In the ten years since this grant was competitively renewed, the complement anaphylatoxins, C3a and C5a have been shown to exhibit major roles in allergic airway hyperresponsiveness and sepsis. During this period, studies supported by this research program included structure-function analyses of both C5a and C3a receptors to identify domains critical to ligand binding and signal transduction. We showed that the N-terminal domain of the C5a receptor forms the ligand-docking site and is distinct from the signaling domain. Similarly, the ligand docking and signaling domains constitute distinct regions of the C3a receptor, identifying structures in the second extracellular loop. Additional studies demonstrated a requirement for tyrosine sulfation on the N-terminus of the C5a receptor and the second extracellular loop of the C3a receptor for binding of the intact anaphylatoxins. We have characterized a third anaphylatoxin receptor, C5L2, with respect to ligand binding and signal transduction in transfected cells and PMNs, and have generated a strain of C5L2 gene deleted mice to complement studies with C5aR-/- and C3aR-/- animals. In this continuation application, we seek to expand our understanding of the signal transduction and structural properties of the anaphylatoxin-anaphylatoxin receptor system. Prelimiary studies demonstrate a role for the sphingosine kinase pathway in C5a receptor signaling.
In Aim 1 we will characterize the concentration dependence of anaphylatoxin responses on the sphingosine kinase pathway in PMNs as well as airway epithelial and smooth muscle cells. We will evaluate the role(s) of the extracellular (EDG) receptors and the intracellular (SCaMPER) receptors for sphingosine-1 -phosphate in anaphylatoxin mediated signaling.
In Aim 2, we will dissect components of the sphingosine kinase and PI3K( pathways involved in anaphylatoxin mediated signaling responses using cells from PISK( -/- and PI3K( KD/KD mice.
In Aim 3 we will characterize the quaternary structures of receptors for both C3a and C5a, and determine the functional role of homo- and hetero-dimers/oligomers. We will also characterize anaphylatoxin receptor associated proteins that may modulate signaling reactions. A thorough analysis of anaphylatoxin receptor-mediated signaling pathways and structural requirements will provide insight necessary to understand the role of the complement anaphylatoxins in health and disease, and to advance therapeutics in man.
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