EXPRESSION AND FUNCTIONAL-ANALYSIS OF A CYTOPLASMIC DOMAIN VARIANT OF THE BETA-1 INTEGRIN SUBUNIT

We have previously described a variant form of the integrin beta1 subunit (beta1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta1B. In these cells beta1B represent approximately 30% of the beta1 and form heterodimers with alpha1 and alpha5 subunits. To investigate the functional properties of beta1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta1A. Immunoprecipitation experiments indicated that the beta1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha5/beta1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta1B and beta1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta1B, in contrast to beta1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta1B has unique functional properties.