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.
Kawaguchi
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Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Hemler
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D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002448:Cell Adhesion; D002460:Cell Line; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA; D006801:Humans; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D016029:Receptors, Very Late Antigen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection
D000911:Antibodies, Monoclonal; D016029:Receptors, Very Late Antigen; D011993:Recombinant Fusion Proteins; D008345:Manganese; D004247:DNA