Fc receptors for immunoglobulin IgG (FcgammaR), upon binding immune complexes, trigger the effector functions of phagocytes, such as phagocytosis and the release of hydrolytic enzymes, activated oxygen intermediates and lipid mediators of inflammation. Little work has been done on the regulation by FcgammaR activation of macrophage transcriptional activity, which is likely to result in altered macrophage effector functions. This topic has been approached by differential display PCR, using a """"""""frustrated phagocytosis"""""""" protocol that results in prolonged and maximal stimulation of FcgammaRIIa. Two resulting cDNA's are the focus of this part of the application. The structure of the extracellular IgG binding domain of FcgammaRs will be elucidated by X-ray crystallography. Preliminary studies of the signaling pathways of FcRs reveal diversity even among members of this closely related family. FcgammaRI, FcgammaRIIIa and FcepsilonRI all require an accessory molecule, the gamma homodimer, which contains a tyrosine activation motif (TAM) similar to that found in T-cell receptor-associated zeta homodimer. FcgammaRIIa, however, has no requirement for gamma chain for either expression of signaling, as it bears a cytoplasmic domain TAM with a larger than consensus spacing between the dyad YXXL motifs. FcgammaRIIIb, expressed exclusively on neutrophils, has a glycan phosphatidyl inositol (GPI) anchor, and the signaling capabilities of this receptor are controversial. Fluorescence spectroscopy, imaging and biochemical methods will be used to test the hypothesis that signaling by RIIIb ligation proceeds via RIIa activation. Both IIa and IIIb ligation trigger a Ca2+ transient when crosslinked. Unlike the receptors that utilize the gamma chain, this Ca2+ increase is inhibited by low concentrations of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-k). The role of PI3-k in the regulation of the activity of phospholipase Cgamma (PLCgamma), and in the translocation of the PLCgamma to the plasma membrane will be studied. The role of the inositol polyphosphates (1,3,4,5)IP4 and IP6, which may lie downstream of PI3-k, in the regulation of membrane recognition and protein complex assembly will be examined in both yeast and mammalian systems. Combined, these related projects will extend our understanding of the specific function of FcgammaRs in immune defenses and will explore fundamental questions in cell biology.
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