Exposure of human polymorphonuclear leukocytes (PMN) to N-formyl peptides (FP) stimulate these cells to migrate in a directed fashion (i.e.., chemotaxis), release a portion of their lysosomal constituents (i.e.., degranulation) and generate oxygen-derived free radicals (i.e. oxidative burst). All these functions are mediated by the binding of fp to structurally specific formyl peptide receptors (FPR) on the PMN membrane. The major objective of the proposed research is to clone, sequence and express the cDNA coding for the FPR present on membranes of differentiated HL-60 cells and PMN. Using techniques established in his laboratory, the applicant has been successful in isolating and purifying a protein exhibiting the known physicochemical characteristics of HL-60 FPR. Sufficient amounts of the purified receptor have been obtained to allow preliminary sequencing. Using synthetic peptides derived from known sequence, we will generate specific monoclonal and polyclonal antibodies. Antibodies will be characterized to determine whether they recognize purified FPR obtained from differentiated HL-60 cells and PMN. Oligonucleotides containing all possible substitutions at each codon will be synthesized and used to amplify HL-60 and PMN FPR cDNA using of the polymerase chain reaction. Experiments will be performed to obtain transient expression of the putative FPR cDNA by transfecting COS-7 cells. Transfected cells expressing FPR will be used to characterize ligand binding. Attempts will be made to generate FPR mutants to delineate important domains in ligand-receptor interaction. Finally, experiments will be performed using antibodies directed against FPR as well as cDNA probes, to determine synthesis and processing of FPR by HL- 60 cells during maturation as well as processing of FPR by resting and stimulated PMN. These studies should provide important and new information regarding the structure of FPR, the interaction of FPR with its ligand(s) and the processing of FPR during formyl peptide-induced PMN chemotaxis.
