The long term goal of the proposed research is to determine the molecular basis of activation and regulation of chemoattractant-induced neutrophil responses initiated by the occupancy of N-formyl chemotactic peptide receptors (FPR). FPR belong to the family of heptahelical receptors which transmits a signal through guanyl nucleotide binding proteins (G proteins). This receptor family has more than 2-3 hundred members and is therefore of central importance for a great many signal transduction pathways in cells. In addition to the interaction of FPR with G proteins, interactions of FPR with the membrane skeletal actin have been implicated in regulation of signal transduction. The research proposed here seeks to identify and structurally characterize the intracellular receptor domains which are interacting with G proteins and membrane skeletal proteins. For this purpose peptides with sequences from the predicted intracellular domains of the FPR will be synthesized and then utilized to study their effect on physical and functional coupling of FPR to signal transduction or regulatory proteins. The physical coupling will be studied with a recently developed reconstitution assay for FPR-G protein complexes which are separated from G protein-free receptors by velocity sucrose density gradients. Functional coupling will be evaluated by measuring peptide perturbation of high affinity agonist binding, receptor-mediated stimulation of U protein GTPase activity, formyl peptide-induced superoxide production and actin polymerization in electroporated cells, and in vitro actin polymerization stimulated by formyl peptides. Peptide mimetics in these studies will also be used in combination to uncover synergistic interactions of peptide pairs which might be expected because of the multi-site nature of these interactions. The three-dimensional structure of peptides identified as being part of regulatory protein- protein contacts will be studied in solution with NMR methods including COSY, TOCSY, ROESY, NOESY, and Tr-NOESY. In addition, the structure of the receptor peptides bound to G protein and regulatory proteins will be determined using Tr-NOESY NMR. The structural information obtained will then be used to identify amino acids critical to the predicted bound structures. These amino acids will then be substituted in recombinant FPR expressed in CHO cells by amino acids that would either interfere with or promote the formation of such three-dimensional conformations, thus providing functional confirmation of these structures. Such studies will be the basis for the development of synthetic peptide derivatives or even non-peptide analogs which may permit the design of new classes of drugs which can specifically inhibit signal transduction pathways important in the pathogenesis of inflammatory diseases.
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