Cloning, physical mapping and expression analysis of the human 5-HT₃ serotonin receptor-like genes HTR3C, HTR3D and HTR3E

Abstract

For more than 50 years the serotonin system has been the subject of intense research. This has provided an exciting insight and led to the discovery of multiple drugs targeting serotonin receptors, metabolising enzymes and re-uptake sites. During the past few years researchers focussed especially on elucidating the complexity of different physiological actions in the serotonergic network. We have identified two novel human serotonin 5-hydroxytryptamine type 3 receptor-like genes, HTR3D and HTR3E, by performing homology searches using the public human sequence databases and subsequently cloned the full length cDNAs by 5′ and 3′ rapid amplification of complementary DNA ends. Mapping of HTR3D and HTR3E by hybridisation, polymerase chain reaction and fluorescence in situ hybridisation revealed that both genes together with HTR3C are clustered in a subinterval of less than 100 kb on chromosome 3q27. Comparative expression analysis of all HTR3 genes, namely HTR3A, B, C, D and E showed HTR3D expression to be restricted to kidney, colon and liver and HTR3E expression to colon and intestine, whereas all other genes are widely expressed in many tissues including brain.

Introduction

Serotonin (5-hydroxytryptamine, 5-HT) controls a variety of physiological functions in the central and peripheral nervous systems. The serotonergic system is organised in a highly complex manner, as serotonin action is mediated by a multitude of 5-HT receptor subtypes. These subtypes can be divided into seven main classes (5-HT₁R–5-HT₇R) based on their structural and functional features (Hoyer et al., 2002). This high degree of receptor variability clearly emphasises the physiological importance of serotonin and points to an extraordinary diversity of functions. The unravelling of the properties of the system leading to this complexity is one of the major goals of serotonin research.

Except for the 5-HT₃ receptor, which is a ligand-gated ion channel, all serotonin receptors represent G-protein coupled binding proteins. However, the 5-HT₃ receptor shares characteristic features with the other members of this ion channel family: a large extracellular domain containing a conserved cysteine loop, four hydrophobic transmembrane segments, a large intracellular loop between the third and fourth transmembrane region and an extracellular C-terminus (Reeves and Lummis, 2002). The ion channel itself is an oligomeric complex composed of five subunits. During the last few years two different human 5-HT₃ receptor subunit genes, HTR3A and HTR3B, have been isolated (Miyake et al., 1995, Davies et al., 1999).

Several studies pointed out the complexity within the 5-HT₃ receptor system based on indications of pharmacological and biophysical variations between tissues and species. Different ligand binding affinities and ion channel conductances of diverse tissues and cell lines have been reported and pharmacologic profiles of homomeric and heteromeric receptors showed differences in the properties of the respective receptors. Only 5-HT_3A subunits can form functional homo-oligomeric complexes after expression in Xenopus oocytes or HEK293 cells, whereas the 5-HT_3B subunits are not able to build a functional receptor on their own (Miyake et al., 1995, Davies et al., 1999, Dubin et al., 1999). Recombinant homo-oligomeric 5-HT_3A receptors show extremely reduced channel conductances compared to native receptors, whereas heteromeric complexes composed of both 5-HT_3A and 5-HT_3B subunits resemble closely the properties of the native receptors (Davies et al., 1999). Variable receptor compositions have also been shown by Western blot experiments which led to the hypothesis that further 5-HT₃ receptor subunits actually should exist (McKernan, 1992, Hussy et al., 1994, Jackson and Yakel, 1995, Fletcher and Barnes, 1998). To verify this hypothesis we have established a 5-HT₃ receptor consensus sequence to screen databases and identified a human BAC clone (RP11-315J22) harbouring additional subunit sequences. We established primers and cloned the entire coding region of two novel HTR3-homologous genes by 5′ and 3′ RACE: HTR3D and HTR3E. Mapping data of HTR3D and HTR3E and of the recently cloned HTR3C gene (accession number AF459285) revealed that they all reside in close vicinity on chromosome 3q27. To get a better insight into the putative role of HTR3D and HTR3E, we carried out expression analysis and compared the expression profiles to those of HTR3A, B and C.