BIOCHEMICAL-CHARACTERIZATION AND TISSUE DISTRIBUTION OF THE A-VARIANT AND B-VARIANT OF THE INTEGRIN ALPHA-6 SUBUNIT

Two cytoplasmic variants of the alpha6 integrin, alpha6A and alpha6B, have been identified previously (Hogervorst, F., I. Kuikman, A. G. van Kessel, and A. Sonnenberg. 1991. Eur. J. Biochem. 199:425-433; Cooper, H. M., R. N. Tamura, and V. Quaranta. 1991. J. Cell Biol. 115:843-850). Using synthetic peptides, containing sequences of their cytoplasmic domains, we have produced mAbs specific for either of the variants. These antibodies reacted with a variety of different epithelial tissues. In some tissues (e.g., salivary gland) both variants could be detected while in others only one of the variants was found (e.g., alpha6A in epidermis and alpha6B in kidney). Among nonepithelial cells and tissues, perineural fibroblasts and Schwann cells in peripheral nerves and platelets reacted with anti-alpha6A, while microvascular endothelia reacted with both anti-alpha6A and anti-alpha6B. From our immunohistochemical results there is no evidence that combination with beta1 or beta4 is restricted to one of the two variants of alpha6. This was confirmed by immunoprecipitation studies which showed that both beta1 and beta4 were coprecipitated by both anti-alpha6A or anti-alpha6B antibodies from cells. Also, the distribution of alpha6A and alpha6B subunits associated with beta1 on cells attached to laminin was similar: both were found in focal contacts colocalizing with vinculin. In contrast, the alpha6A subunit, associated with beta4 in cultures of a squamous cell carcinoma cell line, was found to codistribute with bullous pemphigoid antigen 230 in hemidesmosomal-like structures. The alpha6A and alpha6B variants, immunoprecipitated from various cell lines, exhibited slightly different electrophoretic mobilities. Analysis of the antigens under reducing conditions showed that the mobility of the light chains, but not of the heavy chains, is different. In addition, in some cells the light chains of alpha6A and alpha6B, each are of two different sizes. Treatment with N-glycanase showed that these two light chain variants of alpha6A and alpha6B are not due to differences in N-linked glycosylation, and may therefore represent alternative proteolytic products of the alpha6 precursor. We further demonstrate that alpha6A, but not alpha6B, is a major target for PMA-induced phosphorylation. Phosphorylated alpha6A contained phosphoserine and a small amount of phosphotyrosine. There are also two variants of the integrin alpha3 subunit with different cytoplasmic domains, but in the cell lines examined only alpha3A could be demonstrated by RT-PCR. The alpha3A subunit exhibits increased phosphorylation in cells treated with PMA but to a lesser degree than does alpha6A. Like alpha6A, the alpha3A subunit is phosphorylated both on serine and weakly on tyrosine. Phosphorylation of the A but not of the B variants of the alpha6 subunit may indicate different regulation mechanisms for the two subunits. Furthermore, phosphorylation of alpha subunits as they occur in alpha3 and alpha6 might influence their function.