Abstract
Alternative splicing of the integrin beta 1 subunit mRNA generates a variant form, beta 1C, with a unique cytoplasmic domain that differs from beta 1 for 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)1 cells transiently transfected with cDNAs coding for human integrin beta 1C or beta 1 subunits or mutants containing truncated forms of the beta 1C cytoplasmic domain. A differential effect of beta 1C and beta 1 on cell proliferation was observed. Expression of wild type beta 1 was 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 beta 1C. Cells expressing the beta 1C mutant truncated at Leu794 and lacking the last 31 amino acids of the cytoplasmic domain showed a 12-fold proliferation increase in response to serum. However, three beta 1C deletion mutants, lacking the COOH-terminal 23, 13, and 8 amino acids, which all contained residues Gln795-Gln802 of the variant cytoplasmic domain responded to serum stimulation with a 2-fold increase in [3H]thymidine uptake. The effect of beta 1C expression on cell proliferation was not associated with changes in exposure of integrin functional epitopes, as judged by the finding that CHO transfectants expressing beta 1C, full-length or deletion mutants, or beta 1 equally adhered to a functionally inhibitory monoclonal antibody against human beta 1 integrin. Expression of beta 1C inversely correlated with the mitogenic potential of vascular cells. Absent on growing cultured endothelial cells, surface expression of beta 1C was 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.
Highlights
Alternative splicing of the integrin 1 subunit mRNA generates a variant form, 1C, with a unique cytoplasmic domain that differs from 1 for a 48-amino acid COOHterminal sequence
The translated polypeptide, which showed the expected molecular mass of ϳ100 kDa, was immunoprecipitated by an antiserum against the 785– 808 1C cytoplasmic domain peptide or by P4C10, a monoclonal antibody that recognizes the extracellular portion of human 1 integrin (Fig. 1A), but not by an antiserum directed to the wild type 1(A) cytoplasmic domain (Fig. 1A)
While genetically engineered cell lines stably expressing the 1C integrin could not be obtained, transient surface expression of 1C was detected in Chinese hamster ovary (CHO) cells (Fig. 1B and not shown)
Summary
Alternative splicing of the integrin 1 subunit mRNA generates a variant form, 1C, with a unique cytoplasmic domain that differs from 1 for a 48-amino acid COOHterminal sequence. Selection based on flow cytofluorometric sorting with P4C10 antibody against the human 1 extracellular domain allowed us to obtain transfected cells expressing comparable levels of all the integrin variants (Fig. 2, A and B).
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