PROTEIN(S) INVOLVED IN NEUROTRANSMISSION

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5-hydroxytryptamine (5-HT, serotonin) and its receptors have been implicated in the etiology and therapeutic treatment of human mental depression. A basic knowledge of 5-HT receptors will help to better understand the molecular mechanism of 5-HT mediated neural function and will provide insights into the molecular basis of mental depression. The objectives of this project are to investigate the structure and function relationships, the genomic organization and the regulation of human 5-HT-2 receptor(s). These objectives will be accomplished by using the rat brain 5-HT-2 receptor cDNA cloned recently. The specific aims of the proposed study are outlined below. 1. To clone 5-HT-2 receptor and related cDNAs from human brain. Various 5-HT-2 receptor subtypes and related cDNA clones will be isolated and sequenced. The pharmacological profiles and the second messenger systems linked to the expressed receptors (phosphoinositol (PI) turnover, CA++ release, or adenylate cyclase) will be characterized. 2. To map the chromosomal location of the 5-HT-2 receptor gene. This aim will be accomplished by somatic cell mapping and in situ hybridization of the chromosome. 3. To isolate human genomic 5-HT-2 receptor clones. 4. To construct the restriction map and to sequence human 5-HT-2 receptor genomic clones. 5. To identify and to analyze promoter regions of this gene. This aim will be accomplished by RNase protection, primer extension, and chloramphenicol acetyltransferase (CAT) reporter gene assay. The cis- elements, trans-acting factors and the functions of these elements will be systematically analyzed. 6a. To investigate if 5-HT-2 receptor gene is regulated by protein kinase C (PKC) and/or protein kinase A (PKA). The effects of TPA (12-0- tetradecanoyl-phorbol-14 acetate, activator of PKC) on the 5-HT-2 receptor gene expression will be studied by using CAT report gene assay and footprinting analysis. The ability of 1-(5-isoquinolylsulfonyl)-2- methylpiperazine (H7) to block TPA activation will be studied. Similar studies will be performed using activators of PKA such as forskolin and 8- bromodibutyryl cAMP. 6b. To investigate if antagonist induced 5-HT-2 receptor down regulation is at the transcriptional level.