708 Cloning and analysis of a novel C-Terminal motor domain type kinesin superfamily motor protein, KIFC 3

Abstract

A method for assessing stimulated phosphoinositide turnover by measurement of the liponucleotide CDP-diacylglycerol is presented. The phosphoinositide signal transduction pathway consists of a sequence of reactions in which the second messengers Inositol 1,4,5-triphosphate and diacylglycerol are recycled back to phosphatidylinositol (PtdIns), which then serves to replenish the initial hydrolyzed substrate, phosphatidylinositol 4,5-bis-phosphate. Receptor-stimulated inositol lipid turnover is most commonly assessed by measurement of the accumulation of [3H]inositol-labeled inositol phosphates in the presence of Li+. The latter blocks Inositol monophosphatase and thus can lead to a depletion of intracellular inositol. Because inositol is required for resynthesis of PtdIns, the immediate precursor of PtdIns, CDP-diacylglycerol, also accumulates in the presence of agonist and Li+. Measurement of radiolabeling of this liponucleotide following Incorporation of [3H]cytidine thus forms the basis for an alternative assay for Inositol lipid turnover. The general applicability of this method may be limited, since, In brain slices, not all receptors exhibit CDP-diacylglycerol responses that are consistent with their inositol phosphate responses. In addition, in cultured neural cells, growth in inositol-free, chemically defined medium is required to maximize the Li+ -dependent CDP-diacylglycerol response. A major advantage of this method may be its ability to provide insight Into the regulation of phosphoinositide turnover since this method uniquely reflects slowing of the regenerative cycle. Such in vitro studies may have relevance to the in vivo action of Li+ as a psychotherapeutic agent.