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J. Biol. Chem., Vol. 279, Issue 4, 2772-2780, January 23, 2004

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_v3 and _v5 Integrins Bind Both the Proximal RGD Site and Non-RGD Motifs within Noncollagenous (NC1) Domain of the 3 Chain of Type IV Collagen

IMPLICATION FOR THE MECHANISM OF ENDOTHELIAL CELL ADHESION^*

Vadim Pedchenko¶, Roy Zent||, and Billy G. Hudson**

From the Division of Nephrology, the Center for Matrix Biology, the ^**Department of Biochemistry, and the ^||Veterans Affairs Hospital, Vanderbilt University Medical Center, Nashville, Tennessee 37232

The NC1 domains of human type IV collagen, in particular 3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051–8061). The recombinant 3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to _v3 integrin interactions with non-RGD motifs of the RGD-3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745–23750). This nonfunctionality is surprising given that RGD is a binding site for _v3 integrin in several proteins. In the present study, we used the 3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of _v3 and _v5 integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the _v3 integrin-binding sites of the 3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the 3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-3NC1 domain.

Received for publication, October 30, 2003

^* This work was supported by National Institutes of Health Grants DK18381 (to B. G. H.), DK065123 (to B. G. H.), and P50 DK39261-16 (to R. Z.), a research grant from BioStratum, Inc. (to V. P.), Veterans Affairs Advanced Career Development and Merit Awards (to R. Z.), a grant-in-aid from the American Heart Association (to R. Z.), and Clinician Scientist Award from the National Kidney Foundation (to R. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence may be addressed: Div. of Nephrology, Vanderbilt University Medical Center, S-3223 MCN, 1161 21^st Ave. S., Nashville, TN 37232-2372. E-mail: vadim.pedchenko{at}vanderbilt.edu. To whom correspondence may be addressed. E-mail: billy.hudson{at}vanderbilt.edu.

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