This is a detailed study of an ethanol-responsive potential modulator of GTP-binding protein function which our laboratory has recently isolated by subtractive cloning. Changes in signal transduction, mediated by heterotrimeric GTP-binding proteins (G-proteins), may have an important role in adaption of the central nervous system to chronic ethanol. This phosducin-like protein (PhLP) is prominently induced in NG108-15 neuroblastoma x glioma cells upon chronic exposure to ethanol. Phosducin is a retinal phosphoprotein known to regulate diverse classes of G- proteins. PhLP is widely expressed, highly conserved across species and exists as multiple isoforms, possibly generated through alternative splicing. We hypothesize that ethanol-induced changes in PhLP isoform abundance may mediate at least some of the effects of chronic ethanol on G-protein function. The goal of this project is to identify ethanol- responsive PhLP isoforms and determine their function is possibly mediating ethanol effects on two G-protein coupled signal transduction system: adenylyl cyclase (AC) and phospholipase C (PLC). PhLP isoforms will be characterized by isolating a PhLP genomic clone followed by determining which of these respond to chronic ethanol exposure. In vitro membranae assays will be combined with in vivo overexpression (stable transfection) and underexpression (antisense oligonucleotides) of ethanol-responsive PhLP isoforms to determine whether ethanol induction of these isoforms alters AC and PLC functioning. Finally, mice strains that have been selected for sensitivity or resistance to seizures following chronic ethanol exposure will be used to determine whether PhLP isoforms are regulated by ethanol in selective brain regions and whether there are difference in PhLP expression or regulation between these two lines of mice. These studies could lead to novel therapeutic approaches to alcoholism or new molecular probes for the study of alcoholism.
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