4 research outputs found
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*
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