Molecular cloning and expression of a 5-hydroxytryptamine7 serotonin receptor subtype.

Y Shen,F.J Monsma,M.A Metcalf,P.A Jose,M.W Hamblin,D.R Sibley

doi:10.1016/s0021-9258(17)46830-x

Abstract

We have utilized the polymerase chain reaction technique to selectively amplify a G protein-coupled receptor cDNA from rat kidney proximal convoluted tubule mRNA, which exhibits high homology with previously cloned serotonin receptors. Sequencing of a full-length clone isolated from a rat hippocampal cDNA library revealed an open reading frame of 1,212 base pairs encoding a 404-residue protein with seven hydrophobic regions predicted to represent transmembrane-spanning domains. Within the transmembrane regions, this receptor was found to be 44-50% identical with various members of the 5-HT1, 5-HT5, and 5-HT6 subfamilies with lower (37-40%) homology to the 5-HT2-like receptors. Northern blots revealed a approximately 3.6-kilobase transcript localized in various brain regions with the following rank order of abundance: hypothalamus > hippocampus = mesencephalon > cerebral cortex = olfactory bulb > olfactory tubercle. Expression of this clone in COS-7 cells resulted in the appearance of high affinity, saturable binding of [3H]lysergic acid diethylamide ([3H]LSD; KD = 5 nM) and [3H]serotonin ([3H]5-HT; KD = 1 nM). Among endogenous biogenic amines, only 5-HT completely inhibited radioligand binding. The inhibition of radioligand binding by other serotonergic agents revealed a pharmacological profile that does not correlate with any previously described serotonin receptor subtype. In addition, this receptor exhibits high affinity for a number of tricyclic antipsychotic and antidepressant drugs including clozapine, loxapine, and amitriptyline. In HEK-293 cells stably transfected with this receptor, serotonin elicits a potent stimulation of adenylylcyclase activity. The distinct structural and pharmacological properties of this receptor suggests that it represents a completely novel serotonin receptor subtype, which we propose to designate 5-HT7. Based on its pharmacology and its localization to limbic and cortical regions of the brain, it is likely that this receptor may play a role in several neuropsychiatric disorders that involve serotonergic systems.

Highlights

We have utilized the polymerase chain reaction tech- Serotonin is a ubiquitous neurotransmitter, which is found in nique to selectively amplifya G protein-coupled recep- both the central and peripheranl ervous systems of many spetor cDNA from rat kidney proximal convoluted tubule cies and is involved in a wide variety of behavioral and physimR.NA, which exhibits high homology with previously ological functions
Incompletely or incorrectly spliced mRNA. This possibility was Following the original report of Libert et al (29), we have further investigated by using PCR to amplify this region from used the polymerase chain reaction (PCR) in an attempt to cDNA prepared from rat hippocampal mRNA
Amplification clone novel members of the G protein-coupled receptor family. across this region using primersP1 and P2 (Fig. 2) resulted in Degenerate oligonucleotide primers were prepared using se- two abundant cDNAproducts of approximately 460 and quences from the third and sixth transmembra(nTeM)regions 390 bp in size

Summary

Introduction

We have utilized the polymerase chain reaction tech- Serotonin is a ubiquitous neurotransmitter, which is found in nique to selectively amplifya G protein-coupled recep- both the central and peripheranl ervous systems of many spetor cDNA from rat kidney proximal convoluted tubule cies and is involved in a wide variety of behavioral and physimR.NA, which exhibits high homology with previously ological functions. Sequencing of the plification of a number of cDNA fragments which were deter- smaller PCR product confirmed the predicted splice sites, mined by DNA sequence analysis to represenpt ortions of pre- which indicates that there aretwo forms of PCT-65 mRNA in viously cloned receptors, some novel receptors as well as the hippocampus, only one of which is correctly spliced.

Results

Conclusion