Role of the alpha subunit cytoplasmic domain in regulation of adhesive activity mediated by the integrin VLA-2.

To investigate the role of the alpha subunit cytoplasmic domain in the regulation of VLA-2 functional activity, we expressed several chimeric and deleted forms of the alpha 2 subunit in two different human cell lines, K562 and RD. Each mutant construct formed surface VLA-2 heterodimers as efficiently as wild type alpha 2 subunit, except for a construct (X2CO1127) truncated just before the consensus GFFKR cytoplasmic domain motif, that was not expressed at the cell surface. Truncation of the alpha 2 cytoplasmic domain just after the GFFKR motif resulted in a complete loss of constitutive activity of VLA-2 in RD cells. If the integrin was already constitutively inactive, as in K562 cells, the cytoplasmic domain deletion had no effect. In both K562 and RD cells, cytoplasmic tail deletion eliminated up-regulation of adhesion in response to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In comparison, exchange of the alpha 2 cytoplasmic domain with the alpha 4 or alpha 5 cytoplasmic domains had no effect on constitutive activity (in RD cells), or on constitutive inactivity (in K562 cells) and did not eliminate PMA-stimulated activity (in K562 or RD cells). These results clearly demonstrate that the cytoplasmic domain of an alpha chain (not necessarily from alpha 2 itself) is required to maintain VLA-2 constitutive activity and to allow a responsiveness to PMA stimulation. In cases where VLA-2 was either constitutively inactive (as in K562 cells) or inactive due to cytoplasmic domain deletion (e.g. in RD cells), agents such as Mn2+ or the anti-beta 1 monoclonal antibody TS2/16 caused a marked increase in adhesive function, thus proving that the integrins were not irreversibly inactive, and that cellular regulatory constraints could be bypassed by extracellular stimuli.