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The β1Cintegrin is an alternatively spliced variant of the β1subunit that contains a unique 48-amino-acid sequence in its cytoplasmic domain. We have shown previously that β1Cis a potent inhibitor of cell proliferation and that in vivoits expression is downregulated in prostate and breast carcinoma. In this study, we describe a panel of specific monoclonal antibodies that react with the β1Ccytodomain. We show by immunoblot analysis that the newly generated monoclonal antibodies specifically recognize the β1Ccytodomain expressed as glutathione S-transferase fusion protein. The specificity of the antibodies to β1Cwas confirmed in competition studies by immunoblotting using β1C-specific synthetic peptides. These monoclonal antibodies reacted, in enzyme-linked immunosorbent assays, with the β1C785–808 peptide but failed to bind the β1C778–794, β1C805–825, or β1A765–798 peptides. Thus, the epitope recognized by the antibodies is located within the Q795-F804β1Ccytoplasmic sequence; this region overlaps the previously described Q795-Q802domain necessary for β1Cto inhibit cell proliferation. To our knowledge, these are the first monoclonal antibodies specific for a β1cytoplasmic isoform. The monoclonal antibodies described here will be useful tools for dissecting functional differences, among β1integrin variants, as well as for the study of the role of β1Cin prostate and breast epithelial cell proliferation.

Alternative splicing of the integrin β1subunit mRNA generates a variant form, β1C, with a unique cytoplasmic domain that differs from β1for a 48-amino acid COOH-terminal sequence. The potential role of this unique sequence in modulating cellular functions was investigated using Chinese hamster ovary (CHO)1cells transiently transfected with cDNAs coding for human integrin β1Cor β1subunits or mutants containing truncated forms of the β1Ccytoplasmic domain. A differential effect of β1Cand β1on cell proliferation was observed. Expression of wild type β1was associated with a 6-10-fold increase in cell proliferation in response to serum, as measured by [3H]thymidine incorporation. In contrast, only a 2-fold increase in cell proliferation was observed in transfectants expressing comparable levels of β1C. Cells expressing the β1Cmutant truncated at Leu794and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three β1Cdeletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of β1Cexpression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing β1C, full-length or deletion mutants, or β1equally adhered to a functionally inhibitory monoclonal antibody against human β1integrin. Expression of β1Cinversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of β1Cwas induced in growth-arrested, tumor necrosis factor-stimulated endothelial cells. These findings suggest that integrin alternative splicing may provide an accessory mechanism to modulate cell type-specific growth regulatory pathways during vascular cell injury in vivo.