Colocalization of ankyrin, IP₃R-3, and Sig-1R in NG-108 cells. (A) Immunocytohistochemical colocalization (yellow) of Sig-1R (green) and ankyrin B (red) in reticular perinuclear areas (Top Left), plasmalemmal regions of cell–cell contact (lower panels in low and high magnifications, respectively), and growth cones of processes in NG-108 cells (Top Right; Inset: Nomarski optical image). (B) Immunocytohistochemical colocalization (yellow) of Sig-1R (green) and IP₃R-3 (red) in perinuclear areas (Top Left), plasmalemmal regions of cell–cell contact (lower panels in low and high magnifications, respectively), and growth cones of processes in NG-108 cells (Top Right; Inset: Nomarski optical image).

Identification of IP₃R-3, ankyrin B in NG-108 cells. IP₃R-3 and ankyrin B exist in NG-108 cells. IP₃R type 1 (IP₃R-1) and ankyrin G were not detected. Positive controls of IP₃R type 1 and ankyrin G with the mouse cerebellum (CB) are also shown. Numbers with side bullets indicate positions of molecular weight markers (in kDa).

Hypothetical diagram depicting the regulation of ANK220 by Sig-1R and associated ligands. In the presence of a Sig-1R agonist, such as (+)PTZ, cocaine, or certain steroids, the Sig-1R/ankyrin B complex dissociates from IP₃R-3 on the ER perhaps as a transport vesicle. As a result, IP₃ binding to IP₃R-3 increases and the Ca²⁺ efflux is enhanced. In the presence of the putative Sig-1R antagonist, NE-100, Sig-1R is dissociated from ANK220, which remains coupled to IP₃R on the ER.

Coupling of Sig-1R, ankyrin B, and IP₃R-3 as a trimeric complex. (A) Coimmunoprecipitation (IP) followed by Western blotting of the Sig-1R/ankyrin B/IP₃R-3 complex by respective cognate antibodies. (B) Percentages of IP₃R-3 and ankyrin B, respectively, coimmunoprecipitated with Sig-1R. IP₃R-3 were immunoprecipitated with anti-IP₃R-3 antibodies or anti-Sig-1R antibodies. Ankyrin B isoforms were coimmunoprecipitated with either anti-ankyrin B antibodies or anti-Sig-1R antibodies. Immunoblots were digitally scanned and densitometrically quantified by a Macintosh computer-based image analysis program (image, National Institutes of Health). Note: Anti-IP₃R-3 antibodies and anti-ankyrin B antibodies also immunoprecipitated proteolytic products of IP₃R-3 and ankyrin B, respectively (see lanes 1 and 3, from left).

Effects of Sig-1R ligands on the dissociation of ankyrin B (ANK220) from IP₃R-3 in NG-108 cells. NG-108 cells were incubated with respective drugs and then lysed for coimmunoprecipitation (IP) studies. (A) Time- and concentration-dependent dissociation of ankyrin B (ANK220) from IP₃R-3 by the (+)PTZ treatment. The concentration of (+)PTZ in the top row was 100 nM. For the concentration-dependent curve, immunoblots were digitally scanned and densitometrically analyzed by the National Institutes of Health image program. Data were normalized to total immunoprecipitated IP₃R-3 proteins and expressed as percentage relative to the level of coimmunoprecipitated ANK220 obtained in the absence of (+)PTZ. Note: Ten-minute treatment time for all concentrations. Data represent mean ± SEM from two to four separate determinations. (B) Effects of selective Sig-1R ligands on ANK220 coupling to IP₃R-3. Sig-1R agonists (100 nM, 10 min) decreased the amount of ANK220 coimmunoprecipitated with IP₃R-3 without altering the level of IP₃R-3. NE-100 (100 nM), a Sig-1R antagonist, did not by itself affect the ANK220 dissociation from IP₃R-3. (C) Inhibition of (+)PTZ-induced ANK220 dissociation from IP₃R-3 by NE-100. NE-100 (100 nM) was applied 5 min before (+)PTZ (100 nM, 10 min). Data were analyzed as described in A and represent mean ± SEM from six to seven separate determinations. *, P < 0.05 compared with “control”; ##, P < 0.01 compared with “NE-100 + (+)PTZ”.

Regulation of the level of ANK220 in the P3L fraction. (A and B) Distribution of Sig-1R (immunoblotting) and the relative enrichments of subcellular markers for the plasma membrane (Fas), mitochondria (bcl2), Golgi (GM130) (A), rough ER (BiP/GRB78 and RNA), and smooth ER (P450 reductase) (B) in the P1, P2, P3L, and P3H fractions (see Methods). The total density of each marker at the P1, P2, P3L, and P3H fractions from Western blotting were taken as 100%. (C) Top two and bottom two rows: NE-100 blocked the loss of ANK220, but not Sig-1R, caused by the (+)PTZ (100 nM, 10 min) or cocaine treatment (10 μM, 10 min). The IP₃R-3 levels remained unaltered under all conditions (middle row). (D) Effects of NE-100 treatment (100 nM, 15 min) by itself on the total levels of Sig-1R and ANK220 (top two rows) and on the portion of ankyrin B coimmunoprecipitated (IP) by anti-Sig-1R antibodies in the P3L fraction (lower row). (E) Effects of (+)PTZ treatment (100 nM, 10 min) on the ANK220 and ANK135 that could be coimmunoprecipitated by anti-Sig-1R antibodies in the whole cell lysate.

Relationship between the degree of dissociation of ANK220 from IP₃R-3 induced by Sig-1R ligands and the Sig-1R ligands' abilities to potentiate bradykinin-induced increase in [Ca^{2 +} ]cyt in NG-108 cells. (A) Potentiation of the bradykinin-induced increase in [Ca²⁺]cyt by (+)PTZ (100 nM, 10 min). (B) Temporal correlation of (+)PTZ (100 nM) in increasing the dissociation of ANK220 from IP₃R-3 and in potentiating the [Ca²⁺]cyt increase induced by bradykinin. The numbers below open circles indicate the treatment time (min) of NG-108 cells with (+)PTZ. (C) Efficacy correlation of Sig-1R ligands in their abilities to cause ANK220 dissociation from IP₃R-3 at the 10-min point and in their abilities at the same time point to potentiate bradykinin-induced increases in [Ca²⁺]cyt. Concentrations of Sig-1R ligands were all 100 nM. The total number of cells examined and the number of determinations (in parentheses) for each test drug are indicated (see Methods): NE-100, 23 cells (5); pregnenolone sulfate, 58 cells (7); progesterone, 34 cells (5); PRE084, 91 cells (14); (+)SKF-10047, 36 cells (6); (+)PTZ, 59 cells (8). The value on the x axis represents the mean value from all determinations for each drug. (D) Increase in [³H]IP₃ binding to microsomes of NG-108 cells caused by (+)PTZ. After the (+)PTZ treatment (100 nM, 10 min), cells were harvested and P3 fractions prepared and used for [³H]IP₃ binding assay. Data represent mean ± SEM of three determinations, each assayed in triplicate. *, P < 0.05.