This is a revised application for funding of a new study of chemical sensing and the role of GPI anchored proteins. GPI anchored proteins are important in many signaling pathways (axon guidance, growth factor stimulation, host parasite interactions, T-cell activation and more), but generally they have not been examined much at all for their roles in chemosensation. We study chemosensation in Paramecium because this organism is amenable to multiple techniques for dissecting chemosensory transduction mechanisms in chemoreceptor cells: biochemistry, molecular biology, transmission genetics, electrophysiology, behavioral analysis of populations and individual cells. After we disrupt the cell and study its transduction components in vitro, we can often return our study to the level of the intact cell and ask whether our findings have relevance for the physiology of the cell. Also, we know the identity of stimuli, such as folate, which bind to distinct receptors. In preliminary studies we have shown that some chemoreceptors in Paramecium are likely to be GPI anchored rather than integral membrane proteins, and that we have developed the tools to study GPI anchoring and its role in chemosensory signal transduction. We have 4 specific aims to test the hypothesis that some chemoreceptors in Paramecium are GPI anchored: 1) Transform cells with antisense PIG- A and PIG-K constructs, and characterize these transformants that should have reduced enzyme activity for the first and last steps in GPI anchor synthesis/attachment; combine antisense transformation with inhibitor treatment to more thoroughly reduce GPI anchoring; 2) isolate an endogenous protein that is a candidate for the folate chemoreceptor, clone its gene, and provide evidence for receptor function; 3) analyze by voltage clamp the conductances induced by the stimulus folate and the fate of these conductances in anti-PIG-A and anti-PIG-A transformants; 4) select using alpha-toxin mutants without GPI anchored proteins, and determine whether the folate receptor is among the alpha-toxin receptors. The overall goal is to create lines of cells through transformation and inhibitor treatment that wipe GPI anchored proteins from the cell surface, thereby producing a test system to ask about the effects of loss of GPI anchoring on chemoresponse. We will use electrophysiological, biochemical, molecular biology, and immuno-techniques in a collaborative effort between two laboratories. These studies should inform the research of others who study sensory transduction in olfaction, taste, chemotaxis such as in Dictyostelium and Caenorhabditis, axon guidance host- parasite interaction neurotrophic factors, and other signaling systems as in yeast.
| Yano, Junji; Rachochy, Villa; Van Houten, Judith L (2003) Glycosyl phosphatidylinositol-anchored proteins in chemosensory signaling: antisense manipulation of Paramecium tetraurelia PIG-A gene expression. Eukaryot Cell 2:1211-9 |
