Cytosolic phospholipase A2 (cPLA2) cleaves arachidonic acid from g1ycerophospholipids during intracellular Ca2+ fluxes associated with a variety of biological processes, including cell growth and differentiation. Arachidonic acid and its metabolites act as second messengers in several of these processes. Thus, gaining a molecular understanding of the steps leading to production of these important cellular response mediators could enable intervention in several pathological states, especially cancer, inflammation and autoimmune disease. Ca2+ activates cPLA2 by inducing binding to cellular membranes via its C2 domain, a motif present in several proteins involved in signal transduction. The experiments in this proposal seek to understand the molecular basis of Ca2+ binding to the cPLA2 C2 domain by quantifying the Ca2+ affinity, kinetics and selectivity of the domain, as well as the modulation of these parameters by membrane association. Concurrently, the coordinating structures and the role of specific coordinating residues in tuning the ion binding parameters will be investigated. Metal-binding parameters of the cPLA2 C2 domain, including KD, stoichiometry, k0~ and koff for Ca2+, will be measured to characterize the activation parameters of the domain. In addition, the effect of lipid membranes on the metal-binding parameters will be measured. The KD for other spherical metal ions, and their ability to promote membrane binding, will be measured to characterize the ionic charge and size selectivity of the domain. The residues responsible for Ca2+ coordination in the cPLA2 C2 domain will be investigated by mutating candidate residues and examining the effects on metal binding, which will be interpreted in a structural framework based on the crystal structure of the synaptotagmin C2 domain.
