Membrane cholesterol modulates galanin-GalR2 interaction

The neuropeptide galanin mediates a number of diverse physiological and pat hophysiological actions via interaction with membrane-bound receptors. The role that membrane cholesterol plays in modulating the interaction between galanin and one of the three cloned galanin receptor subtypes (GalR2) expre ssed in Chinese hamster ovary (CHO) cells was examined. Reduction of membra ne cholesterol by treatment with methyl-beta-cyclodextrin (CD) or by cultur ing cells in lipoprotein-deficient serum markedly decreased galanin binding to the receptor. Addition of cholesterol back to CD-treated, cholesterol-d epleted membranes restored galanin binding to control levels. Hill analysis suggests that the GalR2 binds multiple molecules of cholesterol (n greater than or equal to 3) in a positively cooperative manner. This interaction a ppears to be cholesterol-specific as only cholesterol and a limited number of cholesterol analogues were able to rescue, galanin binding. The inabilit y of some of these analogues to rescue the binding activity also suggests t hat binding of galanin to GalR2 is independent of membrane fluidity as, lik e cholesterol, cholesterol analogues generally rigidize membranes. In addit ion, treatment of the membranes with other modulators of membrane fluidity, e.g. ethanol, did not affect galanin binding to the GalR2. In contrast, tr eatment of membranes, with filipin, a molecule that clusters cholesterol wi thin the membranes, or with cholesterol oxidase resulted in markedly reduce d galanin binding. Incubation of membranes with 100 mu M GTP-gamma-S did no t alter the IC50 for CD in the prebinding assay treatment suggesting that t he effect of cholesterol was independent of G protein interaction. Preincub ation of intact cells with CD also drastically impaired the ability of gala nin to activate intracellular inositol phosphate accumulation in GalR2-tran sfected CHO cells. These data detail a new mechanism for the regulation of galanin receptor signaling which may link altered functions of GalRs with a bnormal cholesterol metabolism.