THE NATIVE STRUCTURE OF INTERCELLULAR-ADHESION MOLECULE-1 [ICAM-1] ISA DIMER - CORRELATION WITH BINDING TO LFA-1

In solution, intercellular adhesion molecule-1 (ICAM-1) exhibits extre mely low affinity for its receptor, LFA-1, as direct binding to LFA-1 has not been reported. Furthermore, there are conflicting reports on t he ability of ICAM-1 in solution to inhibit cell adhesion events. Thes e differences could be due to the valency or an oligomeric native bioc hemical form of membrane-bound and soluble ICAM-1, which may correlate with its ability to bind to integrins. To test this, stimulated adeno carcinoma (A549) cells or HUVEC were labeled with S-35-methionine/cyst eine and treated with a chemical cross-linker. A high m.w. form (200 k Da) of ICAM-1 but not ICAM-2 was specifically immunoprecipitated from cross-linked cell lysates and supernatants. Affinity purification of c rosslinked supernatants revealed that the majority of ICAM-1 was dimer ic as opposed to recombinant soluble ICAM-1, which contains a minor fr action of dimer. Gel filtration chromatography was used to isolate mon omeric and dimer-rich fractions of recombinant soluble ICAM-1, and tes ted for direct binding to affinity-purified LFA-1. Dimer-rich fraction s demonstrated an enhanced ability and estimated affinity, compared wi th monomeric protein, to bind to purified LFA-1. These data suggest th at ICAM-1 exists in its native membrane-bound and shed form as a nonco valent dimer, and that dimerization directly correlates with enhanced binding to LFA-1.