Much recent evidence suggests that changes in signal transduction, mediated by heterotrimeric GTP-binding proteins (G-proteins), may have an important role in adaptation of the central nervous system to chronic ethanol. Our laboratory, studying ethanol-responsive gene expression, has recently identified a novel gene, phosducin-like protein (phLP), whose expression is prominently induced in NG108-15 neuroblastoma x glioma cells upon chronic exposure to ethanol. Phosducin is a retinal phosphoprotein that is known to regulate G-protein function and has been shown to modulate the function of diverse classes of G-proteins. PhLP is widely expressed and highly conserved across species. Furthermore, PhLP 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 to determine their function in possibly mediating ethanol effects on two G-protein coupled signal transduction systems: 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 membrane 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 either for sensitivity or resistance to seizures following chronic ethanol exposure will be used to determine whether specific 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. If we find differences in PhLP expression/regulation between these mice lines which have genetic differences in their susceptibility to chronic ethanol, this would suggest a role for PhLP in determining the response of the CNS to chronic ethanol. These studies could thus lead to novel therapeutic approaches to alcoholism or new molecular probes for the study of alcoholism.
