Analysis of α1β1, α2β1 and α3β1 integrins in cell - Collagen interactions: Identification of conformation dependent α1β1, binding sites in collagen type I

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the α1β1 and α2β1 integrins. Different cyanogen bromide (CB) fragments of the α1(1) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed α1β1, primary rat cardiac fibroblasts α1β1 and α2β1, and Rat-1 cells only α2β1. All three cell types expressed α3β1 but this integrin did not bind to collagen - Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with α1(I)CB3 and α1(I)CB8; Rat-1 cells attached to α1(I)CB3 but only poorly to α1(I)CB8-coated substrata. Cardiac fibroblasts and Rat-1 cells spread and formed β1-integrin-containing focal adhesions when grown on substrata coated with native collagen or α1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on α1(I)CBS. The rat α1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to α1(I)CB3 and α1(I)CB8, as well as the attachment of cardiac fibroblasts to α1 (I)CBS, but only partially inhibited the attachment of cardiac fibroblasts to α1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat-1 cells to collagen type I or to α1(I)CB3. These data indicate that binding sites for α1β1 are present in both α1(I)CB3 and α1(I)CB8 and that α1(I)CB3, but not α1(I)CB8, contains a binding site for α2β1, and suggest that collagen type I contains separate binding sites for α1β1 and α2β1. Hepatocyte attachment to heat-denatured collagen type I was inhibited by the hexapeptide GRGDTP. It therefore appears that denaturation both destroys the integrity of conformation dependent binding sites for α1β1 and reveals a cryptic RGD-containing site recognized by the α5β1 of hepatocytes.