Cell Culture and Cloning of Sig-1R from NG-108 Cells.

Culture of NG-108 cells was performed as described (30). Standardcloning procedures were followed for the cloning of Sig-1R in NG-108cells. Briefly, the extracted total RNA of the cells was treated withDNase I and reverse transcribed with random hexamer priming (CLONTECH).By using the products as template, PCR was performed by using5′-CCAGGCTGCCCGCT-3′ as the sense primer and5′-GTGAGTGCATGATCTTACAGTAC-3′ as the antisense primer. These sequencesare 100% conserved between mouse Sig-1R (GenBank accession no.AF030198; sense base 5–18; antisense base 901–923) and rat Sig-1R(GenBank accession no.AF004218; sense base 22–35; antisense base918–940). The PCR products were subcloned into the TA cloning vector(Invitrogen) and sequenced by the dideoxynucleotide chain terminationmethod by using fluorescein donor dyes (Perkin–Elmer). Resultsindicated that the sequence of Sig-1R in NG-108 cells thus cloned is100% identical to that of the rat Sig-1R as previously reported (31).

Subcellular Fractionation of NG-108 Cells.

The subcellular fractionation was performed according to a previousreport (32). Cells were washed twice with ice-cold PBS and harvested.All subsequent steps were carried out at 4°C. After centrifugation(400 ×g, 5 min), cells were suspended in hypotonic TMbuffer (10 mM Tris⋅HCl/1 mM MgCl₂/10μg/ml aprotinin/1 mM PMSF; pH 7.4) and incubated for 20 min. Thecell suspension was diluted with the same volume of TM buffercontaining 0.5 M sucrose and cells disrupted by a Dounce homogenizer(20 strokes). The P1 fraction was obtained as a pellet aftercentrifugation at 900 ×g for 5 min. The supernatantswere centrifuged at 10,000 ×g for 20 min to obtainthe P2 fraction (the pellet). The resultant supernatants werecentrifuged at 105,000 ×g for 60 min to yield the P3fraction (the pellet). The P3 fraction was suspended in the TM buffercontaining 1.35 M sucrose and further separated by centrifugation (SW28rotor at 105,000 ×g, 3 h, with the brake off) ondiscontinuous sucrose gradients (0.25 M, 0.8 M, 1.35 M, 2.1 M) into theP3L (layer between 0.8 M/1.35 M) and the P3H fractions (layer between1.35 M/2.1 M). Each layer was collected separately and diluted with 6vol of TM buffer without sucrose and recentrifuged at 150,000 ×g for 3 h. The pellets were lysed and used forexperiments. The subcellular fractions thus obtained were characterizedby examining the relative enrichment of representative organellemarkers in each fraction by Western blotting. In the present study,only the P3L fraction, containing the highest density of Sig-1R, wasused to examine the dissociation of ANK220 from the subcellularfraction (seeResults).

Immunoprecipitation and SDS/PAGE.

Samples were dissolved in lysis buffer [10 mM Tris⋅HCl (pH7.0)/150 mM NaCl/1% Nonidet P-40/0.5% deoxycholate/5 mMEDTA/1 mM EGTA/1 mM PMSF/10 μg/ml leupeptin/1 mg/mlbenzamidine/15 μg/ml aprotinin/1 mM sodium orthovanadate] andsonicated (2 s × 3). After centrifugation, supernatants weresubjected to immunoprecipitation or SDS/PAGE. For immunoprecipitationof IP₃R type 3 (IP₃R-3),freshly prepared samples containing 500 μg proteins were preclearedand incubated with a mouse monoclonal anti-IP₃R-3antibody (Transduction Laboratories, Lexington, KY) at a dilution of1:50. Immunocomplexes were precipitated by protein A-Sepharose CL-4Bbeads (Amersham Pharmacia) with the addition of the anti-mouse IgGrabbit antibody (Jackson ImmunoResearch) to anchor the mouse IgG to thebeads. When immunoprecipitants were to be used for Sig-1R Westernblotting, however, the anti-mouse IgG rabbit antibody was omitted toavoid the masking of Sig-1R (30 kDa) by the predominant signal from thelight chain of rabbit IgG. Polyclonal Sig-1R antibodies were raised inrabbits by using a peptide antigen corresponding to amino acids144–165 of guinea pig Sig-1R (GenPept accession no.CAA91441). Proteinlysates or immunoprecipitants were separated by SDS/PAGE andtransblotted onto polyvinylidene difluoride membranes without methanol.For immunodetection, membranes were incubated with respectiveantibodies at the dilution of 1:1,500 for Sig-1R, 1:1,000 forIP₃R-3, and 1:150 for ankyrin B (mousemonoclonal; Oncogene Science) and visualized as described (30). Theresultant protein bands were scanned digitally and densitometricallyanalyzed by a Macintosh computer-based analysis system(image, National Institutes of Health) (30). To examine theeffects of different Sig-1R ligands on the coimmunoprecipitation of theprotein complexes, the same procedures described before were followed(30). Briefly, before treatment of cells with Sig-1R ligands, cells inculture wells were deprived of FCS (which may containendogenous steroids) by careful washings with Hanks'balanced salt solution containing 1% BSA. The cells remained culturedin the same buffer for at least 30 min before addition of Sig-1Rligands.

Confocal Microscopy of Cytosolic Free Ca²⁺and Immunostaining.

Cytosolic Ca²⁺ concentrations were measured asdescribed elsewhere in great detail (30). Each experimentaldetermination used a four-well FlexiPERM plate (Heraeus). Theconcentration of bradykinin used to elicit an increase in cytosolicCa²⁺ concentration was 1 μM. An average ofthree to nine cells per culture well was examined in eachdetermination, which always included a control well (30). In eachdetermination, a similar treatment condition was never repeated inother wells, except occasionally the controls may have been repeated.In this report, data were obtained from an average of 5–14determinations by examining an average total of 20–91 cells for eachtest drug. The mean values from multiple determinations are presentedin the study (see Fig.5 legend). Images of immunostaining werecollected by using argon ion laser (488 or 543 nm) and long-passbarrier filter (520 or 590 nm). Sixteen images (512 × 512 pixels)were averaged and collected digitally by using Zeissimagesoftware.

[³H]IP₃-Binding Assay.

The binding of [³H]IP₃ (8nM) to microsomes of NG-108 cells (100 μg) was assayed in 0.5 ml ofice-cold buffer containing 50 mM Tris⋅HCl (pH 8.0) and 1 mM EDTA.Tubes were incubated at 4°C with constant shaking. After 30 min,reactions were terminated by passing the tissue mixture through WhatmanGF/B filters by means of a rapid single manifold filtration, followedby three washings with 5 ml of ice-cold buffer. The filters werepresoaked with ice-cold buffer for 30 min before use. Filters were thensoaked overnight in 6 ml of scintillation mixture (Poly Fluor; Packard,Meriden, CT) containing 0.8% (vol/vol) acetic acid. Theradioactivity trapped on the filters was measured by using a liquidscintillation counter. Nonspecific binding was measured in the presenceof 4 μM IP₃.

Immunostaining.

Immunocytohistochemistry was performed on the 4%paraformardehyde-fixed cultured cells. The anti-Sig-1R IgG was affinitypurified (AminoLink Kit; Pierce). Specificity was verified by using apreimmune serum and a preabsorbed Sig-1R antibody. Fixed cells wereincubated with an anti-ankyrin B monoclonal antibody (1:200) and/oran anti-Sig-1R antibody (1:300) at 4°C for 24 h. ForSig-1R-ankyrin B double staining, Alexa 488 goat anti-rabbit IgGantibody or Alexa 590 goat anti-mouse IgG antibody, respectively(Molecular Probes), was used as the secondary antibody generatingimmunofluorescence. In Sig-1R-IP₃R-3 doublestaining, Alexa 590 goat anti-rabbit IgG antibody for Sig-1R, andFITC-conjugated anti-mouse IgG and Alexa 488 anti-FITC antibody forIP₃R-3 were used for immunofluorescence.

Drugs and Reagents.

NG-108 cells were purchased from American Type Culture Collection.PRE084 was synthesized as described (33). (+)Pentazocine, (+)SKF-10047,and cocaine were from Division of Basic Research, National Instituteson Drug Abuse (Bethesda, MD). Pregnenolone sulfate, progesterone, andbradykinin were purchased from Research Biochemicals (Natick, MA). Theanti-IP₃R type 1 antibodies were from Calbiochemand antiankyrin G antibodies from Oncogene Science. Other antibodiesand their suppliers are: NADPH Cytochrome P450 reductase and bcl2(StressGen Biotechnologies, Victoria, Canada); BiP/GRB78 and GM130(Transduction Laboratories); Fas (Santa Cruz Biotechnology).

IP₃R-3 and Ankyrin B in NG-108 Cells.

Immunoblottings by using specific antibodies indicated that NG-108cells were predominantly expressing IP₃R-3 ratherthan IP₃R type 1 (Fig.1). Isoforms of ankyrin B were detectedin NG-108 cells (Fig.1). Ankyrin G was not detected in NG-108 cells(Fig.1).

The Sig-1R/Ankyrin B/IP₃R-3 Complex and ItsRegulation by Sig-1R Agonists.

First, we examined whether Sig-1R bind to theankyrin/IP₃R-3 complex (7,8,34) in NG-108cells. Sig-1R antibodies coimmunoprecipitated bothIP₃R-3 and ankyrin B isoforms (e.g., ANK220,ANK135) in NG-108 cells (Fig.2A). In analogy to thatreported in rat brain (34), IP₃R-3 antibodiescoimmunoprecipitated ankyrin B isoforms. However, ANK220 was the majorisoform coimmunoprecipitated with IP₃R-3 and notANK135 (Fig.2A). IP₃R-3antibodies also coimmunoprecipitated Sig-1R (Fig.2A). The ankyrin B antibody coimmunoprecipitatedSig-1R and IP₃R-3 (Fig.2A).Thus, Sig-1R form a multicomplex with ankyrin B andIP₃R-3 in NG-108 cells.

Percentages of the total IP₃R-3 and ankyrin B,which may exist in a complex with Sig-1R, were determined. About 43%of IP₃R-3 was coimmunoprecipitated with Sig-1R(Fig.2B). About 20% of ANK220 wascoimmunoprecipitated with Sig-1R (Fig.2B). However,about 97% of ANK135 was coimmunoprecipitated with Sig-1R (Fig.2B). The majority of ANK135 are thus coupled toSig-1R, whereas only a portion of ANK220 is coupled to Sig-1R.

We examined next whether Sig-1R agonists like (+)pentazocine[(+)PTZ], (+)SKF10047, and PRE084 might affect the complex formationbetween Sig-1R, ankyrin B, and IP₃R-3. (+)PTZtreatment (100 nM, 10 min) caused ankyrin B isoforms, predominantlyANK220, to dissociate from IP₃R-3 (Fig.3A). The ANK135 was notsignificantly affected (Fig.3A). It took about 10 min for(+)PTZ to dissociate ≈70% of ANK220 fromIP₃R-3 (Fig.3A). The concentrationsof (+)PTZ (low nM) in causing the ANK220 dissociation were close to theK_d value of (+)PTZ at Sig-1R (11)(Fig.3A). The same results were obtained with PRE084 and(+)SKF10047 (Fig.3B). NE-100, a high-affinity Sig-1Rantagonist (35), although by itself not affecting thecoimmunoprecipitation of ankyrin B with IP₃R-3(Fig.3B), blocked the ANK220 dissociation fromIP₃R-3 caused by (+)PTZ (Fig.3C).

Regulation of Ankyrin Levels in the P3L Fraction by Sig-1R andAssociated Ligands.

IP₃R are ER proteins (7,8). Therefore, resultsfrom the above immunoprecipitation studies might suggest that Sig-1Ragonists can cause the dissociation of ANK220 from the ER. We thusexamined the level of ANK220 in the P3L subcellular fraction (seeMethods), which was characterized in this study andfound to be enriched in Sig-1R and smooth ER with minimalcontaminations from other organelles (Fig.4A andB).

The ANK220 level in the P3L fraction was reduced by the (+)PTZtreatment (Fig.4C, Top Row). This effect by (+)PTZ wasblocked by NE-100 (Fig.4C, Top Row). Sig-1R were alsoreduced in the P3L fraction by (+)PTZ (Fig.4C, Second Row).However, the Sig-1R loss from the P3L fraction caused by (+)PTZ was notblocked by NE-100 (Fig.4C, Second Row). The levels ofIP₃R-3 in the P3L fraction remained unaltered(Fig.4C, Third Row). Because cocaine binds to Sig-1R (14)and because Sig-1R antagonists blocked the behavioral effects ofcocaine (19,20), the effects of cocaine were examined. Like (+)PTZ,cocaine also reduced the levels of both Sig-1R and ankyrin B in the P3Lfraction (Fig.4C, Lower Two Rows). NE-100 again blocked theankyrin loss, but not the Sig-1R loss caused by cocaine (Fig.4C, Lower Two Rows).

These results suggest that Sig-1R agonists cause the dissociation ofANK220, as well as Sig-1R, from the smooth ER. The results with NE-100,however, suggested that NE-100 may uncouple Sig-1R from theANK220/IP₃R-3 complex, thus leaving (+)PTZ ableto dissociate only Sig-1R from the ER. To provide evidence for thispossibility, the effects of NE-100 alone were examined. NE-100 did notapparently affect the total ANK220 or total Sig-1R level in the P3Lfraction (Fig.4D, Top Two Rows). In the presence of NE-100,however, ANK220 could no longer coimmunoprecipitate with Sig-1R (Fig.4D, Bottom Row). NE-100 may thus act as an “uncoupler”between ankyrin B and Sig-1R on the ER to achieve an apparentantagonistic effect against the pharmacological effects of Sig-1Ragonists, such as (+)PTZ and cocaine.

It is interesting to note that the total amount of ANK220 (or ANK135)in NG-108 cells coimmunoprecipitated by anti-Sig-1R antibodies wasfound to be unaltered by the (+)PTZ treatment (Fig.4E).This suggests that Sig-1R and ANK220 remain as a complex afterdissociation from the ER.

ANK220 Dynamics Regulated by Sig-1R Affects Ca²⁺Signaling at the IP₃R-3.

We demonstrated previously that Sig-1R and associated ligands causedthe potentiation of bradykinin-induced increase of cytosolicCa²⁺ concentrations([Ca²⁺]cyt) in NG-108 cells without affectingIP₃ formation (30). Because ankyrin binding toIP₃R inhibited theIP₃R-mediated Ca²⁺ efflux(7,8), the ankyrin dissociation from IP₃R-3 may,by extension, be related to Ca²⁺ efflux. Thus, weexamined whether ankyrin dissociation from ER caused by Sig-1R ligandsmight affect [Ca²⁺]cyt.[Ca²⁺]cyt was monitored by using confocalmicroscopy.

It was found that, although Sig-1R ligands caused dissociation ofankyrin B, specifically ANK220 (see Fig.3A), fromIP₃R-3, the basal[Ca²⁺]cyt was, in agreement with our previousresults (30), not altered. This indicates that dissociation of ankyrinper se from IP₃R-3 does not openIP₃R-3 channels and that the lowIP₃ concentration in resting cells is unable totrigger Ca²⁺ efflux from the ER. However, theresults also suggest a possibility that, given a sufficientconcentration of IP₃, the dissociation of ANK220from IP₃R-3 may participate in the opening ofIP₃R-3 channels. Because bradykinin is known toincrease IP₃ (30,36), and, because we have shownthat Sig-1R ligands potentiate the bradykinin-induced increase in[Ca²⁺]cyt (30), we examined whether thedissociation of ANK220 from IP₃R-3 caused bySig-1R ligands may correlate with their potencies to potentiate thebradykinin-induced increase in [Ca²⁺]cyt.

It took about 10 min for (+)PTZ to reach a near maximal effect incausing both the ANK220 dissociation and the[Ca²⁺]cyt potentiation (Fig.5A andB). Theefficacies of Sig-1R ligands, including certain steroids, to dissociateANK220 from IP₃R-3 at the 10-min time pointcorrelated excellently with their abilities to potentiate thebradykinin-induced increase in [Ca²⁺]cyt (Fig.5C;r = 0.938;P = 0.0057).

To demonstrate a causal link behind these effects, we examined nextwhether (+)PTZ, by causing a dissociation of ANK220 fromIP₃R-3, may increase the binding of[³H]IP₃ toIP₃R, thus causing an increase inCa²⁺ efflux. Indeed,[³H]IP₃ binding to ERmembrane was increased by about 30% in (+)PTZ-treated cells (Fig.5D). Thus, the dissociation of ANK220 fromIP₃R-3 caused by Sig-1R agonists enhances theinteraction between IP₃ andIP₃R-3, leading to a potentiation of thebradykinin-induced increase in [Ca²⁺]cyt.

Colocalization of Sig-1R, Ankyrin B, and IP₃R-3.

Immunocytohistochemistry indicates that ankyrin B and Sig-1R arecolocalized in certain areas in NG-108 cells. Specifically,colocalization is seen in perinuclear areas in reticular patterns (Fig.6A andB). Inconfluent cells, striking colocalizations are seen in plasmalemmalregions of cell–cell contact (Fig.6A). Moreover,Sig-1R and ankyrin are also colocalized in growth cones (Fig.6A). Sig-1R are also colocalized withIP₃R-3 in the same areas where Sig-1R andankyrins are colocalized (Fig.6B). In particular, growthcones exhibit the colocalization of Sig-1R andIP₃R-3 (Fig.6B). These resultssupport the presence of the Sig-1R/ankyrinB/IP₃R-3 complex at the ER and in cellularcomponents implicated in cell-to-cell interaction and cell growth.

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