Cellular internalization and degradation of thrombospondin-1 is mediated by the amino-terminal heparin binding domain (HBD). High affinity interaction 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 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.
Mikhailenko
I
I
Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
Krylov
D
D
Argraves
K M
KM
Roberts
D D
DD
Liau
G
G
Strickland
D K
DK
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D004705:Endocytosis; D006493:Heparin / Q000378:metabolism; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000378:metabolism*; D011973:Receptors, LDL / Q000378:metabolism; D013329:Structure-Activity Relationship; D019699:Thrombospondins
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D008562:Membrane Glycoproteins; D011971:Receptors, Immunologic; D011973:Receptors, LDL; D019699:Thrombospondins; C036053:VLDL receptor; D006493:Heparin