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J. Biol. Chem., Vol. 276, Issue 45, 42401-42408, November 9, 2001

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Stabilization of Cortical Actin Induces Internalization of Transient Receptor Potential 3 (Trp3)-associated Caveolar Ca²⁺ Signaling Complex and Loss of Ca²⁺ Influx without Disruption of Trp3-Inositol Trisphosphate Receptor Association^*

Timothy Lockwich, 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, July 23, 2001, and in revised form, August 23, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ influx via plasma membrane Trp3 channels is proposed to be regulated by a reversible interaction with inositol trisphosphate receptor (IP₃R) in the endoplasmic reticulum. Condensation of the cortical actin layer has been suggested to physically disrupt this interaction and inhibit Trp3-mediated Ca²⁺ influx. This study examines the effect of cytoskeletal reorganization on the localization and function of Trp3 and key Ca²⁺ signaling proteins. Calyculin-A treatment resulted in formation of condensed actin layer at the plasma membrane; internalization of Trp3, G_q/11, phospholipase C, and caveolin-1; and attenuation of 1-oleoyl-2-acetyl-sn-glycerol- and ATP-stimulated Sr²⁺ influx. Importantly, Trp3 and IP₃R-3 remained co-localized inside the cell and were co-immunoprecipitated. Jasplakinolide also induced internalization of Trp3 and caveolin-1. Pretreatment of cells with cytochalasin D or staurosporine did not affect Trp3 but prevented calyculin-A-induced effects. Based on these data, we suggest that Trp3 is assembled in a caveolar Ca²⁺ signaling complex with IP₃R, SERCA, G_q/11, phospholipase C, caveolin-1, and ezrin. Furthermore, our data demonstrate that conditions which stabilize cortical actin induce loss of Trp3 activity due to internalization of the Trp3-signaling complex, not disruption of IP₃R-Trp3 interaction. This suggests that localization of the Trp3-associated signaling complex, rather than Trp3-IP₃R coupling, depends on the status of the actin cytoskeleton.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The Trp family of proteins has been proposed as molecular components of Ca²⁺ entry channels in non-excitable cells that are activated by hormonal stimuli coupled to G protein-dependent activation of phospholipase C (PLC)¹ and phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis (1, 2). Although the exact physiological function of the various Trp proteins has not been established, recent studies have demonstrated functional differences between them. For example, Trp1 can be activated solely by the depletion of internal stores of Ca²⁺, a process downstream from activation of PIP₂ hydrolysis and IP₃ generation (3), whereas Trp3 and Trp6 are activated by PIP₂ hydrolysis per se (4, 5). Thus, these Trp proteins can be expected to be regulated by distinct mechanisms. However, presently, little is known regarding the molecular components which determine the functional specificity and regulation of the various Trps.

The involvement of protein-protein interactions in the regulation of channel function has been most clearly demonstrated for Trp3. It was reported that an interaction between Trp3 and IP₃R was required for channel activation by an agonist (6-8). Direct physical interaction between Trp3 and IP₃R was also suggested by previous studies (9-11) showing that conditions (such as treatment of cells with the phosphatase inhibitor, calyculin-A, or with the actin stabilizing agent, jasplakanolide) that induced condensation of filamentous actin into a tight cortical layer in the subplasma membrane region of the cell, also resulted in loss of agonist-stimulated Trp3 activity and SOCE. The effect of calyculin-A on SOCE and on agonist stimulation of Trp3 activity appeared to be due to the condensation of the cortical actin layer since it was reversed, or prevented, by treatment with the actin depolymerizing reagent, cytochalasin D, or by the protein kinase inhibitor, staurosporine. It was concluded that the cortical actin layer acts as a physical barrier between the ER and plasma membrane and disrupts the interaction between IP₃R and Trp3, thus leading to loss of Ca²⁺ influx. Based on these data, a "secretion-like" coupling model was proposed according to which reversible interaction(s) between the ER and the plasma membrane serves as the key regulatory event in store-operated Ca²⁺ entry (9). Cytoskeletal reorganization was also reported to disrupt Trp1-IP₃R interactions in platelets (12, 13), an observation consistent with the secretion-coupling model. A key assumption in this model is that the localizations of Trp3, or Trp1, and IP₃R in the plasma membrane and ER, respectively, are not altered (9, 13). Only the movement of the ER toward or away from the plasma membrane determines Trp3-IP₃R interactions. These studies implicate a role for cytoskeletal rearrangements in the movement of ER toward, or away from, the plasma membrane. However, important questions that remain presently unaddressed are whether Trp3 directly interacts with the cytoskeleton and whether its localization is altered under conditions used to remodel the actin cytoskeleton. Furthermore, nothing is known about other proteins that might interact with Trp3 and regulate its function. It is possible that the status of the actin cytoskeleton might affect the function of these potential regulatory proteins and thus lead to modulation of Trp3 activity.

There is increasing evidence to suggest that components in a G-protein signaling cascade are tightly coupled (14). Consistent with this, the Drosophila Trp (dTrp), which is activated by a G-protein-mediated signaling mechanism, has been shown to be assembled in a supramolecular complex with a variety of signaling and accessory proteins, such as rhodopsin, protein kinase C, PLC, and NINAC. Localization of this signalplex to the rhabdomers is dependent on the PDZ domain-containing protein, INAD, which provides a scaffold for anchoring the proteins components of the dTrp-signaling complex (15, 16) and protein-protein interactions within this complex regulate the Drosophila visual cascade. Thus, the proteins associated with dTrp are intimately involved in the regulation of its function and targeting to the appropriate cellular locale. Similarly, there is some evidence that mammalian Trps might also be associated with other Ca²⁺ signaling and scaffolding proteins. We previously reported that Trp1, a strong candidate for the store-operated Ca²⁺ entry (SOCE) mechanism in salivary epithelial cells (HSG) (3), is assembled in a signaling complex that is localized in caveolar lipid raft domains (17). We have also shown that disruption of the lipid raft domain induced loss of Trp1 activity, suggesting that Trp1 activity is determined by the status of its microenvironment. More recently, Trp4 and Trp5 have been shown to form a complex with the scaffolding protein NHERF and PLC (18). Thus, it is likely that mammalian Trps, like dTrp, are assembled in macromolecular complexes and regulated by the proteins that are either already localized in, or are recruited into, the complex. Identification of the molecular components involved in the assembly and localization of the Trp-associated signaling complexes in the plasma membrane will be key to understanding their regulation and functional differences.

To address the role of cytoskeleton in the regulation of Trp3 channel activity, we have examined the effects of cytoskeletal rearrangements on the localization and function of Trp3. We have also examined the localization of key Ca²⁺ signaling proteins and some cytoskeletal components under the same conditions as well as possible association between Trp3 and these proteins. Our data demonstrate that Trp3 is assembled in a caveolin-associated multimolecular complex containing key Ca²⁺ signaling proteins from the plasma membrane as well as from the endoplasmic reticulum. Importantly, we report that the calyculin-A-, and jasplakinolide-induced loss of calcium influx is due to internalization of this caveolin-associated Trp3-signaling complex rather than disruption of Trp3-IP₃R interactions. The data suggest that localization of the Trp3-associated Ca²⁺ signaling complex, rather than Trp3-IP₃R coupling, is determined by the status of the actin cytoskeleton.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Culture of HEK-293 Cells and Preparation of Crude Membranes-- hTrp3-expressing and control (vasopressin receptor-expressing) HEK-293 cells were cultured as described (4). Confluent cells were harvested, resuspended in lysis buffer (0.5 ml/plate) containing (in mM): 100 Tris-HCl (pH 8.0), 1 MgCl₂, 0.5 AEBSF (ICN), 0.1 phenylmethylsulfonyl fluoride (Calbiochem), and frozen at 80 °C. Frozen cells (3-5 ml) were thawed on ice, homogenized in a Dounce homogenizer, and diluted to 30 ml with a buffer containing 0.25 M sucrose, 10 mM Tris-HEPES (pH 7.4), 1% (v/v) aprotinin, 1 mM dithiothreitol (Calbiochem Ultrol grade), 0.5 mM AEBSF, 0.167 mM pepstatin A (ICN), and 0.167 mM leupeptin (ICN), and centrifuged at 3,000 × g for 15 min. The resulting supernatant was centrifuged at 50,000 × g for 30 min and the final pellet (crude membrane fraction) was suspended in a desired volume of the sucrose buffer and stored at 80 °C until use. Protein concentration was determined by using the Bio-Rad protein assay (microassay procedure).

Detergent Solubilization of Crude Membranes and Immunoprecipitation-- Unless otherwise noted all steps were carried out at 4 °C. 5 mg of crude membranes were washed by dilution into 5-6 ml of a buffer containing (in mM): 200 KCl, 50 K-MOPS (pH 7.5), 2.5 MgCl₂, and 1 AEBSF followed by centrifugation, 50,000 × g for 30 min. The washed membranes were solubilized (19) with 1.5% octyl glucoside (OG, Calbiochem) in 2.4 ml of a media containing 50 mM K-MOPS (pH 7.5), 20% (v/v) glycerol, 0.5 M KI, 1.5 mM MgCl₂, 1 mM dithiothreitol, 0.5 mM AEBSF, 0.167 mM pepstatin A, 0.167 mM leupeptin, and 10 mg of lipids (Avanti Polar Lipids) from a 50 mg/ml aqueous stock suspension containing 2% (v/v) -mercaptoethanol (Sigma). The lipid stock solution consisted of 60% Escherichia coli ether-washed bulk lipids, 17.5% phosphatidylcholine, 10% phosphatidylserine, and 12.5% cholesterol. The detergent-treated membranes were incubated on ice for 20 min and then centrifuged for 1 h at 145,000 × g. The supernatant (OG + KI solubilized fraction) was stored at 80 °C until used.

50 mg of protein A-Sepharose CL-4B beads (Amersham Pharmacia Biotech) were washed three times with distilled water and then once with solubilization buffer (described above) for 5 min and then resuspended in 150 µl of the solubilization buffer. 50 µl of the washed beads were added to 1 ml of OG + KI supernatant (preclearing), rotated for 30 min, and centrifuged at 1,000 × g for 1 min. The pre-cleared supernatant was then transferred to a new tube and the following antibodies were added (resulting in dilutions ranging from 1:20 to 1:200): anti-HA (Roche Molecular Biochemicals, clone 12CA5), anti-ankyrin (Calbiochem, clone Ank016), anti-actin (Sigma, A2066), anti-ezrin (Santa Cruz, 6407), anti-PLC1 (Santa Cruz, sc-205), anti-caveolin 1 (Transduction Labs, C13630), anti-G_q/11, 06-710), anti-SERCA2 (ABR, MA3-910), anti-IP₃R-I (Santa Cruz, sc-6093), anti-IP₃R-II (Santa Cruz, sc-7278), and anti-IP₃R-III (Transduction Labs, 13122). The samples were rotated for 1 h and added to the remaining Sepharose CL-4B beads. The immunoprecipitated proteins were recovered by centrifugation for 1 min at 1,000 × g. The beads were washed 3 times with buffer containing 500 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10% (w/v) sucrose, 1 mM EDTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 0.5% (v/v) Nonidet P-40 (Sigma), 1 µg/ml aprotinin, leupeptin, and pepstatin A. The washed beads were then incubated with 200 µl of SDS-polyacrylamide gel electrophoresis sample buffer for 5 min at 95 °C. The immunoprecipitated proteins (and co-immunoprecipitated proteins) were detected by SDS-polyacrylamide gel electrophoresis followed by Western blotting as previously reported (17, 20). Primary antibodies used for Western blotting were identical to those used for the initial immunoprecipitations except the anti-HA-Peroxidase High Affinty (Roche Molecular Biochemicals, 3F10) antibody was used to visualize the HA-tagged Trp3 (dilution 1:500). Other dilutions used for Western blotting were: 1:200 (anti-ezrin, anti-ankyrin, and anti-PLC1), 1:500 (anti-actin, anti-caveolin 1, and anti-G_q/11), 1:1000 (anti-IP₃R's), and 1:2500 (anti-SERCA2). For visualization of the low molecular weight proteins caveolin-1 and G_q/11 after immunoprecipitation of the HA-tagged Trp3, the initial immunoprecipitation was performed using 150 µl of anti-HA-linked affinity matrix beads (Covance, AFC-101P).

Confocal Microscopy-- Trp3-expressing HEK293 cells were cultured on poly-L-lysine-coated coverslips for 2 days and then treated with the following as required; calyculin-A, 100 nM for 20 min; staurosporine, 1 µM for 20 min; cytochalasin D, 2.5 µM 20 min; jasplakanolide, 5 µM for 60 min in growth medium at 37 °C. Cells were fixed, permeabilized (20), and treated with the primary antibodies at the indicated dilutions: anti-HA (1:100), anti-caveolin-1 (1:100), rhodamine-phalloidin (1:50), anti-IP₃R-3 (1:25), anti-ezrin (1:100), anti-G_q/11 (1:100), PLC (1:50), anti-SERCA (1:100) and either fluorescein isothiocyantate or rhodamine-linked secondary antibody as described before (20). Confocal images were collected using an MRC 1024 krypton/argon laser scanning confocal system (Bio-Rad) equipped with a Nikon Optiphot II photomicroscope (Melville, NY). Images of control and experimental samples were obtained using identical conditions (laser intensities, gain, iris aperature, black level, and scan speed). Images were collected in the x-y plane using a ×60 oil immersion PlanApo objective (Nikon, Japan; 1.4 N.A.) with a confocal aperture of 2 mm.

[Ca²⁺]_i Measurements-- Fura2 fluorescence in single cells was measured as described earlier (3, 17) by using an SLM 8000/DMX 100 spectrofluorimeter attached to an inverted Nikon Diaphot microscope with a Fluor ×40 oil-immersion objective. Images were acquired using an enhanced CCD camera (CCD-72, MTI) and the Image-1 software (Universal Imaging Corp., Downingtown, PA). Analog plots of the fluorescence ratio (340/380) in single cells are shown.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Effect of Cytoskeletal Modifications on the Localization of Trp3 and IP₃R-3-- Calyculin-A induces condensation of actin into a tight layer in the subplasma membrane region (21). As noted above, this treatment has been proposed to inhibit activation of Trp3 by disrupting the interaction between Trp3 in the plasma membrane and IP₃R in the ER. Fig. 1 shows the effect of calyculin-A treatment on the localization of Trp3, actin, and IP₃R-3 (indicated by white arrows) in HEK293 cells stably expressing HA-tagged hTrp3. Confocal images shown in the figure represent a section from approximately the middle of the cells. Trp3 and actin were detected in the plasma membrane region of the cells while IP₃R was detected inside the cell in the ER region (No treatment, shows untreated cells). After treatment of the cells with calyculin-A, the cells appeared somewhat rounded in shape. Significantly, most of the Trp3 was internalized into the cell although some Trp3 signal was still detected in the plasma membrane region (compare no treatment panels with Caly-A panels). Actin appeared to be tightly condensed in a cortical layer at the plasma membrane in some cells (shown by light arrow). In other cells, actin appeared to be concentrated in a tightly condensed bundle inside the cell as has been shown before (heavy arrow; Refs. 22 and 23). The localization of IP₃R-3 was not altered dramatically by calyculin-A, although some IP₃R-3 appeared to be concentrated in bundles in the cell.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Effect of cytoskeletal modification on the localization of Trp3, IP3R, and actin. A, confocal microscopy was used to detect localization of Trp3, actin, and IP3R-3. Columns are labeled as Control, no primary antibody; No treatment, treated with vehicle alone; caly-A, treated with calyculin-A (100 nM, 20 min); Stauro+caly-A, pretreated with staurosporine (1 µM, 20 min) before then calyculin-A; cyto-D, treated with cytochalasin D (2.5 µM, 20 min); cyto-D + caly-A, pretreated with cytochalasin D before calyculin-A treatment. Details of antibody concentrations, etc. are given under "Materials and Methods." The images shown represent sections from approximately the middle of the cells. White arrows indicate the localization of proteins. Similar results were obtained in at least three to four separate experiments. B, co-localization of Trp3 and IP3R-3 in control (Caly-A) and calyculin-A treated (+Caly-A) cells. Trp3, red; IP3R-3, green; Merged, yellow indicates co-localization of Trp3 and IP3R-3 (indicated by white arrows). Light white arrow indicates some Trp3 and IP3R-3 in the plasma membrane region in a few calyculin-A-treated cells. C, co-immunoprecipitation of IP3R-3 with Trp3. Left panel shows IP3R-3 detected in the fraction immunoprecipitated with anti-HA. Right panel shows Trp3 in fractions immunoprecipitated with anti-IP3R-3. Non-IP shows fraction of IP3R-3 that was not co-immunoprecipated. Immunoprecipitation reactions were carried out using OG + KI solubilized fractions of crude membranes isolated from control (Caly-A) and calyculin-A-treated (+Caly-A) cells. Pull down of IP3R-3 by anti-IP3R-3 antibody is not shown.

The internalization of Trp3 induced by calyculin-A was blocked by preincubating the cells with staurosporine, a Ser/Thr protein kinase inhibitor, or cytochalasin D, an actin depolymerizing agent (24). As shown in the figure, although cytochalasin D, by itself, altered the localization of actin in the cells, it did not alter the localization of Trp3 or IP₃R-3. These results are consistent with previously reported data on the effects of calyculin-A, cytochalasin D, and staurosporine on actin localization (9, 10). Importantly, the present data suggest that Trp3 is not directly linked to actin since cytochalasin D which depolymerizes actin converting it into bundles, has no obvious effect on Trp3 localization.

To determine whether internalization of Trp3 is accompanied by a disruption of Trp3-IP₃R interaction, we examined the co-localization of Trp3 and IP₃R in control and calyculin-A treated cells. Fig. 1B shows the distribution of Trp3 (red) and IP₃R-3 (green) in control and calyculin-A-treated cells. In control cells, Trp3 was localized in the plasma membrane while IP₃R-3 was distributed in the ER region inside the cell. The last panels show the merged fluorescence signal. In untreated cells, overlap of the two signals was detected in the plasma membrane region (yellow signal, indicated by white arrow), indicating co-localization of Trp3 and IP₃R-3 along the plasma membrane of these cells. In calyculin-A-treated cells, the yellow signal was detected in concentrated areas within the cell (indicated by white arrow). Some yellow signal is also seen in the plasma membrane region (indicated by the lighter white arrow). Thus, calyculin-A induces internalization of a substantial amount of Trp3 from the plasma membrane. However, and importantly, the internalized Trp3 remains co-localized with IP₃R-3.

To further demonstrate the interaction between Trp3 and IP₃R in calyculin-A-treated cells, we examined co-immunoprecipitation of Trp3 and IP₃R-3. Experiments were performed using membrane fractions isolated from control and calyculin-A-treated cells (note that calyculin-A was present in the medium during membrane isolation). The data in Fig. 1C clearly demonstrate that co-immunoprecipitation of IP₃R-3 with Trp3 was not affected by calyculin-A treatment. Additionally, similar levels of Trp3 were detected when the immunoprecipitation was performed in the reverse order using anti-IP₃R-3 antibody to pull down IP₃R-3 (immunoprecipitated IP₃R-3 was also detected by Western blotting, data not shown). These data are consistent with the co-localization of Trp3 and IP₃R-3 in both control and calyculin-A-treated cells and demonstrate that only a small part of the total Trp3 and IP₃R in the cells associate with each other. Thus, our data do not support the previous suggestion that calyculin-A treatment leads to the disruption of Trp3-IP₃R interactions. Rather our data suggest that calyculin-A induced internalization of Trp3 results in a decrease in the number of channels in the plasma membrane.

Calyculin-A-induced Decrease in Trp3-mediated Sr²⁺ Influx-- To demonstrate the effect of calyculin-A-induced internalization of Trp3 on Trp3 function, we examined OAG-induced Sr²⁺ influx in control and calyculin-A-treated cells. Sr²⁺ has been previously used to demonstrate Trp3 activity (10). As noted in this previous study, OAG-stimulated Sr²⁺ influx was only seen in cells stably expressing Trp3 protein but not in control HEK293 cells that were stably transfected with the vasopressin receptor (Fig. 2A, fluorescence in control cells is shown by dotted line). Furthermore, OAG did not stimulate any increase in fura2 fluorescence when cells were placed in a nominally Ca²⁺-free medium containing 100 µM EGTA (data not shown). Treatment with calyculin-A (100 nM, 20-30 min, Fig. 2B) significantly reduced the OAG-stimulated increase in fura2 fluorescence in cells maintained in a medium containing 1 mM Sr²⁺, but no Ca²⁺ (p = <0.05, n = >300 fields, each field represents 1-2 cells. The traces shown are average fluorescence values from 64 fields in each case). Treatment of cells with cytochalasin D (1 µM, 20 min) alone did not alter the OAG-induced increase in Sr²⁺ influx (Fig. 2C) while pretreatment with cytochalasin D prevented the calyculin-A-induced loss of Sr²⁺ influx (Fig. 2D). These data demonstrate that calyculin-A treatment induces a significant loss of Trp3-mediated Sr²⁺ influx. These data are not consistent with an earlier report (10) showing that OAG-mediated Ca²⁺ influx was unchanged in calyculin-A-treated cells. While the reason for the discrepancy in the data is not presently clear, it is important to note that the calyculin-A-induced effects on Trp3 function shown in Fig. 2 are fully consistent with its effect on Trp3 localization shown above.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Effect of calyculin-A on cation entry. Trp3-associated Sr2+ influx was measured by addition of 25 µM OAG (shown by arrow) to fura2-loaded cells in Ca2+-free + 1 mM Sr2+ medium (A-D). A, untreated cells, Trp3-expressing, solid trace; control transfected, dotted trace. B, cells treated with calyculin-A. C, cells pretreated with cytochalsin-D then calyculin-A. D, cells pretreated with cytochalasin D. ATP-induced internal Ca2+ release and Sr2+ entry (Sr2+ addition indicated by arrow) in control (E) and calyculin-A-treated (F) cells. Tg-stimulated [Ca2+]i increase in control (G) and calyculin-A-treated (H) cells. Traces shown are averages of fluorescence measured in 64 fields in each case, each field typically contained 1-2 cells. The data represent results obtained with more than 300 fields for each condition, in three to four separate experiments.

We also examined agonist- (ATP) and thapsigargin-induced Ca²⁺ mobilization in control and calyculin-A-treated cells. Figs. 2, E and F, show that calyculin-A treatment attenuated ATP-stimulated internal Ca²⁺ release and Sr²⁺ influx. Fig. 2, G and H, show that Tg-stimulated Ca²⁺ mobilization was also decreased by calyculin-A. However, the Tg-stimulated increase in fura2 fluorescence in a Ca²⁺-free medium was not altered by calyculin-A and was similar to that seen in calyculin-A-treated cells in a Ca²⁺-containing medium (data not shown). These results are consistent with our previous studies (25-27) and demonstrate that calyculin-A (i) inhibits agonist-stimulated Ca²⁺ influx via inhibition of internal Ca²⁺ release, and (ii) inhibits store-operated Ca²⁺ influx via a more direct effect on the influx mechanism per se.

Effect of KI on Detergent Solubilization of HA-hTrp3-- It is widely recognized that proteins which are attached to the cytoskeleton are poorly solubilized by detergents (28). Since Trp3 localization did not appear to be directly associated with actin (see Fig. 1), we examined the solubilization of Trp3 from HEK cell membranes by octyl glucoside (OG). Surprisingly, Trp3 was poorly solubilized by OG (Fig. 3A, and C, control HEK-293 cells; T, Trp3-expressing HEK-293 cells). KI has been used to disrupt cytoskeletal interactions and increase detergent solubility of proteins linked to the cytoskeleton (29). Notably, KI increased detergent solubility of Trp1 from HSG cell plasma membranes (17). Fig. 3A reveals a similar effect of KI on the solubility of Trp3. Presence of 0.5 M KI substantially increased solubilization of Trp3 from the membrane (see Fig. 3B to compare relative levels of Trp3 in crude membrane and the OG + KI-solubilized fraction from control and Trp3-expressing cells).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Detergent solubilization of Trp3 and association with cytoskeletal proteins. A, crude membranes prepared from control-transfected (C) and Trp3-expressing (T) HEK293 cells were treated with either octyl glucoside (OG) or OG + KI and the solubilized fraction was separated by centrifugation as described under "Materials and Methods." Trp3 was detected after SDS-gel electrophoresis and Western blotting using an anti-HA antibody. B, immunoprecipitation of HA-Trp3 using anti-HA antibody. CM, crude membrane; OG + KI, solubilized fraction; Non-IP, fraction remaining after immunoprecipitation; HA-IP, immunoprecipitated fraction. C, association of Trp3 with cytoskeletal proteins. Detection of proteins in immunoprecipitate of Trp3 (lane 2 in each blot) using the antibodies indicated by the bar above each blot. The first lane in each blot contains crude membrane from Trp3-expressing cells as a control. D, reciprocal IP reactions. Ankyrin or actin were immunoprecipitated by using the respective antibodies (presence of these proteins in the IPs are not shown). Trp3 was detected using anti-HA antibody. Note that actin in CM and IP fractions were detected on the same blot.

To further examine possible association between Trp3 and actin, the OG + KI-solubilized fraction was used to immunoprecipitate the expressed Trp3 using an anti-HA antibody. The results of this immunoprecipitation are shown in Fig. 3B. A substantial amount of the protein from the detergent-solubilized fraction was immunoprecipitated by the anti-HA antibody, compare relative recovery of protein in HA-IP (last lane) with that in the Non-IP (fraction of solubilizate after immunoprecipitation). Fig. 3C shows Western blots of anti-HA-immunoprecipitated Trp3, probed with either anti-ankyrin or anti-actin. Importantly, ankyrin or actin were not co-immunoprecipitated with Trp3, although both the proteins were present in the crude membrane fraction. Fig. 3D shows the reverse reaction using anti-actin or anti-ankyrin antibodies. Trp3 was not detected in these immunoprecipitates. However, each of these antibodies immunoprecipitated its respective antigen (data not shown). These data demonstrate that Trp3 is not directly interacting with actin. The KI-induced increase in the detergent solubility of Trp3 might be due to disruption of its interaction via its dystrophin or ankyrin domains, which are also known to be sensitive to KI (29).

Effect of Calyculin-A on the Localization of Ca²⁺ Signaling Proteins and Ezrin-- Since calyculin-A treatment induced loss of agonist-stimulated internal Ca²⁺ release and Sr²⁺ influx, as well as thapsigargin-stimulated Ca²⁺ influx, we examined the localization of key Ca²⁺ signaling molecules, PLC1, G_q/11, and SERCA2, in control and calyculin-A-treated cells (shown by white arrows in each case). In addition, we also investigated the effect of calyculin-A on ezrin, a member of the ezrin/radixin/moesin family of general cross-linkers of actin filaments and plasma membranes (30) known to be involved in the formation of the cortical actin layer. Fig. 4 shows that in untreated cells PLC1, G_q/11, and ezrin were detected in the plasma membrane region of the cells while SERCA2 was detected inside the cell in the ER region. In calyculin-A-treated cells, most of the PLC and G_q/11 were internalized into the cell (compare no treatment panels with Caly-A panels). SERCA localization did not appear to be affected by the cytoskeletal modification. Furthermore, consistent with the proposed role of ezrin in the organization of cortical actin (30), most of the ezrin signal was detected in the plasma membrane region. Our present data suggest that the functional effects induced by cytoskeletal modifications are primarily due to the alterations in the localization of key proteins involved in Ca²⁺ signaling.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effect of calyculin-A on the localization of key Ca2+ signaling proteins. A, confocal microscopy was used to detect localization of SERCA2, Gq/11, PLC, and ezrin in untreated cells (No treatment) and calyculin-A-treated cells (Caly-A). Control represents signal in a sample treated with only the secondary antibody. Details regarding antibody concentrations, treatments etc. are given under "Materials and Methods." The images shown represent sections from approximately the middle of the cells. Localizations of the proteins are indicated by white arrows. Similar results were obtained in at least three separate experiments. B, association of Trp3 with Ca2+ signaling proteins and ezrin. Upper panels, detection of proteins in immunoprecipitate of Trp3 (lane 2 in each blot) using the antibodies indicated by the bar above each blot. The first lane in each blot contains crude membrane from Trp3-expressing cells as a control. Lower panel, reciprocal IP reactions. SERCA2, Gq/11, PLC, and ezrin were immunoprecipitated using the respective antibodies (data not shown). Trp3 was detected in these immunoprecipitates by using anti-HA antibody. In all cases protein in CM and IP were detected on the same blot. In B, lower panel, the blot was exposed for a longer time to detect Trp3 in IP with anti-Gq/11 and anti-PLC.

Since the localizations of G_q/11, PLC, and Trp3 were similarly affected by calyculin-A treatment, we examined possible association between these proteins as has been noted for dTrp and Trp1 (15-17, 31). Fig. 4B shows that SERCA2, G_q/11, PLC, and ezrin were co-immunoprecipitated with Trp3. Immunoprecipitations were done using the anti-HA antibody and Western blots were probed with anti-SERCA2, anti-G_q/11, anti-PLC, or anti-ezrin (upper panel) and confirmed by the reverse immunoprecipitation (lower panel). Consistent with previous findings (8) IP₃R-1 and IP₃R-2 were also co-immunoprecipitated with Trp3 (data not shown). In aggregate, the data in Figs. 1, 3, and 4 confirm that hTrp3 tightly associates with key Ca²⁺ signaling proteins as well as the cytoskeletal-linker protein, ezrin. These data suggest that Trp3 is assembled in a multimeric complex which involves proteins from the plasma membrane as well as the ER. Clearly only a small fraction of the total cellular content of each protein is found in this complex. Further studies will be required to determine how this protein complex is assembled and which of these associations represent functionally relevant protein-protein interactions.

Association of the Trp3-Signaling Complex with Caveolin-1-- Caveolae have been reported to contain a number of Ca²⁺-signaling proteins and suggested to function as platforms for the regulation of receptor-mediated PIP₂ hydrolysis (32). Importantly, caveolae have been reported to be internalized in cells treated with calyculin-A (33, 34). Thus, we examined the localization of caveolin-1, a cholesterol-binding scaffolding protein that is a marker for caveolae (35). Fig. 5A shows the localization of caveolin-1 in Trp3 expressing cells (indicated by white arrows). In untreated cells, caveolin-1 showed a punctate localization in the plasma membrane. Calyculin-A treatment induced internalization of this protein, while pretreatment with either staurosporine or cytochalasin D prevented the internalization. Cytochalasin D by itself did not induce any change in caveolin-1 localization. We also tested the effect of jasplakinolide which causes cortical actin formation in the subplasma membrane region (36) on caveolin-1 and Trp3 localization (indicated by white arrows, Fig. 5B). Jasplakinolide treatment of the cells was also associated with loss of SOCE and agonist-stimulated Trp3 activity (10). As seen with calyculin-A, jasplakinolide caused internalization of both caveolin-1 and Trp3 (Fig. 5B). Thus, caveolin-1 and Trp3 are similarly affected by modifications of the cytoskeleton. Both these proteins are internalized by conditions which induce cortical actin layer formation. The calyculin-A-mediated internalization is prevented by staurosporine or cytochalasin D, although actin depolymerization itself does not affect the localization of either protein.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Localization of caveolin-1 in Trp3-expressing HEK293 cells and effect of calyculin-A. A, confocal microscopy was used to detect localization of caveolin-1. Columns are labeled as in Fig. 1. B, effect of jasplikanolide (5 µM, 60 min) on localization of caveolin-1 and Trp3. Details regarding antibody concentrations, treatments etc. are given under "Materials and Methods." The images shown represent sections from approximately the middle of the cells. Protein localization is indicated by a white arrow. Similar results were obtained in at least three to four separate experiments. C, association of Trp3 and caveolin-1. Immunoprecipitations were done with anti-HA antibody (left panel) or anti-caveolin-1 (right panel). The proteins were detected using antibodies indicated above each panel. Presence of caveolin-1 in the anti-caveolin-IP is not shown. The first lane in each case is the crude membrane fraction, the second lane, the immunoprecipitated fraction.

The association between Trp3 and caveolin-1 was further examined by determining whether these two proteins were co-immunoprecipitated. Fig. 5C (left panel) shows that caveolin-1 was present in crude membranes and a fraction of it was detected in the immunoprecipitate of Trp3. The right panel shows the reciprocal immunoprecipitation reaction; i.e. co-immunoprecipitation of HA-Trp3 with anti-caveolin-1. These data strongly suggest that Trp3 is tightly associated with caveolin-1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The data presented above demonstrate that Trp3 is assembled in a multimolecular signaling complex containing plasma membrane and ER proteins that play key roles in agonist-stimulated Ca²⁺ signaling. Consistent with the idea that proteins assembled in a complex are functionally coupled (14, 37), the proteins that we have identified in the Trp3 complex appear to be appropriate candidates for the regulation of Trp3, which reportedly functions as a Ca²⁺ influx channel activated in response to agonist stimulation of PLC, either by diacylglycerol produced as a result of PIP₂ hydrolysis (1, 2) or by interactions with the IP₃R (6-8, 10, 11). We find that PLC and its activator G_q/11 are present in the Trp3-signaling complex as are IP₃R and SERCA. Importantly, caveolin-1, a cholesterol-binding scaffolding protein which is a component of plasma membrane caveolar domains, is also associated with the Trp3-signaling complex. In aggregate, these data suggest that the Trp3-associated signaling complex is segregated in caveolae. Caveolae are specialized invaginations of the plasma membrane that function as platforms for the assembly and regulation of signal transduction complexes that are recruited into these regions by caveolin-1-dependent interactions (35). Various Ca²⁺ signaling proteins have been identified in caveolae and it has been proposed that specific interaction(s) between these proteins, leading to the activation of PLC, are facilitated by the architecture of this domain (32). Thus, our finding that Trp3 is localized in this domain is highly significant. More importantly, it is tightly associated with proteins that have a role in its regulation, either as mediators of the Ca²⁺ signaling cascade or as anchoring or scaffolding structures.

The molecular mechanism(s) involved in activating the known Trp channels or store-operated Ca²⁺ channels is not yet known. Trp3, Trp6, and dTrp have been reported to be activated in response to PLC stimulation, by PIP₂ hydrolysis, rather than internal Ca²⁺ store depletion per se (4, 5, 38). However, despite the direct activation of Trp3 by OAG, several laboratories have also demonstrated that its regulation is mediated via an interactions with IP₃R (6-8, 10, 11). Support for the suggestion that agonist regulation of Trp3 function is dependent on an interaction between Trp3 and IP₃R was presented in a previous report which showed that calyculin-A treatment prevented agonist activation of Trp3-associated cation influx. Since calyculin-A induced condensation of a cortical actin layer in the subplasma membrane region, it was proposed that interaction between Trp3 and IP₃R was physically disrupted, thus uncoupling store emptying from Ca²⁺ entry (9). These investigators hypothesized that a secretion-like mechanism, involving close but reversible interactions between the ER and plasma membrane mediated the coupling between the two proteins and that cytoskeletal remodeling could be involved in controlling such movements of the ER. Consistent with this, Rosado and Sage (13) have reported that IP₃R and Trp1 are coupled in activated platelets and that this coupling is disrupted by actin-stabilizing reagents. However, they also observed that cytochalasin D decreased Ca²⁺ influx, which is contradictory to the results of Ma et al. (10), Patterson et al. (9), and the present data. More recently, Bakowski et al. (39) reported that in RBL cells cytoskeletal modifications do not affect Ca²⁺ influx via I_CRAC. These latter findings are different from all the others discussed above. Some caution must be excercised when comparing all these data with each other. For example, CRAC, the calcium release-activated calcium channel in RBL-1 cells, has been extensively studied and it has been established that its properties are distinct from those of SOCE in other non-excitable cells or of Trps (40). Furthermore, Rosado and Sage (13) examined Trp1-mediated influx while the present study and that reported by Ma et al. (10) address Trp3 regulation. In our hands, calyculin-A blocked thapsigargin and agonist-stimulated Ca²⁺ influx in three different epithelial cell types HEK-293 (present study), HSG cells (27), and rat parotid acini (25, 26). In addition as shown here, it also blocked Trp3 activity in transfected HEK-293. We have used both whole cell patch clamp (27) and fura2 fluorescence measurements in these studies. Thus, we do not believe that the discrepancies in the effects of calyculin-A or cytoskeletal modifiers are strictly related to methodological differences. Why different cell types exhibit distinct sensitivities to various cytoskeletal reagents is an important point for consideration. Since the molecules associated with the regulation and localization of the various Trps and store-operated calcium channel(s) is not yet known, a conservative suggestion that can be made at present is that different ion channels are regulated differently. An example of this is Trp4 which has a NHERF-binding domain on its C-terminal end which is not present in other Trps (18). Such differences in the interactions of various Trp, or SOCE, channels with regulatory proteins could very well account for the contradictory findings reported by different investigators in different cell types.

Our data provide an alternative and novel explanation for the effects of calyculin-A on agonist-mediated calcium influx. A significant finding of our study is that in untreated cells where actin is present in its filamentous form localized subjacent to the plasma membrane, Trp3 is localized in the plasma membrane and coupled to the IP₃R that are localized in the ER present near the plasma membrane. However, in cells treated with calyculin-A, a substantial part of Trp3 and other plasma membrane Ca²⁺ signaling proteins, e.g. G_q/11 and PLC1, are internalized into the cell. These observations can account for the decrease in ATP-stimulated internal Ca²⁺ release, ATP-stimulated Sr²⁺ influx, and OAG-induced Sr²⁺ influx. Furthermore, we have clearly demonstrated that (i) IP₃R and Trp3 remain co-localized in cells treated with calyculin-A, and (ii) there is no change in the co-immunoprecipitation of IP₃R-3 and Trp3 after calyculin-A treatment. Thus, we conclude that the decrease in agonist-stimulated Ca²⁺ entry in calyculin-A-treated cells is due to internalization of key proteins that regulate Ca²⁺ influx rather than the physical uncoupling of Trp3 from IP₃R. These data reveal that the status of the actin cytoskeleton affects the localization of the Trp3-associated signaling complex. It will be important in future studies to determine whether agonist stimulation of cells can induce dynamic local changes in the actin cytoskeleton which promote ER-plasma membrane coupling and activation of Trp3. Such changes could be mediated via PIP₂ hydrolysis, which would alter actin-PIP₂ interactions, or by phosphorylation, and dephosphorylation, of proteins such as the ERM.

In Fig. 6, we depict how cytoskeletal rearrangements could alter the localization of the Trp3-signaling complex. Based on the data discussed above, we propose that the Trp3-signaling complex is localized in caveolae and that its regulation is dependent upon its interactions with other molecular components that are either already present in the caveolae or are recruited into this domain as a result of agonist stimulation (Fig. 6i). It has been previously reported that after calyculin-A treatment, caveolae detach from the plasma membrane and are internalized via a mechanism that involves the actin cytoskeleton (33, 34, 41). Caveolar internalization is prevented in cells treated with agents that either induce actin depolymerization or prevent actin polymerization (33). The data presented above demonstrate that these cytoskeletal modifications induce similar effects on the localization of Trp3. Therefore, we propose that when cortical actin layer is formed, caveolae containing the Trp3 complex are sealed off and internalized into the cell (Fig. 6ii). When actin is depolymerized, this internalization is prevented (Fig. 6iii). Interestingly, SOCE is also inhibited in calyculin-A-treated cells. Our previous studies demonstrated that an intact plasma membrane lipid raft domain(s) is required for the activation of SOCE and Trp1 (17). In aggregate, these studies place the SOCE mechanism in the same cellular microenvironment as Trp3 and Trp1.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Proposed effects of actin cytoskeleton reorganization on the localization of the caveolar Trp3-signaling complex. See text for details.

In conclusion, we have shown that Trp3 is assembled in multimolecular complex with key Ca²⁺ signaling proteins. We suggest that the caveolar localization of this Trp3-signaling complex likely determines the activation and regulation of the Trp3 channel activity as well as the upstream components involved in Ca²⁺ signaling. Notably, caveolar invaginations can facilitate access of the plasma membrane to regulatory components located further inside the cell, which in this case is most likely the ER (Fig. 6i). Such interactions, reminiscent of the secretion-like model (9), will be determined by the specialized architecture of the microdomain, which includes the localization of caveolae, proximity of the ER to the plasma membrane, strategic localization and/or recruitment of regulatory and signaling proteins, and presence of scaffolding proteins. Importantly, our data demonstrate that the Trp3-signaling complex is internalized by conditions which stabilize the cortical actin cytoskeleton (Fig. 6ii). This internalization, rather than disruption of the Trp3-IP₃R association, accounts for the loss of calcium influx in calyculin-A-treated cells. Caveolae have also been shown to be localized in the subplasma membrane region of cells where they function as vesicular carriers of pre-assembled protein complexes that are recruited into the plasma membrane upon stimulation (34, 35). Interestingly, proteins involved in the docking and fusion of vesicles are enriched in caveolae. Thus, the observed internalization of Trp3-containing caveolae by calyculin-A appears to be consistent with the "vesicle fusion" model (42) for activation of SOCE. Alternatively, caveolar internalization could represent a mechanism for inactivation or desensitization of the signaling complex. Further studies will be required to determine whether agonist activation of Ca²⁺ influx involves protein-protein interactions within a preformed plasma membrane caveolae-ER signaling complex or dynamic recruitment of caveolar vesicles into the plasma membrane.

	ACKNOWLEDGEMENTS

We thank Dr. William Swaim for invaluable assistance with confocal imaging. We also thank Dr. Robert Wellner for his help.

	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.

Contributed equally to the results of this work.

^§ 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: indu.ambudkar@nih.gov.

Published, JBC Papers in Press, August 27, 2001, DOI 10.1074/jbc.M106956200

	ABBREVIATIONS

The abbreviations used are: PLC, phospholipase C; Trp, transient receptor potential; IP₃R, inositol trisphosphate receptor; SERCA, sarcoendoplasmic reticulum Ca²⁺ pump; OAG, 1-oleoyl-2-acetyl-sn-glycerol; OG, octyl glucoside; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; SOCE, store-operated Ca²⁺ entry; ER, endoplasmic reticulum; HA, hemagglutinin; TG, thapsigargin; PIP₂, phosphatidylinositol 4,5-bisphosphate; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

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