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.
Department of Molecular Pharmacology, Physiology and Biotechnology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Zhiping
Z
Wu
Wayne D
WD
Bowen
D000596:Amino Acids / Q000737:chemistry; D002118:Calcium / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017480:Receptors, sigma / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D000097605:Sigma-1 Receptor
D000596:Amino Acids; D002118:Calcium; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D017480:Receptors, sigma; D000097605:Sigma-1 Receptor; C482591:KIDINS220 protein, human