Identification and characterization of a dimeric chicken fibronectin receptor. Subunit-specific monoclonal antibodies to the putative chicken alpha 5 beta 1 integrin.

Abstract

A fibronectin receptor was isolated from chicken embryo fibroblasts using a cell-binding fragment of chicken fibronectin as an affinity matrix. The synthetic peptide GRGDSP specifically eluted the receptor, whereas the GRGESP control peptide was negative. The purified chicken receptor is a heterodimeric molecule as are all described mammalian integrins. It showed an RGD-dependent binding to fibronectin-coated wells after incorporation into liposomes. Monoclonal antibodies were raised against the two subunits of the receptor. Immunoprecipitations with the beta chain-specific monoclonal antibody U2 beta gave the same pattern of multiple bands as with the "avian integrin complex" specific monoclonal antibody JG22. In contrast, the alpha chain-specific monoclonal antibody U1 alpha only precipitated a dimeric fibronectin receptor. On immunoblots, U2 beta recognized the chicken gizzard integrin beta 1 chain, but U1 alpha did not cross-react with the alpha chain of gizzard integrin. Immunofluorescence labeling of cell cultures with U1 alpha revealed an exclusive colocalization with extracellular fibronectin fibrils, whereas U2 beta showed in addition, diffuse staining of the whole cell surfaces. The alpha chain-specific monoclonal antibody for the first time allowed us to exclusively detect the putative chicken alpha 5 beta 1 integrin among the many beta 1 integrin complexes present on chicken cells.