An Alternatively Spliced Exon in the Extracellular Domain of the Human α6 Integrin Subunit-Functional Analysis of the α6 Integrin Variants

Abstract

Variants in the extracellular domain of the integrin α7 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 α7 gene, and homologous exons were found for α6 (Ziober et al., 1993). In this study, we have isolated the region of the α6 gene containing exons X1 and X2 that are, like those of α7, located between stretches of DNA that encode the homologous repeat domains III and IV, proximal to the three divalent cation binding sites of the α6 subunit. We demonstrated by reverse transcriptase polymerase chain reactions and confirmed by sequencing that α6^x1 and α6^×1×2mRNAs are generated by alternative splicing of exon X2. The α6^×1×2 mRNA is expressed in a limited number of tissues and cell lines and it is always co-expressed with the ubiquitous α6^x1 mRNA. Stable transfection of K562 cells with full length cDNAs for the α6A^×1×2 and β4 subunits resulted in cell populations that expressed the α6A^×1×2 variant, in association with either β1 or β4, on their surface. In addition, a population of cells was isolated that expressed the β6A^×1×2 variant at low levels and almost exclusively in association with β1. Comparison of the α6A^×1×2 integrins with α6A^x1 using similarly transfected cells snowed no obvious differences between the alternative extracellular α6A isoforms with respect to ligand specificity and activation-dependency of ligand binding. After treatment with the anti-β1 stimulatory antibody TS2/16, both the α6A^x1β1 and α6A^×1×2β1 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 α6A^x1β4 and α6A^×1×2β4 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 α6 is not involved in defining ligand specificity or affinity.

Key Words:

• integrin variants
• splicing
• laminin