Expression and functional analysis of a cytoplasmic domain variant of the β1 integrin subunit

Abstract

We have previously described a variant form of the integrin β1 subunit (β1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the β1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the β1B 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 β1B. In these cells β1B represent ∼30% of the β1 and form heterodimers with α1 and α5 subunits. To investigate the functional properties of β1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the β1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster β1A. Immunoprecipitation experiments indicated that the β1B was exported at the cell surface in association with the endogenous hamster α subunits. The α5/β1B complex bound to a fibronectin-affmity matrix and was specifically released by RGD-containing peptides. Thus β1B and β1A are similar as far as the α/β association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that β1B, in contrast to β1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that β1B has unique functional properties.