The C5a anaphylatoxin and its seven transmembrane-segment receptor play a broad protective role in host defense, but are also implicated in a variety of disease states from the commonplace to the very rare. Progress towards the goal of understanding mechanisms involved in these reactions has been rapid over the past several years, with cloning the C5a receptor as well as many other seven transmembrane segment receptors and the recognition that they share common structural and regulatory motifs. Progress has also been made in areas of signal transduction. A number of questions remain, however, and with the fundamental basis of knowledge at hand, many of these questions can now be addressed. Our basic hypothesis is that the C5a ligand-receptor pair can serve as a paradigm for other chemotactic receptors with peptide ligands, and is distinct from the well characterized biogenic amine receptors in both specific and general ligand recognition sites, as well as the signalling pathways involved. We further hypothesize that the major differences are not merely reflections of the host cell, but rather depend on integral structural features of the receptor.
Four specific aims are proposed in this continuation application that address these hypotheses.
In Specific Aim 1, we propose specific site-directed mutagenesis and targeted proteolysis of the C5a receptor, and production of chimeric C5a-PAF receptors to probe (a) the role of the N-terminus in ligand recognition, (b) the hypothetical ligand binding pocket formed by juxtaposition of the seven transmembrane helices, and (c) sites of interaction with heterotrimeric G-proteins.
For Specific Aim 2, we will continue to characterize candidate human C3a receptor clones, and identify anaphylatoxin receptor-bearing cells in the lung.
Specific Aim 3 will identify proximal components of the C5a signal transduction pathway.
For Specific Aim 4, we will reconstitute the phosphatidyl inositol signalling pathway in heterologous cells using cloned components. The quantitative and qualitative differences between C5a and PAF receptors will be determined. Taken together, this work will yield important understanding of this important ligand-receptor pair that may provide insight into therapeutic approaches to inflammation.
Showing the most recent 10 out of 43 publications
