The α6β4 Integrin Is a Receptor for both Laminin and Kalinin

Abstract

Previously, we have established K562 transfectants that express either α6Aβ1 or α6Bβ1 (Kα6A or Kα6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the β1-stimulatory antibody TS2/16. Here we introduce the full-length β4 cDNA into the α6A- and α6B-expressing K562 cells and selected stably transfected cells. The β4 subunit was expressed on the surface of both transfectants and it formed dimers with the α6A or α6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed α6β1, in addition to α6β4. While Kα6A and Kβ6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated β4-transfected Kα6A and Kα6B cells to these matrix components was already substantial. This adhesion was mediated by both α6β1 and α6β4 since it was completely blocked by an α6-specific antibody or by a combination of anti-β1 and anti-β4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to β4 than binding to laminin, indicating that the affinity of α6β4 for kalinin is higher than that for laminin. The fact that α6β1 mediated adhesion without TS2/16 stimulation on the β4-transfected Kα6A and Kα6B cells suggests that some activation of α6β1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn2+ induced binding of solubilized α6β4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-α6 mAb GoH3. Together, these results indicate that both α6β1 and α6β4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the α6 subunit when associated with either β1 or β4.