REGULATION OF ALPHA-6-BETA-1-INTEGRIN LAMININ RECEPTOR FUNCTION BY THE CYTOPLASMIC DOMAIN OF THE ALPHA-6-SUBUNIT

The alpha6beta1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha6 subunit cytoplasmic domain in alpha6beta1 integrin activation was examined. The use of P388D1 cells, an alpha6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha6A or alpha6B subunit cDNAs restores their activation responsive laminin adhesion (Shaw, L. S., M. Lotz, and A. M. Mercurio. 1993. J. Biol. Chem. 268:11401-11408). A truncated alpha6 cDNA, alpha6-DELTACYT, was constructed in which the human cytoplasmic domain sequence was deleted after the GFFKR pentapeptide. Expression of this cDNA in P388D1 cells resulted in the surface expression of a chimeric alpha6-DELTACYTbeta1 integrin that was unable to mediate laminin adhesion or increase this adhesion in response to PMA under normal conditions, i.e., in medium that contained physiological concentrations of Ca++ and Mg++. The alpha6A-DELTACYT transfectants adhered to laminin, however, when Ca++/Mg++ was replaced with 150 muM Mn++. We also assessed the role of serine phosphorylation in the regulation of alpha6Abeta1 integrin function by site-directed mutagenesis of the two serine residues present in the alpha6A cytoplasmic domain because this domain is phosphorylated on serine residues in response to stimuli that activate the laminin receptor function of alpha6Abeta1. Point mutations were introduced in the alpha6A cDNA that changed either serine residue #1064 (M1) or serine residue #1071 (M2) to alanine residues. In addition, a double mutant (M3) was constructed in which both serine residues were changed to alanine residues. P388D, transfectants which expressed these serine mutations adhered to laminin in response to PMA to the same extent as cells transfected with wild-type alpha6A cDNA. These findings provide evidence for a novel mode of integrin regulation that is distinct from that reported for other regulated integrins (O'Toole, T. E., D. Mandelman, J. Forsyth, S. J. Shattil, E. F. Plow, and M. H. Ginsberg. 1991. Science (Wash. DC). 254:845-847. Hibbs, M. L., H. Xu, S. A. Stacker, and T. A. Springer. 1991. Science (Wash. DC). 251:1611-1613), and they demonstrate that serine phosphorylation of the alpha6A cytoplasmic domain is not involved in this regulation.