THE ROLE OF LFA-1 (CD11A CD18) CYTOPLASMIC DOMAINS IN BINDING TO INTERCELLULAR-ADHESION MOLECULE-1 (CD54) AND IN POSTRECEPTOR CELL SPREADING/

We have investigated the role of the cytoplasmic domains of LFA-1 in b inding to ICAM-1 and in postadhesion events. Various truncated and chi meric forms of LFA-1 alpha (CD11a) and beta (CD18) chains were generat ed and transfected into murine fibroblast TNR-2 cells. Transfected fib roblasts expressing wild-type LFA-1 adhered only weakly to ICAM-1 immo bilized on plastic, and phorbol ester pretreatment enhanced this adhes ion significantly. In contrast, transfected cells expressing LFA-1 lac king both the alpha and the beta cytoplasmic domains, the beta cytopla smic domain alone, or GPI-anchored LFA-1 adhered to immobilized ICAM-1 without prior activation. Truncation of the alpha cytoplasmic domain alone resulted in much reduced cell adhesion which could be only weakl y upregulated by PMA. The presence of manganese dramatically enhanced the binding to ICAM-1 of LFA-1 lacking the LY cytoplasmic domain or bo th cytoplasmic domains, whereas it had relatively little effect on wil d-type LFA-1 or the mutant lacking the beta cytoplasmic domain. Solubl e LFA-1, generated by phosphatidylinositol-specific phospholipase-C tr eatment of GPI-anchored LFA-1, was capable of binding ICAM-1(+) cells. Although doubly truncated or GPI-anchored LFA-1 mediated cell adhesio n to immobilized ICAM-1, cells expressing these mutants, as well as th ose expressing individual alpha and beta chain truncations, failed to spread out following this adhesion, whereas the wild-type transfectant s did so readily. Manganese had no effect on cell spreading. Fluoresce nt staining of these cells indicated no significant variation in the d istribution of LFA-1 on the cell surface. From these results we conclu de that (1) cells expressing LFA-1 lacking both the alpha and the beta cytoplasmic domains adhere to ICAM-1 without prior stimulation, indic ating the importance of LFA-1 cytoplasmic domains in inside-out signal ing, (2) truncation of the alpha cytoplasmic domain alone inhibits cel l adhesion by making LFA-1 nonresponsive to inside out signaling, and (3) both cytoplasmic domains are required for cell spreading following adhesion to immobilized ICAM-1. (C) 1997 Academic Press.