20 Mole, ular Brain Research, 14 (1992) 20-26 © 1992 Elsevier Science Publishers B V All rights reserved 0169-328X/92/$0500 BRESM 70421 The human 5-HT 2 receptor is encoded by a multiple intron-exon gene Kevin Chen, Wu Yang, Joseph Grimsby and Jean C. Shih Department of Molecular Pharmacology, School of Pharmacy, University of Southern Cahfornta, Los Angeles, CA 90033 (USA) (Accepted 31 December 1991) Key words Serotonln 5-HT2 receptor gene, Human gene; Serotonm, Serotonm receptor, G-protein-coupled receptor, DNA sequence. Amino acid sequence Serotonln (5-hydroxytryptamme" 5-HT) mediates many central and peripheral nervous system functions by Jts interaction with specific neuronal receptors. Here we report the genomac structure of the human 5-HT2 receptor The SacI-EcoRI restriction fragment of rat 5-HT~ receptor cDNA was used as a probe to identify and isolate two posltwe clones of 8 5 and 7 0 kb from an EcoRI restriction digest of a chromosome 13 specific EcoRI fragment X-phage human genomlc hbrary Subclonmg and sequencing of these fragments showed the 8.5 kb fragment (designated XSE-5) contained the first two exons of the 5-HT2 receptor gene The 7 0 kb insert (.;tSE-2) contained an incomplete third exon A HmdIII-EcoRI fragment of this insert was used as a probe to isolate a 9 0 kb clone (XSH-2), which contained the entire third exon, from a chromosome 13 specific HmdIII-fragment X-phage human genomic hbrary The lsolanon of these three clones (2SE-5, XSE-2 and XSH-2) shows that the human 5-HT2 receptor gene consists of three exons separated by two mtrons and spans over 20 kb The deduced amino acid sequence of the human, mouse and rat 5-HT2 receptors are highly conserved and all three share a 90% sequence similarity INTRODUCTION 5-HT 2 receptor actwity has been imphcated in such disorders as migraine headaches 15, anxiety 29. and men- tal depression 23. Physiological functions such as blood vessel contraction5 and platelet shape s are regulated by 5-HT 2 receptors. The pharmacological profiles of hallu- cinogemc drugs lmphcates the 5-HT 2 receptor as their site of action 24'27. These observations suggest that 5-HT 2 receptors have an important role in physiological stasis and in the regulation of normal mental states. It has re- cently been shown 16 that NIH3T3 cells, transfected with 5-HT2 receptor cDNA, formed transformed foci when exposed to a 5-HT receptor agonist. The transformation ability of the activated 5-HT 2 receptor suggests the re- ceptor can also mediate cell growth. The primary structures of 5-HT 2 receptors have been determined by the cloning of rat t6'25 and hamster 3 cDNAs which place the receptors in the G protein re- ceptor supergene family~. The 5-HTld 1'1°'18 5-HTk 17 and 5-HTld ~2 receptors are also members of this family. G-protein-coupled receptors all contain seven hydropho- bic stretches of -20 ammo acids each believed to traverse the membrane to form a tertiary structure similar to that of bacteriorhodopsin t4. Receptors of this family exist m all species from bacteria to man and are believed to have originated from a common ancestral gene 3°. Much of the complexity of the nervous system arises from the dwer- sity of neurotransmitters and hormones which use the conserved functional mechanism of the seven transmem- brane domain structure ~9. Isolation and characterizanon of the genomic structure of G-protein-coupled receptors will bring new knowledge concerning the evolution of the G-protem receptor family. Because of the possible implication ot 5-HT 2 receptors in the enology and therapeutic treatment of mental de- pression, the isolauon of the human 5-HT 2 receptor has special significance We report here the genomic cloning of the human 5-HT 2 receptor from human chromosome 13 genomic DNA libraries. The amino acid sequence of thts receptor shares -90% sequence ldenttty w~th the 5-HT 2 receptors of rat, mouse and hamster indicating it's structure to be highly conserved between species. The human receptor is coded for by a gene greater than 20 kb which consists of three exons separated by two in- trons. MATERIALS AND METHODS Southerti blot analy~t~ Human genomlc DNA samples { ll)/~g each from a normal lym- phoblast cell line) were digested with restriction endonucleases EcoRI, HmdlII and EcoRV separately at 37°C for 16 h The dl- Correspondence J C Shlh, Department of Molecular Pharmacology and Toxicology, School ot Pharmacy. University of Southern Cahfornla. 1985 Zonal Avenue, Los Angeles, CA 90033, USA Fax (1) (213) 342-3229