Leukocytes play important roles in host defense against invading microorganisms and also participate in the formation of numerous pathological conditions such as autoimmune diseases and tissue injuries. The long-term objective of the proposed work is to understand the molecular mechanisms of leukocyte activation. Current research in this laboratory is focused on the mechanisms by which occupancy of a single type of chemoattractant receptor invokes diverse cellular functions.
The specific aims of this application are to identify the structural basis for (1) interaction of the N-formyl peptide receptor (NFPR) with ligand and, (2) association and activation of guanine nucleotide regulatory proteins (G proteins). This work will utilize the cloned human NFPR cDNA exogenously expressed in a mammalian cell line and in insect Sf9 cells. To map the ligand binding pocket, various NFPR segments will be replaced with the counterparts of p39, a putative leukocyte receptor that shares 69% sequence identity with NFPR but does not bind formyl peptides. Binding studies of these chimeric receptors is expected to identify NFPR domains bearing putative ligand-interacting residues, which will be individually mutated to test their functions. A reciprocal approach will be adopted to confirm the roles of these residues by converting p39 to a receptor capable of binding formyl peptides with amino acid substitutions. Ligand binding properties of the mutant receptors will be further analyzed by spectrofluorometric methods using various fluorescein-labeled ligands. To identify receptor domains and residues interacting with G proteins, a series of mutants will be constructed with truncations of the cytoplasmic tail, which has been shown by our preliminary data to interact with G proteins. These mutants will be tested for ligand-specific calcium mobilization and GTPase activity, as well as for their physical interaction with G protein by sucrose density sedimentation of the NFPR-G protein complex. Residues in these domains that interact and/or activate G protein will then be identified by site- directed mutagenesis. The wild-type as well as mutant NFPRs, expressed in Sf9 cells, will be reconstituted in lipid vesicles to further test their interaction with G proteins, and to examine which G protein alpha- subunit preferentially interact with NFPR. Information obtained from the proposed studies is expected to extend our knowledge of signal transduction by chemoattractant receptors and by other stimulatory receptors in general, and to provide a basis for the development of potential therapeutic means.
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