The goal of this project is to determine the effects of acute and chronic exposure to ethanol on the 5-hydroxytrypamine (serotonin) type 2A receptor, or 5HT-2A receptor. An extensive literature documents the involvement of serotonin in alcoholism, particularly with respect to comorbidity factors such as hostility, aggression, impulsivity, and tendency to commit suicide. Both general anesthetics and n-alcohols inhibit the activity of 5HT-2A receptors via a mechanism that may depend upon protein kinase C. A number of mutagenesis studies confirm that the structure of this receptor conforms to the arrangement of the 7 a-helices, typical of the G protein-coupled receptor family. However, little or no information exists regarding the structural features that regulate the binding of agonists or antagonists for the family of 5HT-2 receptors. In this project, human 5HT-2A receptors are being expressed in insect cells (Sf9 cells). Optimum conditions for high-level expression and growth are being determined. The next stage of the project will consist of studying agonist and antagonist binding and G protein activation in purified native membranes. Finally, the receptor will be purified free of lipid and reconstituted it into phospholipid bilayers of defined composition. This will make it possible to conduct detailed investigations of its structure and function, interaction with the lipid bilayer, and interaction with ethanol under controlled and defined conditions. A distinct advantage of working with the receptor in isolated or recombinant membranes is the ability to directly monitor, in real time, all aspects of receptor activity; agonist/antagonist binding, activation, and coupling to G protein. This will be accomplished via a variety of non-invasive spectroscopic techniques. The 5HT binding pocket is reported to consist of three highly conserved tryptophan residues, thus the binding of 5HT, and perhaps other agonists and antagonists, will be monitoring via changes in intrinsic tryptophan fluorescence of the 5HT-2A receptor. The binding of fluorescent ligands will be monitored by both fluorescence and CD assays of agonist binding. This will be conducted with a stopped flow instrument that is capable of simultaneously collecting CD and fluorescence, as well as fluorescence polarization, stopped flow data. Both the fluorescence and CD techniques have the added advantage of requiring very little sample in order to make accurate measurements, which will provide information relative to the structural changes associated with ligand binding to the 5HT-2A receptor.
