title: Alpha(v)beta3 and alpha(v)beta5 integrins bind both the proximal RGD site and non-RGD motifs within noncollagenous (NC1) domain of the alpha3 chain of type IV collagen: implication for the mechanism of endothelia cell adhesion.
abstract: The NC1 domains of human type IV collagen, in particular alpha3NC1, 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 alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. 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 alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 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 alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 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 alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 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 alpha3NC1 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-alpha3NC1 domain.
authors: Division of Nephrology, Veterans Affairs Hospital, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA. vadim.pedchenko@vandebilt.edu
authors: Vadim
authors: V
authors: Pedchenko
authors: Roy
authors: R
authors: Zent
authors: Billy G
authors: BG
authors: Hudson
mesh_terms: D000595:Amino Acid Sequence; D001324:Autoantigens / Q000378:metabolism* / Q000502:physiology; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D024141:Collagen Type IV / Q000378:metabolism* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000378:metabolism* / Q000502:physiology; D016023:Integrins / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D009842:Oligopeptides; D011485:Protein Binding; D019030:Receptors, Vitronectin / Q000378:metabolism* / Q000502:physiology
chemical_list: D001324:Autoantigens; D024141:Collagen Type IV; D039302:Integrin alphaVbeta3; D016023:Integrins; D009842:Oligopeptides; D019030:Receptors, Vitronectin; C064660:integrin alphaVbeta5; C057112:type IV collagen alpha3 chain; C047981:arginyl-glycyl-aspartic acid