This grant application is a request for an ADAMHA Scientist development award (SDA). We propose a program for a clinically trained research scientist whose career goals require training in molecular biology. This program will consist of a firm grounding in molecular biological research techniques as well as participation in courses and functions of the UCSF Program in Biological Sciences. It will encompass relevant courses given at the Cold Spring Harbor Laboratories, and selected short-courses at meetings such as the Annual Meeting of the Society for Neuroscience. The major objective of the proposed project is to understand how biogenic amines such as serotonin (5HT) modulate cellular function. 5HT exerts its physiological effects by binding to a family of pharmacologically distinct cell surface receptor subtypes. I will focus on a particular serotonin receptor subtype, namely the 5HT3 receptor (5HT3R). This proposal addresses the specific question of how the molecular structure of the 5HT3 receptor relates to its physiological role in the nervous system. Isolation of the gene(s) encoding the 5HT3R will permit structure-function analysis and will provide the necessary molecular probes to investigate the cellular and subcellular localization of the receptor. Serotonin (5HT) acts as a neurotransmitter, but also has hormonal and mitogenic actions in non-neuronal tissues. Until recently the variety of cell surface receptors that respond to 5HT were all believed to act by modulating intracellular second messenger systems. Recently, however, 5HT3R subtypes were shown to act as ligand-gated ion channels that promote rapid depolarizing responses in neurons. Although little is known about the specific function of these receptors, selective 5HT3R antagonists have clinical value as antiemetic agents and are thus used to prevent the drug- induced emesis associated with chemotherapeutic drug regimens.
The specific aims of this proposal are to pursue three independent yet overlapping strategies for the elucidation of the primary structure of the 5HT3R. Using a variety of cell lines and tissues that express 5HT3R, we will develop PCR, protein purification and functional expression strategies to isolate a cDNA clone encoding a member of the 5HT3R family. The long- term goal of the proposed experiments is to examine the physiological properties of this receptor in heterologous cell systems and to characterize sites of 5HT3R expression within the central and peripheral nervous systems.
