Isolation of a novel integrin receptor mediating ARG-GLY-ASP-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel /β1-related subunit with αv

Abstract

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the α/β complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against β1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against β3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-β1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and β1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-β3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "β1-like" integrin subunit that can associate with αv and can bind to RGD. We propose to name this β1-like subunit βn. The data reported here thus demonstrate that in these two cell lines αv associates with two β subunits, βn and β3, forming two heterodimers. The αv βn complex mediates binding to fibronectin and type I collagen, whereas the αv β3 complex mediates binding to vitronectin.