Abstract

Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-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 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.