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Proc Natl Acad Sci U S A. 2001 September 11; 98(19): 10692–10697. Published online 2001 September 4. doi: 10.1073/pnas.191360198. | PMCID: PMC58528 |
Copyright © 2001, The National Academy of Sciences Cell Biology Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform X.-Z. Shawn Xu,*† Fabian Moebius,*‡ Donald L. Gill,§ and Craig Montell*¶ *Departments of Biological Chemistry and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; and §Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201 Received June 1, 2001; Accepted July 16, 2001. The TRP (transient receptor potential) superfamily includes a group of subfamilies of channel-like proteins mediating a multitude of physiological signaling processes. The TRP-melastatin (TRPM) subfamily includes the putative tumor suppressor melastatin (MLSN) and is a poorly characterized group of TRP-related proteins. Here, we describe the identification and characterization of an additional TRPM protein TRPM4. We reveal that TRPM4 and MLSN each mediate Ca2+ entry when expressed in HEK293 cells. Furthermore, we demonstrate that a short form of MLSN (MLSN-S) interacts directly with and suppresses the activity of full-length MLSN (MLSN-L). This suppression seems to result from the inhibition of translocation of MLSN-L to the plasma membrane. We propose that control of translocation through interaction between MLSN-S and MLSN-L represents a mode for regulating ion channel activity. The transient receptor potential (TRP) superfamily of Ca 2+-permeable cation channels includes a diverse group of channel-like proteins that resemble the prototype Drosophila TRP channel ( 1– 4). Members of the TRP superfamily function in processes ranging from vasorelaxation ( 5) to phototransduction ( 6), thermal hyperalgesia ( 7, 8), mechanosensation ( 9, 10), and the acrosomal reaction and osmosensation ( 11– 13). Among the six subfamilies of TRP proteins, the three with the greatest structural and sequence homology to Drosophila TRP are TRP-classic (TRPC), TRP-vanilloid (TRPV), and TRP-melastatin (TRPM) ( 3). Whereas most members of the first two subfamilies have been expressed and functionally characterized, the majority of TRPM proteins have not. Members of the TRPM subfamily share ≈20% amino acid identity with TRP over a 325-aa segment that includes the C-terminal five transmembrane domains and a highly conserved 25-residue TRP domain ( 3). The founding member of the TRPM subfamily, melastatin (MLSN), is encoded by a gene that was isolated in a screen for genes that were down-regulated in mouse melanoma tumor-cell lines. As such, it has been suggested to be a tumor-suppressor gene ( 14, 15). The expression of MLSN inversely correlates with the severity of the melanocystic tumors isolated from patients ( 16). Moreover, treatment of melanoma cells with an agent that induces differentiation and reduces features associated with metastatic melanomas greatly potentiates MLSN expression ( 17). A recent study of patients with localized malignant melanomas indicates that down-regulation of MLSN RNA is a prognostic marker for metastasis ( 18). MLSN is alternatively spliced, and one of the major isoforms is predicted to encode a short protein (MLSN-S) that includes only the N-terminal segment but not any transmembrane domain ( 17). Consequently, MLSN-S would be incapable of functioning independently as an ion channel. Two other TRPM proteins have been described that map to chromosomal positions associated with human diseases or that display alterations in expression levels in tumor cells. One such protein, MTR1, maps to a region on chromosome 11 associated with Beckwith–Wiedemann syndrome and several neoplasms ( 19, 20). TRP-p8 is a prostate-enriched gene that is up-regulated in a variety of tumors. The gon-2 gene in Caenorhabditis elegans encodes a TRPM protein, and mutations in this locus delay or eliminate mitotic divisions of gonadal precursor cells ( 21). Despite the potential importance of each of these TRPM proteins, and despite the fact that they share channel-like structural motifs with the TRP superfamily, no expression studies have been undertaken to examine their cellular function or the mode by which these putative channels are regulated. Recently, two unusual members of the TRPM family have been shown to consist of TRP channel domains fused to C-terminal enzyme domains. One such protein, TRPC7, is a candidate gene for several diseases, including bipolar affective disorder and nonsyndromic hereditary deafness ( 22). This protein is referred to here as “TRPM2,” because “TRPC7” is also used to designate a TRPC subfamily protein ( 23). Interestingly, TRPM2 includes an ADP-ribose pyrophosphatase domain and seems to be activated by ADP-ribose ( 24). Another protein, TRP-PLIK (also LTRPC7), contains a C-terminal serine/threonine kinase domain ( 25). According to one report, TRP-PLIK requires protein kinase activity for channel function. However, another study does not invoke a requirement for the kinase domain for channel activity. Rather, TRP-PLIK may be regulated by Mg 2+-ATP ( 26). Given that none of the other TRPM proteins have been functionally expressed, the question arises as to whether those TRPM proteins lacking a C-terminal enzyme domain are ion channels capable of functioning in the absence of a regulatory C-terminal enzyme domain. In the current work, we functionally expressed two members of the TRPM subfamily, MLSN and a previously undescribed TRPM protein, TRPM4. Each of these proteins lacks a linked enzyme domain, and yet each protein mediates cation influx in HEK293 cells. Moreover, we determined that MLSN-S, which lacks the channel-forming transmembrane domains, interacts directly with MLSN-L and suppresses the functional channel activity of the full-length form (MLSN-L) by inhibiting its translocation to the plasma membrane. We propose that the interaction between MLSN-S and MLSN-L provides a mechanism for regulating channel activity. Cloning of TRPM4. The expressed sequence tag clone H18835, encoding a fragment of TRPM4, was used to screen human brain, placenta, and testis cDNA libraries. Several positive clones were recovered, including a 4.0-kb cDNA encoding a predicted protein of 1,040 aa. The TRPM4 cDNA was subcloned into the vector pcDNA3 (Invitrogen) to create pTRPM4. The coding region was fused to an N-terminal FLAG tag. Construction of MLSN Clones. The coding regions of the human cDNAs encoding MLSN-L (1,533 aa) and MLSN-S (500 aa) were fused to N-terminal MYC tags and subcloned in pcDNA3 to create pcDNA3-hMLSN and pcDNA3-N-MLSN-500, respectively. RNA Blot Analyses. Multiple-tissue RNA blots (CLONTECH) were probed according to standard procedures with a 32P-labeled 1.5-kb fragment generated from the 3′ end of the TRPM4 cDNA. The fetal RNA was pooled from various developmental stages (9–26 weeks). Transfections and Ca2+ Imaging. Transfections of HEK293 cells were performed with Lipofectamine (BRL) according to the manufacturer's instructions. A plasmid encoding yellow fluorescent protein (YFP) was cotransfected at ≤1/15 of the total DNA. The ratio of pMLSN-S and pMLSN-L DNA used for the transfections was 2:1. The cells were replated on coverslips coated with polylysine 24 hr after transfection, and the Ca2+ imaging experiments were performed 12–24 hr later. The 340- and 380-nm images were acquired with a cooled charge-coupled device camera (CoolSNAPfx, Roper Scientific, Trenton, NJ) and processed with RATIOTOOL software (Inovision, Raleigh, NC). Cells were loaded with 5 μM fura-2-AM (Calbiochem) at 37°C for 30 min in a Ca2+-containing solution (140 mM NaCl, pH 7.4/1.2 mM MgCl2/2.5 mM CaCl2/10 mM glucose/14 mM Hepes/4 mM KCl). After the fura-2-AM solution had been removed, cells were maintained in the Ca2+ solution until the experiments were initiated. To select cells for the analyses, fields were randomly chosen before the UV light was turned on. All YFP-positive cells in a randomly chosen field (typically 10–20 cells) were selected for data collection. Individual traces selected for display (Figs. E and F and A and B) were similar to the average trace in a given experiment. The Ca2+-free solution was identical to the Ca2+ solution except that 2.5 mM EGTA was substituted for the Ca2+. The Sr2+- or Ba2+-containing solutions were prepared by adding 5 mM SrCl2 or BaCl2 to the Ca2+-free solution. To prepare the hypotonic solution (hOs), the Ca2+ solution was diluted 33% with H2O, and additional CaCl2 was added to maintain the Ca2+ concentration at 2.5 mM. Sucrose (10%) was added to the Ca2+-containing solution to generate the hypertonic solution. Immunofluorescence. A plasmid encoding green fluorescent protein (GFP) was cotransfected in HEK293 cells at ≤1/15 of the total DNA. Cells were replated on polylysine-coated coverslips 24 hr after transfection and incubated for ≈12 hr at 37°C. Cells were fixed with 3.7% (vol/vol) formaldehyde in PBS. The cells then were permeabilized in PBS containing 0.5% Triton X-100, blocked in PBS containing 2% (vol/vol) newborn goat serum and 1% BSA, incubated with the primary antibodies, and then incubated with the secondary antibodies. The confocal images were acquired with an Oz confocal laser scanning microscope (Noran Instruments, Middleton, WI) and processed for three-dimensional deconvolution. To select cells for analyses, one investigator performed the immunostainings and a second investigator, who was unaware of the plasmids used for the transfections, selected and photographed random fields. All of the stainings were repeated at least three times. Glutathione S-Transferase (GST) Pull-Down Assays. The cDNA encoding MLSN-S was inserted in pGEX-KG and the GST-MLSN-S fusion protein was expressed in Escherichia coli (BL-21) by induction with isopropyl β-d-thiogalactoside. The fusion protein or GST alone was purified with glutathione beads, as described by the manufacturer (Amersham Pharmacia), by taking advantage of the specific affinity of GST for glutathione. The purified GST-MLSN-S (0.5 μg) or GST (1 μg) was immobilized on glutathione beads (25 μl) and incubated with MLSN-L or MLSN-S translated in vitro with [35S]methionine (10 μl, Promega) in TBST [20 mM Tris/150 mM NaCl/0.5% Triton X-100 containing protease inhibitors (Roche Molecular Biochemicals), pH 7.5] for 1.5 hr. After three washes, the bound beads were eluted with SDS sample buffer, and the eluates were fractionated by SDS/PAGE followed by autoradiography. TRPM4, a Member of the TRPM Family. To identify new members of the TRPM family, we scanned the Expressed Sequence Tags database (at http://www.ncbi.nlm.nih.gov/dbEST/) and identified a human cDNA clone ( H18835) that included a 105-aa deduced sequence that exhibited significant homology to known TRPM proteins. Subsequently, we screened several human cDNA libraries and isolated a cDNA encoding a predicted protein of 1040 aa with six putative transmembrane domains (Fig. ). We refer to this protein as TRPM4 (TRP-MLSN-4) because it displays a high level of sequence identity (≈30–40%) to members of the TRPM subfamily over nearly the entire protein. Within the TRPM subfamily, TRPM4 was most related to MTR1 and least similar to TRP-p8 (Fig. A). As is the case with other TRPM proteins, TRPM4 displayed ≈20% identity to members of the TRPC subfamily over a 325-aa region that included the C-terminal five transmembrane segments and the TRP domain. The TRPM4 RNA was expressed in most adult tissues, but at the highest levels in the heart, prostate, and colon (Fig. B). No transcripts were detected in leukocytes. In the fetus, TRPM4 RNA was most abundant in the kidneys. There were at least two distinct bands detected on the Northern blot (6.2 kb and 4.2 kb), indicating alternative splicing of TRPM4. In addition to the two major RNA species, a much smaller but relatively abundant 2.4-kb RNA was detected in the testes (Fig. B). Examination of the Human Genome database (at http://www.ncbi.nlm.nih.gov/genome/guide/human) indicates that the TRPM4 gene maps to 19q13.32. Two human diseases map to this position: orofacial cleft-3 and autosomal dominant spastic paraplegia. TRPM4 Mediates Ca2+ Entry. To determine whether the expressed TRPM4 protein may have Ca2+ channel properties, we transiently expressed TRPM4 in HEK293 cells and undertook protein localization studies as well as fura-2 ratiometric Ca2+-imaging analyses. To identify successfully transfected cells, we cotransfected cells with pTRPM4 and pYFP. The TRPM4 protein was localized very close to the plasma membrane, as indicated by confocal microscopy (Fig. A and B). We found that transient expression of TRPM4 increased Ca2+ entry activity in HEK293 cells. Cells expressing YFP (Fig. C; top three traces) typically had a higher basal concentration of Ca2+ than nontransfected cells (Fig. C; bottom trace). Moreover, removal of Ca2+ from the bath solution triggered a decrease in Ca2+ in the YFP-positive cells but not in the nontransfected cells (Fig. C) or in the control cells cotransfected with the empty pcDNA3 vector and pYFP (Fig. D). Reintroduction of Ca2+ to the bath solution resulted in rapid Ca2+ entry in most of the cells cotransfected with pTRPM4 and pYFP (72% ± 4%, n = 180). The nonresponding cells (28%) may represent cells that did not express TRPM4 or expressed TRPM4 at very low levels. After Ca2+ entry, the Ca2+ concentration in the pTRPM4-transfected cells transiently rose above the basal Ca2+ level, suggesting that TRPM4 may undergo Ca2+ feedback regulation. No such entry of Ca2+ was observed in nontransfected cells (Fig. C, bottom trace) or in transfected control cells (Fig. D). To characterize the TRPM4-dependent influx further, we tested the effects of cations that inhibit or permeate known channels within the TRP superfamily. The TRPM4-mediated Ca 2+ entry was rapidly blocked by 80 μM La 3+ (Fig. E, n = 38) as well as by 80 μM Gd 3+ (data not shown). When Sr 2+ rather than Ca 2+ was present in the bath solution, there was no change in fluorescence, indicating that the TRPM4 channels were impermeable to Sr 2+ (Fig. F, n = 25). Ba 2+ entered the cells efficiently (Fig. F, n = 25), although the high level of Ba 2+ accumulation is, in part, caused by the inability of pumps to remove Ba 2+ from the cytosol. Nontransfected cells did not exhibit Ba 2+ influx over a similar time scale; however, low levels of Ba 2+ influx were detected after ≈50 min (data not shown). Unlike some TRPV proteins, TRPM4 did not respond to alterations in osmolarity (Fig. F, n = 25). These data indicate that TRPM4 is able to function as a divalent cation channel with a specificity for ions of Ca 2+ ≥ Ba 2+ ![[dbl greater-than sign]](/corehtml/pmc/pmcents/x226B.gif) Sr 2+. MLSN Is a Plasma Membrane Protein Mediating Ca2+ Entry. To obtain additional evidence that members of the TRPM subfamily promote cation influx, we expressed the founding member of the TRPM subfamily, MLSN, in HEK293 cells and performed Ca 2+ imaging. Expression of MLSN in these cells led to a pronounced Ca 2+ entry upon switching the cells from the Ca 2+-free to the Ca 2+-containing solution; however, alterations in osmolarity had no effect (Fig. A, n = 280). Nontransfected cells or cells cotransfected with pYFP and the empty pcDNA3 vector did not display changes in Ca 2+ concentration upon switching from the Ca 2+-free to the Ca 2+-containing bath solutions (Fig. D). As was the case with TRPM4, Ca 2+ entry mediated by MLSN was blocked by La 3+. There was no Sr 2+ permeation, and Ba 2+ entry was considerably slower than Ca 2+ entry (Fig. B, n = 32), indicating that MLSN channels have a divalent cation selectivity with Ca 2+ > Ba 2+ Sr 2+. To determine the subcellular localization of MLSN, we performed immunofluorescent microscopy on transfected HEK293 cells. The confocal images indicated that the MLSN protein was localized at or in close proximity to the plasma membrane (Fig. C and D). Direct Protein Interaction Between MLSN Isoforms. MLSN is alternatively spliced, resulting in the production of a long form (MLSN-L; 1533 residues; ref. 15) and a short, N-terminal form devoid of any putative transmembrane segments [MLSN-S; 542 residues in the mouse ( 14) and 500 residues in humans ( 17)]. Whereas expression of MLSN-L promoted Ca 2+ influx (Fig. A and B), introduction of MLSN-S in HEK293 cells did not (data not shown). We considered the possibility that this short isoform may play a regulatory role. To test this possibility, we first examined whether MLSN-S could directly interact with MLSN-L by performing in vitro pull-down assays. We found that 35S-labeled MLSN-L interacted with GST-MLSN-S but not with GST (Fig. E). MLSN-S interacted strongly with itself as 35S-labeled MLSN-S bound efficiently to GST-MLSN-S (Fig. F). MLSN-S and MLSN-L also coimmunoprecipitated after expressing the proteins in vitro (data not shown). MLSN-S Suppresses the Activity of MLSN-L. The observation that MLSN-S interacted directly with MLSN-L raised the possibility that MLSN-S may regulate the activity of MLSN-L. Therefore, we tested the functional consequences of coexpressing MLSN-S and MLSN-L in HEK293 cells. We found that introduction of MLSN-S along with MLSN-L significantly suppressed the MLSN-L-dependent Ca2+ entry (Fig. ). The percentage of cells exhibiting Ca2+-influx activity was reduced by 65.5% [from 61% ± 3% (n = 208) to 21% ± 1% (n = 192), Fig. E] and the overall Ca2+-influx activity was suppressed by 75.5% (Fig. F). In addition to the reduction in the level of Ca2+ influx, those cells coexpressing MLSN-S and MLSN-L displayed a longer lag between introduction of Ca2+ to the bath solution and production of the peak levels of intracellular Ca2+ (162 sec to 288 sec; Fig. C and D). MLSN-S Alters the Localization of MLSN-L. Having identified an inhibitory effect of MLSN-S on MLSN-L activity, we attempted to explore its underlying molecular mechanism. One possibility was that MLSN-S could potentially block transport of MLSN-L to the plasma membrane. To test this hypothesis, we compared the spatial distribution of MLSN-L in the presence and absence of MLSN-S. Expression of just MLSN-L resulted in a localization juxtaposed to or in the plasma membrane (Fig. C and D and Fig. A and B). Introduction of MLSN-S alone in HEK293 cells resulted in a cytosolic-staining pattern (Fig. C). Strikingly, upon coexpression of MLSN-S with MLSN-L, we found that MLSN-L was no longer enriched in the plasma membrane. Instead, MLSN-L was detected primarily in the cytosol and displayed a spatial distribution that overlapped extensively, but not completely, with MLSN-S (Fig. D–F). Whereas MLSN-S appeared to be uniformly distributed in the cytoplasm, the spatial distribution of MLSN-L had a more punctate appearance. Members of the TRPM family are candidate proteins for involvement in several types of human disease. However, functional characterization of TRPM family members has been limited. In this article, we demonstrate that expression of either TRPM4 or MLSN in HEK293 cells elicited Ca2+ influx, indicating that these members of the TRPM subfamily function as Ca2+-permeable channels. MLSN is expressed in melanocytes and has been described as a tumor suppressor that may regulate the metastatic potential of melanomas ( 14, 16). However, it is unclear how reduction of MLSN function may promote metastasis. Our observation that MLSN mediates Ca 2+ entry suggests a role for Ca 2+ in regulating the metastatic potential of melanomas. Although the properties of the MLSN and TRPM4-dependent cation entry are not identical, they seemed to be similar. Expression of either of these proteins promoted entry of Ca2+ and Ba2+, but not Sr2+. Moreover, the channels (when expressed) seemed to be at least partially opened and subject to a negative feedback effect by Ca2+. The Ca2+ influx mediated by either of these two proteins was inhibited by either 80 μM La3+ or 80 μM Gd3+. Thus, MLSN and TRPM4 seemed to function autonomously as cation channels, although neither of these proteins included a linked enzyme domain like TRP-PLIK and TRPM2 (TRPC7). Given that neither MLSN nor TRPM4 contains a regulatory enzyme domain, we attempted to identify an alternative process that might regulate these TRPM proteins. The observation that one of the major MLSN RNAs encodes an isoform, MLSN-S, that is devoid of predicted transmembrane domains and channel activity, prompted us to speculate that MLSN-S might regulate the activity of MLSN-L. Consistent with this proposal, we found that MLSN-S interacted directly with MLSN-L. The interaction seemed to occur through the N terminus of MLSN-L because MLSN-S, which consists of the N-terminal 500 residues of MLSN-L, can also form homomeric interactions. The Drosophila TRPC proteins TRP, TRP-like (TRPL), and TRPγ also interact in various pairwise combinations through their N termini ( 27, 28). However, the N termini of TRPM and TRPC proteins do not display sequence homology. In further support for a regulatory role for MLSN-S, we found that coexpression of the two proteins interfered with transport of MLSN-L to the plasma membrane. Consequently, there was a reduction in the activity of MLSN-L. We suggest that the function of MLSN-S is to regulate transport of MLSN-L to the plasma membrane. According to this model, MLSN-L is retained in intracellular compartments through association with MLSN-S under basal conditions. Upon stimulation of the relevant as-yet-unidentified signaling pathway, MLSN-S is then released, thereby permitting transport of MLSN-L to the plasma membrane, thus permitting Ca2+ entry. Retention of MLSN-L in an intracellular compartment may be the critical mode regulating Ca2+ influx, as localization of MLSN-L in the plasma membrane leads to constitutive Ca2+ influx. Alternative splicing also occurs with other TRPM members. In the case of MTR1, an alternatively spliced isoform encodes a shorter protein, which is unlikely to possess channel activity because it truncates the last two transmembrane segments and the entire C-terminal domain ( 20). The short form of MTR1 includes the highly conserved N-terminal portion that mediates channel interaction in MLSN. Thus, it is possible that this alternatively spliced MTR1 isoform may also play a regulatory role. Although we do not know the structure of the protein encoded by the testis-specific 2.4-kb transcript of TRPM4, it would be predicted to be considerably shorter than the TRPM4 protein characterized in this article and could potentially serve a regulatory role as well. We suggest that interactions between alternative isoforms and retention in the cytoplasm may be a common mechanism for regulating the activity of those TRP proteins that form constitutively active influx channels. We thank V. Setaluri for providing the cDNAs encoding MLSN-S and MLSN-L. F.M. was supported by Human Frontier Science Program Fellowship LT0518/1998-M. This work was supported by Grant EY10852 from the National Eye Institute (to C.M.). | | GST | glutathione S-transferase | | | GFP | green fluorescent protein | | | MLSN | melastatin | | | MLSN-L | full-length MLSN | | | MLSN-S | short form of MLSN | | | TRP | transient receptor potential | | | TRPC | TRP-classic | | | TRPM | TRP-melastatin | | | YFP | yellow fluorescent protein |
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