AN ALTERNATIVELY SPLICED EXON IN THE EXTRACELLULAR DOMAIN OF THE HUMAN ALPHA-6 INTEGRIN SUBUNIT - FUNCTIONAL-ANALYSIS OF THE ALPHA-6 INTEGRIN VARIANTS

Variants in the extracellular domain of the integrin alpha 7 subunit which arise as a consequence of alternative splicing of mRNA have recently been reported. Two alternative exons, X1 and X2, have been identified in the alpha 7 gene, and homologous exons were found for alpha 6 (Ziober et al., 1993). In this study, we have isolated the region of the alpha 6 gene containing exons X1 and X2 that are, like those of alpha 7, located between stretches of DNA that encode the homologous repeat domains III and IV, proximal to the three divalent cation binding sites of the alpha 6 subunit. We demonstrated by reverse transcriptase polymerase chain reactions and confirmed by sequencing that alpha 6(X1) and alpha 6(X1X2) mRNAs are generated by alternative splicing of exon X2. The alpha 6(X1X2) mRNA is expressed in a limited number of tissues and cell lines and it is always co-expressed with the ubiquitous alpha 6(X1) mRNA. Stable transfection of K562 cells with full length cDNAs for the alpha 6A(X1X2) and beta 4 subunits resulted in cell populations that expressed the alpha 6A(X1X2) variant, in association with either beta 1 or beta 4, on their surface. In addition, a population of cells was isolated that expressed the alpha 6A(X1X2) variant at low levels and almost exclusively in association with beta 1. Comparison of the alpha 6A(X1X2) integrins with alpha 6A(X1) using similarly transfected cells showed no obvious differences between the alternative extracellular alpha 6A isoforms with respect to ligand specificity and activation-dependency of ligand binding. After treatment with the anti-pr stimulatory antibody TS2/16, both the alpha 6A(X1)beta 1 and alpha 6A(X1X2)beta 1 integrin variants mediated cell adhesion to EHS tumor laminin (laminin-1), kalinin (laminin-5), human placental (laminin-2 and -4) and bovine kidney laminins. In contrast, the alpha 6A(X1)beta 4 and alpha 6A(X1X2)beta 4 integrins also mediated cell adhesion to laminin and kalinin without stimulation. Furthermore, the different transfectants did not differ in their ability to spread on kalinin. The presented data indicate that the X2 region in alpha 6 is not involved in defining ligand specificity or affinity.