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J. Biol. Chem., Vol. 283, Issue 42, 28198-28215, October 17, 2008

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Role of Sigma-1 Receptor C-terminal Segment in Inositol 1,4,5-Trisphosphate Receptor Activation

CONSTITUTIVE ENHANCEMENT OF CALCIUM SIGNALING IN MCF-7 TUMOR CELLS^*

From the Department of Molecular Pharmacology, Physiology and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912

Sigma-1 receptor (sigma-1R) agonists enhance inositol 1,4,5-trisphosphate (IP₃)-dependent calcium release from endoplasmic reticulum by inducing dissociation of ankyrin B 220 (ANK 220) from the IP₃ receptor (IP₃R-3), releasing it from inhibition. MCF-7 breast tumor cells express little or no sigma-1R and were used here to investigate the effect of receptor overexpression and the role of its N- and C-terminal segments in function. We stably expressed intact sigma-1R (amino acids (aa) 1–223; lines 11 and 41), N-fragment (aa 1–100; line K3), or C-fragment (aa 102–223; line sg101). C-fragment expressed as a peripheral membrane-bound protein that was removable from the endoplasmic reticulum membrane by chaotropic salt wash, consistent with lack of a putative transmembrane domain. The expressed sigma-1R, N-fragment, and C-fragment exhibited normal, low affinity, and no [³H](+)-pentazocine binding activity, respectively. All transfected lines showed constitutive enhancement of bradykinin (BDK)-induced calcium release, because of a decrease in BDK ED₅₀ values. Interestingly, sigma-1R and C-fragment had high activities, whereas the N-fragment was much less active. The antagonist BD1063 behaved as an inverse agonist in sigma-1R cells, whereas C-fragment was insensitive to ligand regulation. Like BDK, vasopressin- and ATP-induced calcium release was enhanced with the same pattern in cell lines. Anti-IP₃R-3 immunoprecipitates from cells expressing sigma-1R or C-fragment contained significantly less ANK 220 compared with untransfected or N-fragment cells, indicating a higher amount of ankyrin-free IP₃R-3. Anti-ankyrin B immunoprecipitates contained sigma-1R or C-fragment, with markedly lower levels of N-fragment present. These results suggest that sigma-1R overexpression drives sigma agonist-independent dissociation of ANK 220 from IP₃R-3, resulting in activation. The C-terminal segment plays a key role in the interaction.

Received for publication, March 17, 2008 , and in revised form, June 6, 2008.

^* This work was supported by a 2007 Salomon award (to W. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Physiology, and Biotechnology, Box G-B389, Division of Biology and Medicine, Brown University, Providence, RI 02912. Tel.: 401-863-3253; Fax: 401-863-1595; E-mail: Wayne_Bowen{at}brown.edu.

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