D-myo Inositol 1,4,5-triphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of intracellular Ca++. The objective of this proposal is to obtain information on the molecular mechanism, regulation and subcellular structures involved in IP3 action using membranes derived from brain as experimental material. Studies on the mechanism of IP3 action will be directed towards the functional reconstitution of a purified receptor for IP3 from cerebellum microsomes, a region of the brian particularly rich in this protein. On the basis of preliminary data, the receptor protein incorporated into phosphatidylcholine liposomes can mediate stimulation of Ca++ transport in the presence of IP3. Experiments will be performed to optimize this reconstitution procedure. Phospholipid requirements for IP3 stimulation of Ca++ fluxes will be investigated. In addition the relationship between ligand binding and Ca++ release will be explored using the proteoliposomal preparation. Protease and glycosylation studies will be used to study the relationship between structure and function of this protein. Proteoliposomes will be fused with planar lipid bilayers in order to study the conductance properties of IP3 gated divalent cation channels. Regulation of IP3 action will be focussed on investigating: a) feed-back regulation by Ca++ of steps involved in Ca++ release and their role in generating oscillatory behavior; b) the mechanism of inhibitory action of heparinoids; c) the possible involvement of protein phosphorylation. IP3 can be phosphorylated to I(1,3,4,5)P4 and the latter molecular releases CA++ from cerebellum at relatively low concentrations. The mechanism of this effect and functional interactions between IP3 and other inositol polyphosphates, particularly I(1,3,4,5)P4 will be investigated. Subcellular fractionation studies will be used to determine if the IP3 binding site copurifies with discrete vesicle structures containing Ca++ binding proteins. Antibodies raised to the purified IP3 receptor protein will be used to study the biosyntheses and processing of this protein in cultured neuronal cells. These studies are intended to more clearly define the role of inositol phosphates in hormone action.
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