; CCh, carbachol; Trp, transient receptor potential protein; PCR, polymerase chain reaction.

	REFERENCES

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