1Departments of Pediatrics and 2Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033; 3The Henry Hood Research Program, The Sigfried and Janet Weis Center for Research, The Geisinger Clinic, Danville, Pennsylvania 17822; 4Division of Cellular and Molecular Biology, Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada; and 5Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153
Submitted 2 June 2004 ; accepted in final form 18 August 2004
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ABSTRACT |
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transient receptor potential protein channels; erythropoietin receptor; calcium channels
The TRP protein superfamily comprises a diverse group of voltage-independent Ca2+-permeable cation channels that are related to the archetypal Drosophila TRP and expressed in mammalian cells (15, 3537). Many mammalian TRP channels (TRPC) are activated after stimulation of receptors, and most of these receptors activate different isoforms of phospholipase C (PLC) (15, 36, 37). Activation of PLC results in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Some TRPC are Ca2+ store release operated: one proposed mechanism of activation is that IP3-mediated release of Ca2+ from internal stores results in a conformational change in IP3 receptors (IP3R), inducing a conformational change and opening of the store-operated TRPC (36). However, a direct interaction between TRPC and IP3R was recently demonstrated for all TRPC and for all IP3R (6, 19, 20, 29, 42). High IP3 concentrations in the vicinity of IP3R, resulting from close association of IP3R with PLC-coupled receptors, have been proposed to directly activate IP3R and the associated TRPC (13). For TRPC2, interaction with IP3R type III in the vomeronasal (VMO) organ has been reported (7). Some TRPC (TRPC3, TRPC6) can also be activated by DAG (16, 28, 30, 45).
Epo binding activates Epo-R by inducing a conformational change in Epo-R, resulting in activation of Jak2 through transphosphorylation (21). Jak2 subsequently phosphorylates some or all of the eight tyrosines in the intracellular domain of Epo-R, and phosphorylation of tyrosine residues on Epo-R attracts other intracellular proteins to bind to Epo-R via SH2 domains (21, 47). Epo stimulation of its receptor induces tyrosine phosphorylation and activation of both PLC1 and PLC
2 (5, 31, 32, 40). In addition, Epo stimulation results in the production of IP3 (40) and DAG (2).
In the present study, we examined the mechanism of activation of TRPC2 by Epo. In both primary erythroblasts and Chinese hamster ovary (CHO)-S cells transfected with Epo-R and TRPC2, Epo stimulated a significant increase in [Ca2+]i. Epo-induced [Ca2+]i increase was inhibited by pretreatment with the PLC inhibitor U-73122 and by downregulation of PLC1 with RNA interference (RNAi), demonstrating the requirement for PLC activity in Epo-modulated Ca2+ influx. In cells transfected with Epo-R and TRPC2 mutants with deletions of IP3R binding sites, Epo failed to stimulate a significant rise in [Ca2+]i, demonstrating the requirement for IP3R interaction with TRPC2 in Epo-induced Ca2+ influx. TRPC2 constitutively associated with Epo-R, PLC
1, and IP3R. These data suggest the existence of a signaling complex consisting of Epo-R, PLC
, IP3R, and TRPC2. They support the conclusion that after Epo stimulation, PLC
activation produces IP3, which activates IP3R, and IP3R interaction with TRPC2 contributes to and is required for channel opening.
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EXPERIMENTAL PROCEDURES |
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Tissue and cells lines. CHO-S cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS) and 0.1 mM nonessential amino acids. Human embryonic kidney (HEK)-293T cells were cultured in DMEM with 10% FCS. Splenic erythroblasts were obtained by injecting C57Bl/6 mice (812 wk of age) with phenylhydrazine (60 mg/kg) intraperitoneally on days 1 and 2 (1, 22). Mice were killed on day 5 by cervical dislocation, the spleens removed, and single-cell suspensions were prepared. To separate splenic erythroid cells into different stages of maturation, the spleen cell suspensions were washed and labeled with TER-119 MicroBeads (10 µl/1 x 107 cells; Miltenyi Biotech, Auburn, CA). TER-119 (erythroid progenitors and early precursors) and TER-119+ cells (more mature nucleated erythroblasts and erythrocytes) were selected by magnetic sorting with the VarioMACS (Miltenyi Biotech) as described previously (12, 18). Wright's staining and immunofluorescence confirmed that >90% of TER-119 and TER-119+ nucleated cells were erythroid (12).
Transfection of mTRPC and Epo-R into CHO-S cells. TRPC2 clone 14 (c14) (44), TRPC2 with IP3R binding mutants, and rat TRPV1 (8) were subcloned into pcDNA3, pQBI50 (QbioGene, Carlsbad, CA), or pcDNA3.1/V5-His TOPO (Invitrogen, Carlsbad, CA). Other constructs were used in some experiments (see below). CHO-S cells at 5070% confluence were transfected with these vectors and/or pTracer-CMV expressing Epo-R with the use of Lipofectamine Plus (Invitrogen) or Lipofectamine 2000 in accordance with the manufacturer's recommended transfection protocols (11). CHO-S cells were routinely studied 48 h after transfection.
Measurement of [Ca2+]i with digital video imaging.
The fluorescence microscopy-coupled digital video imaging system used to measure changes in [Ca2+]i was described previously (911, 33, 34). After isolation and separation with the VarioMACS, TER-119 splenic erythroblasts were incubated in Iscove's modified Dulbecco's medium (GIBCO-BRL, Grand Island, NY) containing 2% FCS and 50 µM -mercaptoethanol for 24 h without growth factor. Splenic erythroblasts were then adhered to fibronectin-coated glass coverslips and loaded for 20 min with 0.1 µM fura-2 AM (Molecular Probes, Eugene, OR). For some experiments, the active PLC inhibitor U-73122 (0.12.5 µM; Sigma, St. Louis, MO) or the inactive inhibitor U-73343 (Sigma) were included in the buffer during fura-2 loading and during Epo stimulation. Fura-2-loaded cells were visualized with digital video imaging, and fluorescence was quantitated by using the fluorescence intensity ratio (R) of the emission (510 nm) measured after excitation at 350 nm divided by the emission after excitation at 380 nm. The free intracellular calcium concentration ([Ca2+]i) was calculated from fura-2 fluorescence signals using the in vivo calibration method and the equation
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We were not able to use fura-2 as the detection fluorophore to study changes in [Ca2+]i in transfected cells, because its excitation and emission wavelengths overlap with those of green fluorescent protein (GFP). Instead, we used the fluorescent indicator fura red (excitation, 440 and 490 nm; emission, 600 nm long pass), a dual-wavelength excitation probe (12, 23, 48). Transfected CHO-S cells grown on glass coverslips were loaded at 48 h with 5 µM fura red for 2025 min at 37°C in the presence of Pluronic F-127. Cells were treated with 0 or 40 U/ml of Epo. In some experiments, 680 µM MnCl was substituted in the extracellular buffer for CaCl. In others, cells were exposed to 3.5 µM thapsigargin after baseline [Ca2+]i measurements or to 0.110 µM U-73122 or U-73343 during fura red loading and during Epo stimulation. [Ca2+]i was measured by determining of the fluorescence intensity ratio R (F440/F490), which was measured at intervals of 5 s for the first 30 s, intervals of 515 s for the next 24 min, and at 2- to 5-min intervals over the remaining 20-min time course after treatment with PBS or Epo. We calibrated the constants Sf2, Sb2, and Kd of fura red and measured Rmin and Rmax for fura red as described previously (12). [Ca2+]i was calculated using the same formula used for calculations with fura-2.
Statistical significance of results was analyzed by performing one-way analysis of variance. In experiments with HEK-293T cells, the cells were transfected under conditions identical to those used for CHO-S cells. HEK-293T cells were loaded 48 h after transfection with 2.5 µM fura red and [Ca2+]i measured as described for CHO-S cells.
Downregulation of PLC with RNAi.
To reduce endogenous expression of PLC
, PLC
1 SMARTpool small interfering RNA (siRNA) reagent (no. 60034; Upstate, Charlottesville, VA) targeted to human PLC
1 was cotransfected into HEK-293T cells with Epo-R in pTracer-CMV and TRPC2 c14 in pQBI50 (24). NonSpecific Control SMARTpool siRNA reagent was cotransfected in control cells. SiRNA reagents were transfected using the manufacturer's recommended protocol at a final concentration of 200 pmol/35-mm dish, with Lipofectamine Plus used as the transfection reagent. At 48 h, cells were used for [Ca2+]i measurements or for Western blotting.
Immunolocalization of TRPC2, Epo-R, PLC, and IP3R in primary erythroid cells and transfected CHO-S cells.
TER-119 splenic erythroblasts (3 x 105 cells/chamber) were placed in each well of Lab-Tek Permanox chamber slides precoated with fibronectin. After 45 min, cells were washed twice with PBS, fixed in methanol at 20°C for 10 min, and permeabilized in 0.5% Triton X-100 in PBS for 2 min. Blocking was performed by incubating cells for 10 min in 5% milk. Cells were then stained with the primary antibodies anti-TRPC2 c14 (11), anti-Epo-R (sc-697, Santa Cruz Biotechnology, Santa Cruz, CA), anti-PLC
1 (1249; Santa Cruz Biotechnology), anti-IP3R type II (sc-7278; Santa Cruz Biotechnology), and/or anti-IP3R type III (sc-7277; Santa Cruz Biotechnology) at 1:50 for 45 min at room temperature, followed by the appropriate secondary antibody (donkey anti-rabbit Alexa 488, donkey anti-goat Alexa 594; Molecular Probes) for 2 h in the dark. Cells were stained with 4,6-diamidino-2-phenylindole in Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for visualization. Images were acquired with the Leica TCS SP2 confocal microscope.
For immunolocalization experiments with TRPC2 IP3R binding mutants, CHO-S cells were cotransfected with TRPC2 or TRPC2 IP3R binding mutants in pQBI50 or in pcDNA3.1/V5-His TOPO, IP3R type II in pcDNA3, Epo-R in pTracer-CMV, or combinations of these plasmids. For these experiments, a blue fluorescent protein (BFP) tag was substituted for the GFP tag in pTracer-CMV so that GFP would not interfere with Alexa 488 fluorescence. Cells were stained as described above for primary erythroblasts. Either goat or donkey anti-rabbit Alexa 488, donkey anti-goat Alexa 594, or goat anti-mouse Alexa 594 antibodies (1:200 and 1:50 dilutions, respectively; Molecular Probes) were used as the secondary antibodies where appropriate.
Immunoblotting of whole cell lysates and crude membrane preparations.
Cell pellets from TER-119 and TER-119+ splenic erythroblasts, CHO-S cells, or HEK-293T cells were removed from storage at 80°C. For Western blotting of whole cell lysates, cell pellets were suspended in lysis buffer, and Western blotting was performed as previously described (12). After blocking, nitrocellulose membranes were incubated with anti-TRPC2 c14 (1:400), anti-Epo-R (1:200), anti-PLC1 (1:200), or anti-IP3R type II (1:100 dilution) antibodies. Blots were then washed and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-goat antibodies (1:2,000 dilution). Enhanced chemiluminescence (ECL) was used for signal detection.
For crude membrane preparations, cell pellets were suspended in buffer I (10 mM Tris·HCl, pH 7.4, and 1x protease inhibitor cocktail) and sonicated. An equal volume of buffer II (10 mM Tris·HCl, pH 7.4, 300 mM KCl, 20% sucrose, and 1x protease inhibitor mixture) was added. The suspension was then centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was spun at 100,000 g for 1 h at 4°C. Crude membranes were solubilized in buffer containing 62 mM Tris·HCl (pH 6.8), 2% SDS, and 10% glycerol. Protein was quantified and Western blotting was performed as described above.
Immunoprecipitation.
To determine whether mTRPC2 associates with Epo-R, PLC, or IP3R, CHO-S cells were transfected with mTRPC2 c14 (in pcDNA3.1/V5His TOPO), mEpo-R (in pcDNA3), rat PLC
1 (in pcDNA3), rat IP3R type II (in pcDNA3) (39), or combinations of these vectors. Lysates were prepared from cell pellets with lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton-X, 1 mM EDTA, and 1x protease inhibitor cocktail. Protein lysates were incubated with preimmune rabbit serum, anti-V5 (Invitrogen), anti-Epo-R, anti-PLC
1, or anti-IP3R type II antibodies for 2 h at room temperature. Protein A/G PLUS Agarose beads (Santa Cruz Biotechnology) were then added for 1 h at room temperature with mixing, and immunoprecipitates were washed three times. Sample buffer (2x) was added to the pellets, and the samples were boiled. Western blotting was performed as described above, and blots were probed with anti-V5 (1:2,000 dilution; Invitrogen), anti-Epo-R, anti-PLC
1, anti-IP3R type II, or anti-actin (Sigma) antibodies, followed by the appropriate HRP-conjugated secondary antibodies and ECL.
As a control for the specificity of immunoprecipitation, a mutant rat TRPV1 gene (8) (gift from Dr. Jan Teisinger, Institute of Physiology, Academy of Sciences, Prague, Czech Republic) was prepared in which the NH2 terminus including the single putative PLC SH3 binding domain at amino acid (aa) 38 was deleted. A nucleotide sequence encoding methionine was substituted for that of proline at aa 38, and the normal TRPV1 protein coding sequence began with aa 39. This sequence was subcloned into the pcDNA3.1/V5His TOPO vector. Cells were cotransfected with mutant TRPV1, mEpo-R, and rat PLC in control immunoprecipitation studies.
For immunoprecipitation with TER-119 erythroblasts, protein lysates were incubated with preimmune serum, anti-PLC1 (4 ug), or anti-Epo-R (4 µg) antibodies for 3 h at 4°C with mixing. Protein A/G PLUS-Agarose beads were then added for 30 min, followed by centrifugation of beads and washing. Sample buffer was added to the pellets, followed by boiling and loading of the supernatant onto gels for protein electrophoresis.
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RESULTS |
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Subcellular localization of TRPC2, PLC, and IP3R in primary erythroblasts.
We previously demonstrated that Epo stimulation regulates Ca2+ influx through TRPC2 (11, 12). To examine the subcellular localization of TRPC2, Epo-R, PLC
, and IP3R in primary murine erythroid cells, TER-119 erythroblasts were costained with anti-TRPC2 c14 or anti-Epo-R or anti-PLC
1 (rabbit) antibodies as well as anti-IP3R type II (goat), followed by appropriate secondary antibodies. Representative results of immunofluorescence observed with confocal microscopy are shown in Fig. 1. Endogenous TRPC2 (Fig. 1A), IP3R type II (Fig. 1, B, E, and H), and PLC
1 (Fig. 1G) were found at or near the plasma membrane but also throughout the cell. Epo-R (Fig. 1D) localized at or near the plasma membrane in primary erythroid cells. TER-119 cells were also stained with anti-IP3R type III antibody; IP3R type III was not detected and was not studied further. Colocalization studies were limited because the majority of available antibodies to these proteins are produced in rabbits (anti-TRPC2, anti-Epo-R, and anti-PLC
1) or are murine and thus could not be used in the present study. However, the merger of confocal images obtained when costaining with anti-TRPC2, anti-Epo-R, or anti-PLC
1, and anti-IP3R type II antibodies, demonstrated that TRPC2 (Fig. 1C), Epo-R (Fig. 1F), and PLC
1 (Fig. 1I) all partially colocalized with IP3R type II. These data demonstrate that TRPC2, Epo-R, PLC
1, and IP3R have similar localization in primary erythroid cells.
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PLC is required in Epo-modulated Ca2+ influx through TRPC2. To examine the mechanisms by which Epo modulates TRPC2, CHO-S cells transfected for 48 h with Epo-R and TRPC2 c14 were loaded with fura red in the presence of 010 µM of the active PLC inhibitor U-73122 or the inactive analog U-73343. Pretreatment with the active PLC inhibitor U-73122 inhibited the Epo-stimulated rise in [Ca2+]i through TRPC2 at concentrations of 1 µM or greater (Table 1; P < 0.01), whereas the inactive analog U-73343 did not. These data strongly suggest that PLC is required for Epo-stimulated Ca2+ influx through TRPC2.
Depletion of PLC by RNAi suppresses Epo-stimulated Ca2+ influx.
An alternative approach to using PLC inhibitors to examine the role of PLC
in Epo-stimulated Ca2+ influx through TRPC2 is to use RNAi to downregulate PLC
. For these experiments, we used HEK-293T cells and siRNA targeted to human PLC
, the effectiveness of which was previously validated (24). HEK-293T cells were transfected with TRPC2 c14 and Epo-R as well as either siRNA directed to PLC
or nonspecific control siRNA. The effectiveness of RNAi in altering expressed protein levels was monitored with Western blotting. Cotransfection of HEK-293T cells with siRNA directed to PLC
resulted in significant suppression of endogenous PLC
protein, compared with cells transfected with nonspecific control siRNA (Fig. 5). In contrast, expression of TRPC2 and Epo-R was equivalent in cells transfected with PLC
siRNA and control siRNA, demonstrating the specificity of siRNA directed to PLC
. The higher (165 kDa) molecular mass of TRPC2 c14 in Fig. 5 is the result of linkage to BFP.
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IP3 receptors are involved in Epo activation of TRPC2. PLC activation results in production of IP3. TRP channels were previously shown to interact with all IP3R, and direct interaction with IP3R may be a common mechanism for the activation of TRP channels (6, 42). TRPC2 has been reported to have two conserved IP3R binding domains in its COOH terminus: one corresponding to aa 10011,036 of TRPC2 c14 and a second, less well defined site at aa 1,0441,172 (42). To determine whether IP3R binding sites are involved in Epo stimulation of TRPC2, two COOH-terminal mutants of TRPC2 were prepared (Fig. 6). Mutant 1 (M1) had a complete deletion of TRPC2 after aa 1,000 (aa 1,0011,172), including both potential IP3R binding sites. The second mutant (M2) had a deletion of the IP3R binding site (aa 1,0011,036) in the TRPC2 COOH terminus common to all TRPC proteins (42). Both mutants retained all six transmembrane domains and the putative Ca2+ pore (Fig. 6). CHO-S cells were cotransfected with Epo-R in pTracer-CMV and with BFP-tagged TRPC2 (in pQBI50) with or without deletion of the IP3R binding sites. In cells transfected with Epo-R and TRPC2 c14 mutants with complete or partial deletion of the COOH-terminal IP3R binding domains, no significant rise in [Ca2+]i was observed in response to Epo stimulation, compared with control cells transfected with wild-type TRPC2 (Table 3). Expression of each of these channels in digital video imaging experiments was confirmed by BFP fluorescence. These data demonstrate a requirement for IP3R binding domains in Epo-modulated Ca2+ influx through TRPC2.
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Coassociation of TRPC2 and IP3R. To determine whether TRPC2 interacts with IP3R, immunoprecipitation experiments were performed. CHO-S cells were transfected with Epo-R (pcDNA3), IP3R type II (pcDNA3), and/or V5-tagged TRPC2 c14 (pcDNA 3.1/V5-His TOPO). Immunoprecipitation was performed with antibodies to V5 or IP3R type II using cell lysates of CHO-S cells expressing IP3R, V5-TRPC2 c14, and Epo-R or CHO-S cells transfected with single plasmids as controls for specificity. Western blotting demonstrated that in cells transfected with wild-type V5-TRPC2, IP3R type II, and Epo-R, anti-V5 antibody coimmunoprecipitated IP3R and Epo-R (Fig. 9). Anti-IP3R also immunoprecipitated TRPC2 and Epo-R in cotransfected cells. In contrast, IP3R was not immunoprecipitated by anti-V5 antibody in cells expressing IP3R alone, nor was V5-TRPC2 immunoprecipitated by anti-IP3R antibody in cells expressing V5-TRPC2 alone.
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DISCUSSION |
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A common characteristic of many TRPC channels is that they are activated through pathways involving PLC (36). Because Epo also activates PLC1 and PLC
2 (5, 26, 31, 40), Epo activation of TRPC2 through a PLC-dependent pathway was explored. We have demonstrated that PLC activation by Epo is required for stimulation of Ca2+ influx in primary erythroid cells and for TRPC2 activation in a CHO-S model. Our evidence includes 1) abolition of the Epo-induced [Ca2+]i increase by either a specific PLC inhibitor (U-73122) or specific downregulation of PLC
with RNAi and 2) PLC
association with Epo-R and TRPC2. Immunoprecipitation confirmed direct binding of PLC
to TRPC2, which was predicted on the basis of a domain search (the Massachusetts Institute of Technology online Scansite Menu, available at: http://scansite.mit.edu/) indicating two potential PLC
SH2 binding domains and two PLC
SH3 binding domains on TRPC2. Our data are consistent with reports that demonstrated that PLC
is involved in the activation of, and directly associates with, other TRPC. B cell receptor-induced activation of TRPC3 requires PLC (45), and PLC
1 directly interacts with TRPC3 through the PLC
1 SH3 domain (38). Further investigation is required to determine whether PLC modulates its effect on Epo-R regulated Ca2+ influx strictly by hydrolysis of PIP2 to release IP3 or whether it may also have a conformational or adapter role, as suggested in other studies in which catalytic phospholipase activity was not needed (38).
TRPC2 can be activated either by Ca2+ store release and/or by receptor-operated mechanisms (17, 44). Store depletion occurs after activation of PLC by appropriate receptor agonists, followed by production of IP3, which then signals internal Ca2+ release from the endoplasmic reticulum through IP3R. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+. However, Ca2+ store release does not appear to play an important role in the activation of TRPC2 by Epo-R. In primary erythroid cells and in CHO-S cells transfected with TRPC2, a significant increase in [Ca2+]i was not observed in the first 2 min after Epo addition, arguing against activation of TRPC2 by Ca2+ store release in these cells (Fig. 4) (12). Furthermore, when extracellular Ca2+ was substituted with Mn2+, a slow quenching of fura red fluorescence, indicative of Mn2+ influx, was observed that was maximal after 10 min of Epo stimulation (Fig. 3). In addition, after thapsigargin depletion of intracellular Ca2+ stores, a significant rise in [Ca2+]i was observed after treatment with Epo (Fig. 4). Consistent with these observations, in primary human erythroblasts in which Epo-responsive, voltage-independent Ca2+ channels have been demonstrated by single-channel recording (10), the increase in [Ca2+]i after Epo stimulation required external Ca2+, and no significant increase in [Ca2+]i was observed in its absence (9). Similarly, the increase in [Ca2+]i after Epo stimulation of CHO-S cells transfected with Epo-R and TRPC2 was dependent on extracellular Ca2+ (11). Although the lack of global increase in [Ca2+]i in response to Epo in cells incubated in Ca2+-free medium does not rule out a local release of Ca2+ that was effectively buffered, our experiments strongly suggest that the increase in [Ca2+]i observed with Epo was mediated primarily through Ca2+ influx. These data are consistent with observations in the VNO (27), where TRPC2 regulates VNO sensory transduction independently of depletion of internal Ca2+ stores, which are absent in the sensory microvilli. The increase in [Ca2+]i after Epo activation of TRPC2 was delayed, and this may be secondary to the interplay of several factors, including channel conductance, channel open probability, channel open time, and the rate at which Ca2+ is extruded from the cell or buffered in intracellular organelles.
TRPC interact with all IP3R, and direct interaction of activated IP3R with TRPC, altering TRPC conformation, has been proposed to be a common activation mechanism (4, 6, 13, 19, 20, 29, 42). In the present study, we have demonstrated that Epo-dependent activation of TRPC2 requires IP3R binding to TRPC2. This is consistent with the report of Brann et al. (7), who demonstrated that IP3R type III interacts with TRPC2 in the VNO. Previously, researchers at our laboratory (9) and others (32) were unable to demonstrate a global rise in IP3 in primary human erythroid cells in response to Epo stimulation, although an increase in IP3 in response to Epo stimulation was observed in UT-7 cells, a human Epo-dependent cell line (40). In the present study, we have demonstrate that IP3R are in close proximity to PLC-coupled TRPC2. This would allow IP3R exposure to a localized increase in IP3 near the cell membrane that might escape detection in whole cell studies.
In this report, we demonstrate the colocalization of TRPC2, Epo-R, PLC1, and IP3R type II at or near the plasma membrane of primary murine erythroid cells. We also demonstrate the coassociation of TRPC2 with Epo-R, PLC
1 and IP3R type II in erythroblasts and in transfected cells. In previous studies, Epo-R was shown to interact with PLC
1 (31), and TRPC2 was shown to interact with IP3R (7), in nonerythroid cells. An unexpected finding in the present study was the association of Epo-R with TRPC2 and IP3R. Future studies are required to determine whether these associations represent direct interactions or whether adapter proteins such as PDZ domain-containing proteins serve to link the complex (25, 43, 46). The importance of the linkage of IP3R to specific receptors, allowing IP3 to be produced at required concentrations and precise locations, was demonstrated in neuronal cells for the B2 bradykinin receptor (13).
Our data demonstrate that Epo-R modulates TRPC2 activation through PLC and that interaction with IP3R is required. They suggest that a signaling complex including Epo-R, TRPC2, PLC
, and IP3R is involved in the regulation of TRPC2. These data are consistent with a mechanism in which Epo stimulation of Epo-R results in activation of PLC
and production of IP3. Whether a conformational change in PLC
has any direct influence on TRPC2 opening is unknown. In any case, IP3 then interacts with IP3R, which is in close proximity, resulting in a conformational change in IP3R and contributing to activation of TRPC2.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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