The migration of neutrophils towards invading bacteria is partly dependent on their ability to recognize and bind bacterial N-formylated peptides (such as fMLF.) Upon binding of the bacterial peptide, the N-formyl peptide receptor (FPR) activates a guanyl nucleotide binding protein (G protein) which transduces the signal to intracellular effector molecules causing a cascade of cellular events including chemotaxis, lysosomal enzyme secretion and production of superoxide. In addition to normal host defense functions, neutrophils also play a central role in chronic inflammatory processes. By regulating the behavior of the neutrophils, much of the tissue damage caused by superoxide and its metabolites could be prevented. Understanding the molecular mechanism of formyl peptide- binding to FPR, as well as FPR-G protein interaction might facilitate the design of receptor antagonists that could be useful in controlling chronic inflammatory diseases. The broad goal is to develop site directed photoaffinity scanning in conjunction with mass spectral analysis as a generally applicable tool for studies of membrane proteins. In addition methods will be developed to elucidate possible structural changes caused by site specific mutagenesis using phage display libraries and affects of photoactive agonist labeling. We have developed a working hypothesis of N- formyl-Met-Leu-Phe (fMLF) binding to the formyl peptide receptor (FPR) based upon a structural model of G-protein coupled receptors as proposed by Baldwin, the known 3D structure of fMLF bound to a specific immunoglobulin, and the structural similarity between retinal and fMLF. We propose to test and refine this working hypothesis using site directed photoaffinity labeling in concert with site directed mutagenesis. We have previously photoaffinity labeled the formyl peptide receptor (FPR) and have used this labeled receptor to monitor its interaction with both G- protein and actin. The present studies will greatly extend the photoaffinity agonist approach by preparing more compact photoaffinity analogues which can be used to map the agonist binding site of FPR. We will use a wide variety of photoaffinity analogues that fully probe the agonist site and sequence the photoaffinity crosslinked sites to provide structural information on the transmembrane organization of the receptor. In addition, using site directed mutagenesis, we will determine those residues which are important in agonist binding and protein folding. This work should provide useful information about the structure of FPR and the primary events in the chemotaxis of phagocytes. It should also serve as a conceptual framework for the study of other heptahelical receptors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI040108-02
Application #
2457879
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1996-08-01
Project End
2000-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Montana State University Bozeman
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
City
Bozeman
State
MT
Country
United States
Zip Code
59717