STRUCTURAL REQUIREMENTS FOR ALPHA-1-BETA-1 AND ALPHA-2-BETA-1 INTEGRIN-MEDIATED CELL-ADHESION TO COLLAGEN-V

A large variety of cells adhere to and spread on specific regions with in the triple helix of collagens, mainly via alpha 1 beta 1 and alpha 2 beta 1 integrins, Disruption of collagen triple helical integrity ge nerally affects the efficiency of cell adhesion on different collagens including collagen V. This report addresses the question of the impor tance of the linear sequence of the constitutive a-chains versus the t riple helical conformation in the recognition of collagen V binding si tes. To investigate this question, in vitro renaturation of the isolat ed alpha 1(V) and alpha 2(V) chains was performed according to the ann ealing procedure and formation of the triple helix was monitored by ro tary shadowing and by mild trypsin digestion followed by electrophoret ic analysis, The results indicate that the alpha 1(V) and alpha 2(V) h omotrimeric reassociation can occur up to a full-length triple helix b ut intermediate forms of 50-200 mn long rod-like segments are also obs erved. We have previously shown that alpha 1 beta 1 and alpha 2 beta 1 integrins, the major collagen receptors, are also involved in cell ad hesion to native collagen V. Therefore we chose the following two diff erent cell lines for this study: HT1080 (a human fibrosarcoma cell lin e) expressing alpha 2 beta 1 and HBL100 (a human mammary epithelial ce ll line) containing significant amounts of alpha 1 beta 1 and alpha 2 beta 1 integrins, We showed that both alpha 1(V) and alpha 2(V) homotr imers induced cell adhesion but refolded alpha 2(V) chains were more e fficient and promoted cell adhesion as well as native collagen V. Ther mal stability of refolded alpha-chains was monitored by adhesion promo ting activity and showed that cell adhesion was dependent on triple he lical conformation of the substrates, Adhesion in all cases was strong ly Mg2+ and Mn2+-dependent and Ca2+ ions alone were ineffective, Antib odies against alpha 2 and beta 1 integrin subunits completely inhibite d HT1080 cell adhesion to all substrates, Moreover, addition of cyclic RGD peptides, which had been shown to interact with alpha 2 beta 1, d ramatically affected HT1080 cell adhesion to native collagen V and to the refolded alpha-chains. Antibody to beta 1 subunits abolished HBL10 0 cell adhesion to all substrates, A complete inhibition of HBL100 cel l adhesion to native collagen V was achieved only by simultaneous addi tion of function-blocking specific monoclonal antibodies against alpha 1 and alpha 2 integrin subunits, However, only alpha 2 beta 1 was eng aged obviously in HBL100 cell adhesion to refolded alpha-chains. These data indicate that triple helical conformation is particularly critic al for alpha 2 beta 1- and alpha 1 beta 1-dependent adhesion and that the integrin alpha 2 beta 1 is a dominant functional receptor for refo lded alpha-chains, We conclude that alpha 2 beta 1-dependent adhesion seems to involve multiple different conformational binding sites while alpha 1 beta 1-dependent adhesion is more restricted to the heterotri meric native form of the molecule.