Group IVA cytosolic phospholipase A2alpha (cPLA2alpha) mediates agonist-induced release of arachidonic acid, the precursor for eicosanoids. These potent lipid mediators, which include prostaglandins and leukotrienes, are required for many normal physiological processes and also promote acute inflammation. Eicosanoids produced as a result of cPLA2alpha activation have been implicated in the development of acute lung injury and pulmonary fibrosis. The objectives of this proposal are to elucidate the mechanisms involved in targeting cPLA2alpha to membrane and regulation of its hydrolytic activity, cPLA2alpha is regulated by phosphorylation and calcium, which binds to the C2 domain of cPLA2alpha and induces its translocation to Golgi, ER and nuclear membrane.
The specific aims of this proposal are to identify residues in the C2 domain and catalytic domain that determine membrane targeting and stable association with membrane. The lipid binding specificity of cPLA2alpha will also be determined, and residues in the C2 domain and catalytic domain that interact with anionic phospholipids identified. The role of calcium and phosphorylation in regulating (1) translocation of cPLA2alpha in CHO cells by agonists that act through distinct signaling pathways, and (2) the interaction of cPLA2alpha with Golgi membranes isolated from CHO cells will be studied. Another Group IV PLA2, cPLA2beta was identified in lung fibroblasts from cPLA2alpha-/- mice, and it provided an alternative pathway for arachidonic acid release and PGE 2 production. The enzymatic properties of cPLA2beta and characteristics of its C2 domain will be studied. The results will provide detailed information about the regulation of cPLA2alpha, the first key regulatory enzyme in eicosanoid production, and new information about the properties of cPLA2beta.
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