αvβ3 and αvβ5 Integrins Bind Both the Proximal RGD Site and Non-RGD Motifs within Noncollagenous (NC1) Domain of the α3 Chain of Type IV Collagen

Abstract

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 antiangiogenic activity is attributed exclusively to ␣ v ␤ 3 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 ␣ v ␤ 3 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 ␣ v ␤ 3 and ␣ v ␤ 5 integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the ␣ v ␤ 3 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 antiangiogenic activity of the RGD-␣3NC1 domain.Type IV collagen is the major constituent of basement membranes, a specialized form of extracellular matrix underlying all epithelia, that compartmentalizes tissues and provides molecular signals for influencing cell behavior. The type IV collagen family is comprised of six ␣-chains (␣1-␣6) that assemble into three kinds of triple-helical protomers of different chain composition. Each protomer has three functional domains: a 7 S domain at the N terminus, a long triple-helical collagenous domain in the middle of the molecule, and a trimeric noncollagenous (NC1) domain at the C terminus. Protomers selfassemble into networks by end-to-end associations that connect four 7 S domains at one end and connect two NC1 trimeric domains at the other end, forming an NC1 hexamer configuration (1). Three types of networks are known: an ␣1⅐␣1⅐␣2 network, present in the basement membranes of all tissues and animal phyla and ␣3⅐␣4⅐␣5 and ␣1⅐␣2⅐␣5.␣6 networks that have a restricted tissue distribution. These networks are essential for tissue development and function. They provide mechanical stability, a scaffold for assembly of other macromolecular components, and act as a ligand for integrins, receptors that mediate cell adhesion, migration, growth, and differentiation.Cell ...

Human Podocytes Adhere to the KRGDS Motif of the α3α4α5 Collagen IV Network

“…The adhesion is mediated by integrin ␣v␤3, as shown in other cell types. 15,16 Thus, podocytes do not adhere to the GBM by interactions with the NC1 domain proper of the ␣3␣4␣5(IV) network. Although podocytes do not adhere to ␣3(IV) NC1 monomers, Podocyte adhesion to simian NC1 hexamer (80 g/ml) is blocked by mAb M3/1, specific for the RGD region of ␣3 NC1 domain, but not by mAb EB3, which binds to ␣3NC1 residues 127 to 141.…”

“…As shown in Figure 2B, podocyte adhesion is significantly increased by the addition of the collagenous domain containing the KRGDS motif. Thus, this motif, which is only found in primates, 16 was critical for podocyte adhesion.…”

“…12,13 Although the integrin binding sites to the ␣1␣1␣2(IV) network have been extensively studied, it is not known which integrins bind to the triple-helical domain of the ␣3␣4␣5 collagen IV network. There is evidence that ␣v␤3 and ␣3␤1 integrins bind monomeric ␣3 NC1 domain, [13][14][15][16] but no integrin binding has thus far been shown for the ␣4NC1 and ␣5NC1 domains. 13 Integrin-␣3 NC1 domain interactions are the most studied because of the potential role of the ␣3NC1 domain as an antiangiogenic agent.…”

“…Whereas current technology is unavailable to purify the full ␣3␣4␣5(IV) network, recombinant ␣3(IV), ␣4(IV), ␣5(IV) NC1 monomers and native ␣3␣4␣5(IV) hexamers are available to determine integrin binding sites. Based on our data demonstrating that various cell types bind and adhere to collagen IV NC1 domains, 15,16 we investigated how conditionally immortalized human podocytes interact with the NC1 domains of the ␣3␣4␣5(IV) network in either their monomeric or hexameric states. We demonstrate that podocytes do not adhere to NC1 domains per se; however, they do adhere to the KRGDS site of the collagenous domain located adjacent to the ␣3(IV) NC1 domain via integrin ␣v␤3.…”

“…Its binding to integrin ␣v␤3 is highly dependent on the RGD site located in the amino-terminus of the collagenous domain, whereas the interaction with integrin ␣3␤1 is RGD-independent. 15,16 The integrity of the glomerular filtration barrier requires normal interactions between the podocytes and the GBM (Figure 1). The predominant integrin expressed by podocytes is ␣3␤1, 17 and in vivo deletion of the ␣3 subunit, specifically in podocytes, results in a marked glomerular phenotype.…”