Chemoattractants and their receptors are key components of our immune defense system with a critical role in phagocyte migration and activation. The """"""""classical"""""""" chemoattractants include formylated peptides, the complement fragments C3a and C5a, platelet-activating factor and leukotriene B4. They bind receptors that share significant sequence homology and structural features, and together they constitute a subfamily of G protein-coupled receptors (GPCR). This subfamily of receptors inhibit each others function through cross-desensitization. In the hierarchy of receptor cross-desensitization, the formyl peptide receptor (FPR) is the dominant receptor, i.e., FPR can cross-desensitize the other chemoattractant receptors to higher extent than they can cross-desensitize FPR. Thus, we may be able to take advantage of this information in treatment of chronic inflammatory diseases. By understanding the molecular mechanism of receptor activation and desensitization we may find ways to blunt the neutrophil response to activating signals. We have previously characterized a mutant FPR that exhibits normal ligand binding and G protein coupling, but shows functional defects in signaling and induction of chemotaxis. Based on this finding, we propose that cytoplasmic proteins, other than G proteins, interact and regulate the function of FPR. We will test this hypothesis by analyzing the ligand binding-induced membrane translocation of certain cytoplasmic proteins that have been previously shown to interact with other GPCRs. This will be carried out in CHO cells expressing wild-type FPR and various mutant FPRs. We will also attempt to identify additional interacting proteins by comparing which proteins are co-immunoprecipitated with wild-type, but not with mutant FPRs. To identity previously uncharacterized interactions, we will carry out yeast two-hybrid analysis. We will examine interactions with cytoplasmic regions of FPR and C5aR. The positive interactions will be confirmed by mammalian two-hybrid analysis and co-immunoprecipitation. The function of the molecules, if unknown, will be examined by overexpression of full length or truncated proteins in CHO cells expressing FPR. Finally, we will examine the mechanism by which FPR cross-desensitizes C5aR. We will test our hypothesis that homologous desensitization of FPR is required for cross-desensitization of C5aR. We will also test our hypothesis that co-endocytosis of C5aR with FPR is an important mechanism of cross-desensitization and down-regulation of C5aR, whereas FPR's dominance over C5aR may in part be due to its resistance to co-endocytosis upon activation of C5aR. This work should provide novel information regarding the regulation of chemoattractant receptor-mediated cell activation.
