Cellular Internalization and Degradation of Thrombospondin-1 Is Mediated by the Amino-terminal Heparin Binding Domain (HBD)

doi:10.1074/jbc.272.10.6784

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*

From the Holland Laboratory, Departments of ^‡ Biochemistry and
^∥Molecular Biology, American Red Cross, Rockville, Maryland 20855, and the
Laboratories of ^§ Biochemistry and
^¶ Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892

** To whom correspondence should be addressed:
American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855.
Tel.: 301-738-0726; Fax: 301-738-0794; E-mail: strickla{at}usa.redcross.org

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 K_D 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 K_D 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.

Footnotes

↵* This work was supported by Grants HL50787 (to D. K. S.), GM42581 (to D. K. S.), and HL37510 (to G. L.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TSP-1

thrombospondin-1

LDL

low density lipoprotein

VLDL

very low density lipoprotein

LRP

low density lipoprotein receptor-related protein

RAP

receptor-associated protein

HBD

amino-terminal heparin binding domain of thrombospondin generated by proteolytic cleavage of human TSP

rHBD

recombinant heparin binding domain of murine thrombospondin containing residues 1-214

GST

glutathione S-transferase

Ad-VLDLR

adenovirus containing the human VLDL receptor cDNA

Ad-LacZ

adenovirus containing lacZ cDNA

BSA

bovine serum albumin

ELISA

enzyme-linked immunosorbent assay

TBS

Tris-buffered saline.
↵² I. Mikhailenko and D. K. Strickland, unpublished experiments.
- Received June 6, 1996.
- Revision received November 6, 1996.