Lymphocyte function-associated (LFA) molecules and intercellular adhesion molecules (ICAMs) are important both in antigen-specific interactions in immune responses and in interactions with endothelial cells during leukocyte localization in inflammation. LFA1 is an ?? heterodimer and a member of the integrin family. It binds ICAMS, including ICAM1, ICAM2, ICAM3, and ICAM5 that are members of the Ig superfamily (IgSF). The physiologic importance of these molecules is demonstrated by effect of deficiency of LFA-1 in an inherited disease, LAD;and previous approval of an antagonist of LFA-1 to treat autoimmune disease in patients. Studies of the function of these molecules in health and disease, the regulation of adhesiveness through them, and the structure of their interaction sites is of great importance, and will lead to the development of further anti-inflammatory agents. LFA-1 transduces signals bidirectionally across the plasma membrane that regulate binding to extracellular ligands and intracellular signaling. The conformational changes in the extracellular and cytoplasmic domains and the mechanisms that couple them are of great interest. We will use crystal, SAXS, and EM structures to study how conformational change is relayed in the LFA-1 ectodomain to the headpiece, and between the hybrid, ?I, and ?I domains to activate binding of ICAM to the LFA-1 ?I domain. Structures of allosteric small molecule and Fab antagonists and agonists bound to LFA-1, together with measurements of how conformational equilibria affect affinity, will provide information on affinity regulation and advance drug development. In cells, cytoplasmic domain regions will be tested for effect on LFA-1 affinity and LFA-1- dependent cell migration. How is integrin activation coordinated with the cytoskeleton to regulate directed lymphocyte migration? We hypothesize that inherent in LFA1 structural transitions is a mechanism for linking the high affinity, open conformation to engagement with ICAM1 and the cytoskeleton. We will test the innovative concept that a lateral pull on the integrin ? cytoplasmic domain by the cytoskeleton, resisted by ICAM, is important in maturation of LFA-1 to high affinity. Multiple fluorescent microscopy techniques will examine the link between LFA-1 structure and function in migration of intact cells. These include measurements on single molecules and adhesive foci that correlate LFA-1 activation with actin cytoskeleton and cellular motion, dynamic FRET to measure integrin force transmission and two fluorescence polarization techniques to measure effects of the cytoskeleton on the dynamics and alignment of the LFA-1 headpiece. Uniting molecular and cellular studies, we will determine the orientation of LFA1 as it provides cellular traction by binding to ICAM1 and the cytoskeleton. The orientation of LFA-1 will be determined relative to actin retrograde flow, to test a key tenet of te traction (lateral) force model of integrin activation.
We will determine the mechanism of activation of an important adhesion molecule on white blood cells that enables them to leave the bloodstream and accumulate at sites of infection, and to mount immune responses to protect us from microbes and tumors. This molecule is the target of a new class of therapeutics termed selective adhesion molecule inhibitors, which were discovered over thirty years ago in the first project period of this grant. The research may aid further development of these inhibitors, which have applications in a wide range of autoimmune diseases and transplantation.
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