CHEMISTRY OF TUMORICIDAL MACROPHAGE SURFACE ANTIGENS

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We will characterize the ligand specificity, ligand binding sites, regulation of affinity for ligand, and cytoskeletal association of the leukocyte adhesion receptors Mac-l and p150,95. Mac-l is both a complement receptor (CR3) and a cell adhesion receptor; p150,95 is a closely related adhesion receptor expressed on myeloid cells and abundantly on hairy leukemia cells. Genetic deficiency of these receptors results in recurring bacterial infections which are often fatal in childhood. The alpha subunits of these receptors control ligand specificity, and we hypothesize that tandem repeats which contain putative divalent cation binding sites are the ligand binding sites. To define ligand binding sites, we will cut and paste alpha subunit cDNA clones to prepare hybrid Mac-l x LFA-l and p150,95 x LFA-l alpha subunits which will be expressed using retroviral vectors in association with the common beta subunit on the surface of T lymphoma cells. MAb epitopes will be localized and correlated with MAb effect on function. Binding to iC3b in solution or on E; and binding to novel cellular ligands in solution, artificial planar membranes, and on intact cells will define the binding sites specific for different ligands, and the contribution of these sites to affinity. Similarly expressed truncated alpha subunits will be used to define the importance of transmembrane and cytoplasmic domains in regulating affinity for ligand and cytoskeletal association. We hypothesize that cells dynamically regulate adhesion receptor affinity for ligand. This will be tested for native molecules on neutrophils and for recombinant hybrid and truncated molecules expressed on T lymphoma cells by Scatchard binding of monomeric iC3b and cell surface ligands, after stimulation with fMLP or phorbol esters. Cytoskeletal association will be measured in parallel by detergent solubilization. In parallel with the above studies, novel ligand molecules recognized by Mac-l in neutrophil aggregation and adhesion to endothelial cells will be defined with MAb. Ligand molecules will be characterized and primary sequence defined at the protein and cDNA levels. Recognition sequences, hypothesized to be RGD-like, will be defined. Purified natural and recombinant secreted molecules will be used in the shove ligand binding studies. The intracellular storage site of fMLP-regulatable Mac- l in neutrophils and s murine Mae-l cDNA clone will also be characterized.