Members of the 5-HT2 serotonin receptor family are thought to play key roles in a number of physiological and behavioral processes, including neuronal excitability, feeding behavior, circadian rhythms, and hallucinations. In addition, both the 5-HT2A and 5-HT2C receptors have been implicated in mental abnormalities such as psychotic depression, anxiety and schizophrenia. Recent studies have indicated that the generation of multiple 5-HT2c receptor isoforms is regulated by a novel RNA processing event referred to as RNA editing. This post- transcriptional modification may represent an additional mechanism by which cells modulate their response to extracellular signals by altering the efficacy of receptor: G-protein interactions; the ling term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of serotonergic signal transduction in the central peripheral nervous systems. We propose to examine the function of 5-HT2c receptor isoforms generated by RNA editing in a transfected NIH-3T3 fibroblast model system. Pharmacological characterization of multiple receptor isoforms will include ligand binding affinities, constitutive receptor activation, desensitization kinetics, phosphoinositide hydrolysis and direct examinations of receptor: G-protein interactions and coupling specificity. Site-directed mutagenesis will be performed to localize the key residue(s) responsible for observed changes in receptor function. Identification of the the cis-active regulatory sequences responsible for dictating the site-specific patterns of 5-HT2c receptor RNA processing will take advantage of tissue culture model systems which exhibit RNA processing patterns analogous to those observed in vivo. Analyses of RNA from the rat C6 glioma cell line, transfected with a variety of mutant 5-HT2cR transcription units, will serve as the primary methodology for these mapping studies. Development of an in vitro RNA editing reaction utilizing nuclear extracts from rat brain will also be used as a mapping technique by testing the ability of in vitro transcribed RNA transcripts to be accurately modified. This in vitro assay system will also serve as a direct bio-chemical approach allowing characterization and purification of the cellular machinery involved in such post-transcriptional processing reactions. To determine if RNA transcripts derived from the 5-Ht2b receptors undergo editing events similar to those observed for the 5-HT2cR, nucleotide sequence comparisons will be made between genomic and cDNA clones. Sequence analysis of individual cDNA isolates and primer- extension strategies, similar to those developed for studies of 5-HT2c transcripts, will be employed to assess 5-HT2A and 5-HT2b RNA processing. Should RNA transcripts encoding 5-HT2A and 5-HT2b receptors undergo such RNA editing events, these studies will be extended to examine the effect to such modifications on receptor function. It is anticipated that these studies will provide new insights concerning the regulation of cellular processes involved in the transduction of serotonergic signals and the role(s) of multiple serotonin receptors in the nervous system.
