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Abstract
An integral component of the biological response of the neutrophil is its migration, guided by chemotactic peptides such as N-formyl-Met-Leu-Phe, to sites of bacterial infection. The neutrophil membrane contains a specific chemotaxis receptor for N-formylated peptides. We have generated several anti-formyl peptide chemotaxis receptor monoclonal antibodies in an attempt to characterize further the formyl peptide chemotaxis receptor. These monoclonal antibodies were screened for their reactivity with neutrophil membranes by ELISA and FACS analysis. They were also screened for anti-receptor activity by their ability to react with detergent-soluble receptor previously complexed with f-Met-Leu-($\sp3$H) Phe and for their ability to immunoprecipitate material that retained receptor binding activity. Additionally, these monoclonal antibodies were shown to react with a protein of Mr 29,000. The protein that these monoclonal antibodies recognize is not the ligand binding chain of the receptor. This protein was shown to be a member of the family of small molecular weight GTP binding proteins. The experiments that established this were: co-migration of a GTP binding protein and the protein recognized by the anti-receptor monoclonal antibodies on SDS-PAGE and immunoprecipitation of GTP binding activity by the anti-receptor monoclonal antibodies. This GTP binding protein was shown to be physically coupled to the formyl peptide chemotaxis receptor both by immunoprecipitation of receptor binding activity and by co-migration of the receptor ligand binding chain and the protein recognized by the anti-receptor monoclonal antibodies on sucrose gradients. Finally, this Mr 29K GTP binding protein was shown to be functionally coupled to the formyl peptide chemotaxis receptor as determined by the ability of the anti-chemotaxis receptor monoclonal antibodies to partially inhibit the metabolic burst generated in neutrophils in response to f-Met-Leu-Phe. This finding is significant because the interaction between a small molecular weight GTP binding protein and a receptor ligand binding chain has been presumed to exist, however, the data described in this thesis establishes for the first time that such an interaction does exist.
