Human neutrophils serve a critical role in defending the body against bacterial infection. Inappropriate and/or excessive neutrophil activity can also cause much of the tissue injury resulting from the inflammatory response. The underlying mechanism of neutrophil activation by chemoattractant factors involves signal transduction events between their receptors (R) and GTP-binding (G) proteins. The rationale for our studies is that understanding the specific molecular interactions underlying R-G signal transduction will lead to the development of agents able to specifically and effectively disrupt neutrophil activation. The interaction of the N-formyl peptide (NFP) chemoattractant receptor with heterotrimeric, pertussis toxin-sensitive G proteins is well documented. The sites on the NFPR and G responsible for their specific interactions have not yet been defined. We will evaluate the ability of the human NFPR to couple to specific G protein alpha subunits using phospholipid vesicle and soluble reconstitution systems. Sites involved in the interactions of R and G will be probed using sequence-specific peptides and antibodies, as well as NFPR-derived fusion proteins. Site-directed mutagenesis of NFPR and G-alpha will be pursued to verify these results. Procedures which will enable us to assess R-G-E pathway specificity in vivo will be developed to confirm and extend the data obtained with in vitro experiments. These novel approaches will involve the use of chimeric and mutated G-alpha subunits which act as dominant negative inhibitors of R-G signal transduction. Finally, there is evidence that the NFPR may directly interact with GTP-binding proteins of the low molecular weight variety (LMWG). We will evaluate this hypothesis using reconstitution approaches, as well as direct measures of NFPR effects on LMWG regulatory protein activities. Identification of LMWG able to interact at the level of the NFPR will provide the basis for studies of interaction sites between this important second class of signal transducing proteins. In total, this grant describes an integrated approach to the elucidation of the molecular basis for signal transduction by chemoattractant receptors in human neutrophils.
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