Discrete neutrophil functions can be selectively evoked by the binding of various chemoattractant ligands to specific cell surface receptors. The long-term objective of this proposal is to understand the molecular mechanisms for the initiation of neutrophil functions through these receptors. Interest will be focused on (1) the definition of domains and residues on these receptors important to ligand binding; (2) the localization of receptor domains interacting with relevant G-proteins (Gn); and (3) the mechanisms by which binding of different ligands, or the same ligand at different concentrations, induces distinct cellular responses. The human receptors for N-formyl peptides (NFPR), whose gene has been cloned, is chosen as a model system for most of the proposed studies. The predicted membrane topology of NFPR will be confirmed by mapping with anti- peptide antibodies which have been prepared. Residues involved in ligand binding will be defined by exogenous expression of site-directed mutant receptors combined with measurement of changes in binding affinity of the mutants to fluorescent ligands, with the use of spectrofluorometric and flow cytometric methods. Domains on the NFPR that interact with Gn will be initially defined by the inhibitory effects of NFPR-derived synthetic peptides and antibodies against these peptides on receptor-Gn (R-G) coupling, as being measured by (1) the ligand-induced mobilization of intracellularly bound Ca2+; (2) the conversion of binding affinity states of the receptor in permeabilized cells or in membrane preparations. Key residues in these domains will be further identified by experiments with site-specific mutants. In addition to the novel receptor gene we recently isolated, genes for several other receptors in this class will be cloned by the use of cross-hybridization and expression screening methods. Ligand binding and R-G coupling mechanisms in these receptors will be analyzed as above. The results will be related to the ability of these receptors to initiate respective neutrophil responses. Chimeric receptors, with domains switched among these receptors, will be constructed for detailed analysis of structural requirement for ligand binding and R-G coupling. Information obtained from the proposed studies will be of great value in understanding signal transduction via cell surface receptors and in developing therapeutic means for the treatment of inflammatory and autoimmune diseases.
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