This is a study on the N-formyl peptide receptor (FPR), a leukocyte chemoattractant receptor that recognizes a bacterially derived agonist. While previous research on FPR has contributed greatly to our understanding of phagocyte activation, much of the molecular mechanisms for chemoattractant-triggered receptor activation and its regulation remain to be understood.
The specific aims of this project are: (1) Characterization of FPR, formyl and non-formyl peptides. (2) Characterization of FPR interaction with intracellular signaling molecules. (3) Identification of the structural basis for FPR internalization. The proposed studies will address several specific and fundamental questions: (1) How does an agonist, such as N-formyl-Met- Leu-Phe (fMLF), bind and activate FPR? To be tested will be the hypotheses that certain positively charged residues in the receptor extracellular loops and transmembrane domains are responsible for binding of formyl and non-formyl peptides, and that ligand composition but not N-terminal modification plays a more important role in determining agonistic vs. antagonistic activities. Potentially novel ligands for FPR will be identified using a phage display approach. (2) How does a receptor such as FPR transduce signals across the plasma membrane? Mutant receptors will be tested for their ability of interacting with G proteins, and anti-G protein antibodies will be used to detect specific G proteins that are activated upon agonist stimulation. Using FPR carboxy terminal tail fusion proteins, potentially novel intracellular components that interact with the receptor will be identified and characterized. (3) How is the receptor activation regulated after agonist binding? Can antagonist trigger certain receptor activities such as receptor internalization? Interaction of the FPR intracellular domains with beta-arrestins will be examined in order to identify the structural basis of receptor internalization. These studies are an integral part of our long-term objective of understanding the molecular mechanisms of leukocyte activation, thereby developing new approaches to enhance host defense and to control inappropriate activation of phagocytes which can lead to tissue damage associated with many inflammatory and autoimmune diseases.
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