Leukocytes express a family of receptors, CR3, LFA-1, and p150, 95 that function in adhesion reactions. CR3 mediates the binding of C3bi-coated particles to phagocytes, LFA-1 mediates the binding of killer T cells to targets, and all three receptors appear to function in the binding of leukocytes to endothelial cells, unopsonized E. coli, and certain yeasts. While the structure of these receptors has been well studied, the nature of the ligands recognized by the receptors is obscure. This proposal focusses on the ligands recognized by CR3, LFA1, and p150,95, and on the biological consequences of receptor ligation. We will first extend our recent observation that all members of the CR3/LFA family recognize E. coli by binding to lipopolysaccharide (LPS). We will employ analogs and precursors of LPS to determine the structural aspects of LPS recognized, and whether CR3, LFA-1, and p150,95 recognize identical or different portions of LPS. Since binding of LPS is followed by sharply enhanced synthesis of IL-1 and TNF, we will observe whether ligation of CR3, LFA-1, and p150,95 by antibodies mimics the effects of LPS on macrophages. Members of the CR3/LFA family exhibit structural features similar to a newly recognized class of receptors that bind the amino acid sequence, Arg-Gly-Asp (RGD). Since C3bi contains RGD, we will use synthetic peptides to find if this is the region of C3bi recognized by CR3. Preliminary experiments indicate that CR3 does recognize an RGD-containing sequence, and further experiments will determine the precise structural requirements for recognition and whether LFA-1 and p150,95 also recognize peptides. We will next observe whether CR3 has separate binding sites for peptides and LPS. Preliminiary experiments with monoclonal anti-CR3 Abs suggest that CR3 does possess two binding sites. Further experiments will observe the effect of soluble competitive inhibitors of both sites and will determine functions (phagocytosis, release of H202, synthesis of IL-1, killing microbes) initiated by ligation of each site. Our previous experiments indicate that the ability of CR3 to bind C3bi and initiate phagocytosis is regulated by the proteins, IFN and fibronectin. We will observe whether the binding of LPS and triggering of functions unique to LPS are also regulated in a similar fashion. Finally, we will explore the possibility that the function of CR3, LFA-1, and p150,95 is regulated by posttranslational modification of the receptors (myristylation, phosphorylation) or by the binding of an additional receptor subunit.
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