Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity.
Bottom Line: This association is robust since it is preserved in Triton X-100, can be detected with multiple anti-TSP-1 and anti-STIM1 antibodies, and is detected in a wide range of cell types.Thus, this interaction could occur in the ER under conditions of normal or low calcium concentration.These data indicate that the TSPs regulate STIM1 function and participate in the reciprocal regulation of two channels that mediate calcium entry into the cell.
Affiliation: The Division of Experimental Pathology, Department of Pathology, Beth Israel Deaconess Medical School, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215, United States.Show MeSH
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Mentions: Having established the co-association between the TSPs and STIM1, we next undertook experiments to determine if this association is due to direct binding. A solid-phase binding assay demonstrated the direct interaction of the N-terminal domain of STIM1 with TSPs (Fig. 4). The wells of the plate were coated with TSP-1 or COMP, and BSA was used as a negative control. The addition of increasing amounts of labeled N-terminal domain of STIM1 resulted in increased binding to the TSPs with little or no binding to BSA observed (Fig. 4A and B). The binding of fluorescently labeled N-terminal domain of STIM1 to TSP-1 or COMP was inhibited by the non-labeled N-terminal of the STIM1 domain, indicating that the observed binding was not an artifact of the labeling procedure. Binding of the N-terminal domain of STIM1 to the TSPs was also observed when the assay was performed in the presence of EDTA (Fig. 4C and D). This result was consistent with the earlier experiments in which STIM1 and TSP-1 co-immunoprecipitate in the presence or absence of calcium. Moreover, the N-terminal domain of STIM1 also bound to recombinant versions of the signature domains of TSP-1 (E3T3C1) and COMP (E4T3C5) in a saturable manner (Fig. 4C and D). These data demonstrate that STIM1 co-association with TSPs is the result of direct binding.
Affiliation: The Division of Experimental Pathology, Department of Pathology, Beth Israel Deaconess Medical School, Harvard Medical School, 99 Brookline Ave., Boston, MA 02215, United States.