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Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity.

Duquette M, Nadler M, Okuhara D, Thompson J, Shuttleworth T, Lawler J - Matrix Biol. (2014)

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.

View Article: PubMed Central - PubMed

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.

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Binding of the N-terminal domain of STIM1 to TSPs. A. Human platelet TSP-1 and BSA were adsorbed by the wells of an Immulon II plate overnight in 2 mM CaCl2. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. B. Recombinant COMP and BSA were adsorbed to the wells of an Immulon II plate overnight in 2 mM CaCl2. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. C. Human platelet TSP-1 and E3T3C1 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. D. Human recombinant COMP and E4T3C5 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. In all cases, the wells were washed, and the bound protein then was quantified using a fluorescent plate reader. Each point shown is the mean and standard deviation of four determinations.
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Figure 4: Binding of the N-terminal domain of STIM1 to TSPs. A. Human platelet TSP-1 and BSA were adsorbed by the wells of an Immulon II plate overnight in 2 mM CaCl2. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. B. Recombinant COMP and BSA were adsorbed to the wells of an Immulon II plate overnight in 2 mM CaCl2. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. C. Human platelet TSP-1 and E3T3C1 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. D. Human recombinant COMP and E4T3C5 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. In all cases, the wells were washed, and the bound protein then was quantified using a fluorescent plate reader. Each point shown is the mean and standard deviation of four determinations.

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.


Members of the thrombospondin gene family bind stromal interaction molecule 1 and regulate calcium channel activity.

Duquette M, Nadler M, Okuhara D, Thompson J, Shuttleworth T, Lawler J - Matrix Biol. (2014)

Binding of the N-terminal domain of STIM1 to TSPs. A. Human platelet TSP-1 and BSA were adsorbed by the wells of an Immulon II plate overnight in 2 mM CaCl2. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. B. Recombinant COMP and BSA were adsorbed to the wells of an Immulon II plate overnight in 2 mM CaCl2. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. C. Human platelet TSP-1 and E3T3C1 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. D. Human recombinant COMP and E4T3C5 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. In all cases, the wells were washed, and the bound protein then was quantified using a fluorescent plate reader. Each point shown is the mean and standard deviation of four determinations.
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Figure 4: Binding of the N-terminal domain of STIM1 to TSPs. A. Human platelet TSP-1 and BSA were adsorbed by the wells of an Immulon II plate overnight in 2 mM CaCl2. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. B. Recombinant COMP and BSA were adsorbed to the wells of an Immulon II plate overnight in 2 mM CaCl2. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 2 mM CaCl2. C. Human platelet TSP-1 and E3T3C1 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. Fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. D. Human recombinant COMP and E4T3C5 were adsorbed by the wells of a FluoroNunc Maxisorb plate overnight in the presence of 5 mM EDTA. The fluorescently labeled, recombinant N-terminal domain of STIM1 was incubated in the wells overnight in 5 mM EDTA. In all cases, the wells were washed, and the bound protein then was quantified using a fluorescent plate reader. Each point shown is the mean and standard deviation of four determinations.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus