CELLULAR INTERNALIZATION AND DEGRADATION OF THROMBOSPONDIN-1 IS MEDIATED BY THE AMINO-TERMINAL HEPARIN-BINDING DOMAIN (HBD) - HIGH-AFFINITYINTERACTION OF DIMERIC HBD WITH THE LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN

Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly sy nthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 cat abolism is mediated by the low density lipoprotein receptor-related pr otein (LRP), a large endocytic receptor that is a member of the low de nsity lipoprotein receptor family. Using adenovirus-mediated gene tran sfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP 1. An objective of the cur rent investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino te rminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, wh en prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent K-D ranging from 10 to 25 nM. R ecombinant HBD (rHBD) purified following proteolytic cleavage of GST-H BD, also bound to purified LRP, but with an apparent K-D of 830 nM. Th e difference in affinity was attributed to the fact that GST-HBD exist s in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, I-125 -labeled GST-HBD or I-125-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both I-125-labele d GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa r eceptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of I-125-labeled TSP-1 in a do se dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-termi nal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside withi n the same TSP-1 domain. Further, high affinity binding of TSP-1 to LR P likely results from the trimeric structure of TSP-1.