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.

Integrin activation is crucial for numerous cellular responses, including cell adhesion, migration, and survival. Recent studies in mice have specifically emphasized the vital role of kindlin-3 in integrin activation. Kindlin-3 deficiency in humans also has now been documented and includes symptoms of bleeding, frequent infections, and osteopetrosis, which are consequences of an inability to activate beta1, beta2, and beta3 integrins. To date, kindlin-3 was thought to be restricted to hematopoietic cells. In this article, we demonstrate that kindlin-3 is present in human endothelial cells derived from various anatomical origins. The mRNA and protein for KINDLIN-3 was detected in endothelial cells by reverse transcription-PCR and Western blots. When subjected to sequencing by mass spectrometry, the protein was identified as authentic kindlin-3 and unequivocally distinguished from KINDLIN-1 and KINDLIN-2 or any other known protein. By quantitative real time PCR, the level of kindlin-3 in endothelial cells was 20-50% of that of kindlin-2. Using knockdown approaches, we show that kindlin-3 plays a role in integrin-mediated adhesion of endothelial cells. This function depends upon the integrin and substrate and is distinct from that of kindlin-2. Formation of tube-like structures in Matrigel also was impaired by kindlin-3 knockdown. Mechanistically, the distinct functions of the kindlins can be traced to differences in their subcellular localization in integrin-containing adhesion structures. Thus, the prevailing view that individual kindlins exert their functions in a cell type-specific manner must now be modified to consider distinct functions of the different family members within the same cell type.

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655-1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316-326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with beta3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.