Leukocytes play an important role in host defense against invading microorganisms. Their ability to generate superoxide radicals and release degradative enzymes following migration to sites of inflammation is essential for this function. However, these same responses can also participate in the formation of numerous pathological conditions. The re-introduction of blood flow to ischaemic tissues following myocardial thrombosis, stroke or frostbite is responsible for the observed tissue damage, which can be alleviated by the depletion of neutrophils. Chemoattractants elicit their effects on neutrophils by binding to cell surface receptors coupled to guanine nucleotide-binding regulatory proteins (G proteins). The goal of this work is to understand the molecular mechanisms involved in the activation of leukocyte G protein-coupled chemoattractant receptors as they pertain to receptor signaling and processing, receptor interactions with the cytoskeleton and chemotaxis.
The specific aims of this proposal include the determination of the role of the N-formyl peptide receptor (FPR) activation I the physical binding of the receptor to G protein. The proposed experiments will also determine residues responsible for the interaction of the receptor with the cytoskeleton and one of its major components, actin. We will generate mutations in the intracellular domains of the recombinant FPR and express these in tissue culture cell lines for functional analysis. The roles of specific residues, including potentially phosphorylated residues, will be determined with respect to receptor processing and chemotaxis utilizing a novel system we have recently developed. These complex activities likely require interactions following receptor phosphorylation. Utilizing this system, we will also examine the role(s) of the low MW G proteins rho, rac and cdc42 in leukocyte chemotaxis. Information obtained from the proposed studies is expected to extend our knowledge of the activation of signal transduction pathways by chemoattractant receptors with the long term goal that such knowledge will lead to the development of therapeutic means to control neutrophil activation and prevent the tissue damage resulting from the excessive activation of neutrophils following reperfusion.
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