Integrins are a widely distributed supergene family of integral membrane proteins currently comprised of 15 members which mediate cell/cell and cell/substratum interactions. Integrins play important roles in the immune response, lymphocyte targeting, regulation of metalloproteinase genes, and in the migratory and invasive properties of cells, and therefore may be diagnostic and therapeutic targets in rheumatic disease. Continued support is requested for molecular analysis of the ligand binding to B3 integrins. This will be achieved by site directed and random mutagenesis of regions of the B3 integrins critical to ligand binding function. The functions of these mutants, when transiently expressed in COS cells or in stable cell lines in Chinese hamster ovary cells, will be evaluated by; 1) conformation specific monoclonal antibodies which preferentially recognize occupied forms of B3 integrins, 2) binding of fluorescent latex beads bearing bound adhesive proteins, and 3) quantitative binding of ligands to purified integrins in vitro. The alpha subunits control ligand recognition specificity of integrins. Domain """"""""swapping"""""""" experiments will pinpoint regions which control this specificity. Additional regions of integrins involved in ligand recognition will be identified by sequencing of naturally occurring mutants of B3 integrins by use of polymerase chain reaction. Novel regions identified in this way will be subjected to the same intensive mutagenic analysis proposed for the already identified regions. A second proposed method of identification of novel regions of integrins involved in recognition function is by scanning linker mutagenesis with selection by conformation specific antibodies. A complementary strategy will be to prepare synthetic peptides from regions of integrins thought to be involved in ligand binding, to evaluate the capacity of these peptides to inhibit ligand binding, and to bind the ligands themselves. Monoclonal antibodies against these peptides will also be used as probes of the ligand recognition site. These studies will provide a detailed molecular analysis of ligand binding regions of prototype integrins, and will provide fundamental information concerning ligand recognition by all integrins.
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