Thrombospondins (TSPs) are secreted glycoproteins found in organisms as diverse as Drosophila (lgene), Ciona (several genes), and vertebrates (5 genes called TSP-1 through TSP-5). The signature piece of a TSP subunit is at its C-terminus and comprised of tandem EGF-like modules, a series of aspartate-rich Ca2+-binding loops, and approximately 230 residues with long blocks of identity among """"""""SPs. This part of TSPs binds many (>20 per human TSP-2 subunit) Ca2+. Such binding is cooperative over the range of 50-500 micromolar Ca2+ and associated with major conformational changes. Missense mutations and non-synonymous single nucleotide polymorphisms of DNA encoding the C-terminal region of TSPs have been associated with human musculoskeletal, cardiovascular, and respiratory diseases. We hypothesize that TSPs function in the endoplasmic reticulum as Ca2+-sensitive escorts of cell surface and extracellular proteins being processed through the secretory apparatus and that disturbances of these functions contribute to disease. These hypotheses will be evaluated by experiments focused on 3 specific aims. (1) Determine structure of the C-terminal portions of human TSP-1, TSP-2 and TSP-4. We recently obtained crystals of a recombinant TSP-2 construct that are suitable for such a determination. Successful completion of this aim will give unprecedented insight into the structure and function of TSPs. (2) Determine consequences of the N700S polymorphism in a Ca2+-binding loop of TSP-1. This polymorphism has been associated with familial premature coronary artery disease and poor outcome after acute lung injury. The experiments will build on recent studies suggesting that TSP-1 S700 is more labile than TSP-1 N700. Studies will be extended to the A387P polymorphism in the EGF region of TSP-4 to explore whether the TSP-4 polymorphism, also a presumptive determinant of cardiovascular disease, causes changes similar to the TSP-1 polymorphism. (3) Compare secretory trafficking of TSP-1 S700 and N700. Transfected cells expressing these proteins as GFP chimeras will be subjected to heat shock, manipulations of ER [Ca2+], and other treatments that perturb trafficking. We will identify conditions in which the proteins are arrested in the ER and ask whether the S700 protein is more sensitive to a given perturbation. ? ?
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