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: We used a proteomic approach to identify proteins that associate with the signature domain of TSP-1 in human blood platelets. TSP-1 was immunoprecipitated from Brij 99 solubilized human blood platelets using a monoclonal antibody to the N-terminal domain, designated MA-IV. We postulated that an antibody to this domain would be least likely to displace proteins that are bound to the signature domain because the two domains are at opposite ends of the molecule. The immunoprecipitates were electrophoresed and various molecular weight ranges were excised and analyzed by mass spectroscopy. The greatest number of peptides identified include those from fibrinogen (18 peptides), the integrin αIIbβ3 (6 peptides), coagulation factor XIII (6 peptides) and GRP78/BiP (4 peptides), and all are known to associate with TSP-1, validating our experimental approach (Bale and Mosher, 1986; Prabakaran et al., 1996; Panetti et al., 1999). Notably, several novel binding partners, such as STIM1 (6 peptides), were identified. We next performed immunoblotting of TSP-1 immunoprecipitates for STIM1 to confirm the mass spectrometry data. Platelets were solubilized in TBS containing either 1% Brij 99 or Triton X-100 and immunoprecipitations were preformed with the anti-TSP-1 monoclonal antibody MA-IV or the anti-TSP-1 polyclonal antibody R1. Immunoblotting of these immunoprecipitates with a monoclonal anti-STIM1 antibody (clone 44) identified an 84,000-dalton band that corresponds to the molecular weight of intact STIM1 (results with R1 are shown in Fig. 1A, lane 2). No STIM1 was detected when the immunoprecipitations were performed with isotype matched IgG (for MA-IV) or pre-immune serum (Fig. 1A, lanes 1, 3 and 5). In addition, an extremely weak band with a molecular weight of 68,000 Da was variably detected in these immunoprecipitates (Fig. 1A, lane 2). We also performed the immunoprecipitation with an anti-STIM1 antibody, designated 5A2, followed by western blotting with a rabbit polyclonal antiserum that was raised against a fusion protein containing the type 1 repeats of TSP-1 designated R3. These experiments specifically identified the 180,000-dalton subunit of TSP-1 in the STIM1 immunoprecipitates (Fig. 1B). Taken together, these data demonstrate a specific interaction between STIM1 and TSPs, and are consistent with a recent report by Ambily et al. (2014) in which TSP-1 was detected in STIM1 immunoprecipitations from human platelets.
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.