Structural requirements for α1β1 and α2β1 integrin mediated cell adhesion to collagen V

ABSTRACT A large variety of cells adhere to and spread on specific regions within the triple helix of collagens, mainly via α1β1 and α2β1 integrins. Disruption of collagen triple helical integrity generally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the importance of the linear sequence of the constitutive α-chains versus the triple helical conformation in the recognition of collagen V binding sites. To investigate this question, in vitro renaturation of the isolated α1(V) and α2(V) chains was performed according to the annealing procedure and formation of the triple helix was monitored by rotary shadowing and by mild trypsin digestion followed by electrophoretic analysis. The results indicate that the α1(V) and α2(V) homotrimeric reassociation can occur up to a full-length triple helix but intermediate forms of 50-200 nm long rod-like segments are also observed. We have previously shown that α1β1 and α2β1 integrins, the major collagen receptors, are also involved in cell adhesion to native collagen V. Therefore we chose the following two different cell lines for this study: HT1080 (a human fibrosarcoma cell line) expressing α2β1 and HBL100 (a human mammary epithelial cell line) containing significant amounts of α1β1 and α2β1 integrins. We showed that both α1(V) and α2(V) homotrimers induced cell adhesion but refolded α2(V) chains were more efficient and promoted cell adhesion as well as native collagen V. Thermal stability of refolded α-chains was monitored by adhesion promoting activity and showed that cell adhesion was dependent on triple helical conformation of the substrates. Adhesion in all cases was strongly Mg2+ and Mn2+-dependent and Ca2+ ions alone were ineffective. Antibodies against α2 and β1 integrin subunits completely inhibited HT1080 cell adhesion to all substrates. Moreover, addition of cyclic RGD peptides, which had been shown to interact with α2β1, dramatically affected HT1080 cell adhesion to native collagen V and to the refolded α-chains. Antibody to β1 subunits abolished HBL100 cell adhesion to all substrates. A complete inhibition of HBL100 cell adhesion to native collagen V was achieved only by simultaneous addition of function-blocking specific monoclonal antibodies against α1 and α2 integrin subunits. However, only α2β1 was engaged obviously in HBL100 cell adhesion to refolded α-chains. These data indicate that triple helical conformation is particularly critical for α2β1- and α1β1-dependent adhesion and that the integrin α2β1 is a dominant functional receptor for refolded α-chains. We conclude that α2β1-dependent adhesion seems to involve multiple different conformational binding sites while α1β1-dependent adhesion is more restricted to the heterotrimeric native form of the molecule.