The complement activation fragment anaphylatoxin C5a is the most potent plasma-derived chemotactic factor known. This humoral factor induces leukocyte activation at low nonamolar concentrations. Investigating the molecular requirements for the interaction of C5a with its cellular receptor, present on polymorphonuclear granulocytes (PMNs) and monocytes, has been a long time interest of the P.I. While modelling studies up to now focused mainly on the ligand, i.e. C5a molecule, recent cloning of the PMN C5a-receptor (C5aR) and publication of the amino-acid sequence makes it possible to investigate the receptor-ligand binding interactions and receptor triggering events at the molecular level. Analyses of ligand binding site of this membrane-spanning receptor, and molecular arrangement of the seven transmembrane domains can also be explored. The goals of this study are to collect preliminary data and show feasibility for developing a program to investigate C5a-C5aR interaction, using site-specific receptor peptides and poly- and monoclonal antibodies. Selective blocking of ligand binding by polyclonal and monoclonal antibodies will identify and map molecular regions on the receptor that participate in C5a binding. Identifying these local sites on the receptor will not only facilitate an understanding of the molecular requirements for ligand-receptor interactions, but provide a model for receptor- ligand interactions as well. This work will also identify regions of special interest for site-specific mutagenesis studies. %%% C5a is a principal mediator in eliciting both local and systemic inflammatory responses. It interacts with a membrane bound receptor, C5aR, which has recently been cloned. Understanding these receptor-ligand interactions and receptor triggering events at the molecular level will provide a model for receptor-ligand interactions in general; and in addition might generate potential inhibitors of C5aR-mediated cell activation.
