Elsevier

Neuropharmacology

Volume 42, Issue 1, January 2002, Pages 60-73
Neuropharmacology

Cloning and characterization of a novel human 5-HT4 receptor variant that lacks the alternatively spliced carboxy terminal exon. RT-PCR distribution in human brain and periphery of multiple 5-HT4 receptor variants

https://doi.org/10.1016/S0028-3908(01)00154-XGet rights and content

Abstract

We have cloned a novel C-terminal splice variant of serotonin 5-HT4 receptors from human hippocampus. The deduced protein extends only one aminoacid past the splicing point. We propose to call the novel variant h5-HT4(n) since it contains none of the C-terminal exons alternatively spliced in other variants. The pharmacological profile of h5-HT4(n) stably expressed in HeLa cells is in agreement with other reported variants. Stably transfected cells showed increased basal levels of intracellular cAMP in absence of agonist, indicating constitutive activity of the expressed receptors. 5-HT induced robust increases of intracellular cAMP. The 5-HT4 receptor antagonist GR 113808 blocked the effects of 5-HT and brought intracellular cAMP below basal constitutive levels, indicating inverse agonism of this compound in this system. The RT-PCR distribution of all known human C-terminal splice variants in human brain regions and periphery showed complex patterns of variant expression, with the novel variant h5-HT4(n) being widely and abundantly expressed.

Introduction

The existence of 5-HT4 receptors was proposed in 1988 as a non-classical 5-HT (5-hydroxytryptamine) receptor positively coupled with adenylate cyclase and presenting a differential pharmacology (Dumuis et al., 1988, Dumuis et al., 1989). Since this initial proposal, the characteristics and distribution of this receptor have been studied extensively in brain and in peripheral tissues thanks to the development of several selective agonists and antagonists (Ford and Clarke, 1993, Eglen et al., 1995, Hegde and Eglen, 1996). Structural information on 5-HT4 receptors became available in 1995 with the cloning of two cDNAs from rat brain that encoded what appeared to be two splice variants of 5-HT4 receptors differing in the length and sequence of the carboxy-terminal (C-terminal) tail (Gerald et al., 1995). Since this initial report, several C-terminal variants of 5-HT4 receptors have been described by cDNA cloning in human and other species. Following the recommendations of the Serotonin Receptor Nomenclature Committee (Hoyer and Martin, 1997), the different putative C-terminal splice variants have been named alphabetically and chronologically. Thus, variants 4(a), 4(b), 4(c), 4(d) and 4(e) have been described in human (Claeysen et al., 1997, Claeysen et al., 1999; Van den Wyngaert et al., 1997; Mialet et al., 2000a; Blondel et al., 1997, Blondel et al., 1998a), variants 4(a), 4(b) and 4(e) in rat (Gerald et al., 1995, Claeysen et al., 1999), and variants 4(a), 4(b), 4(e) and 4(f) in mouse (Claeysen et al., 1996, Claeysen et al., 1998, Claeysen et al., 1999). Within each species, these variants are identical up to aminoacid Leu358 after which they diverge in sequence and length (Blondel et al., 1998a, Claeysen et al., 1999). More recently, the structure of the human 5-HT4 receptor gene has been reported (Bender et al., 2000). It spans more than 130 kb and contains at least 10 exons. Exons 2–5 encode most of the common part shared by all variants up to Leu358. Downstream of exon 5, several other exons have been identified and also termed alphabetically to match the previous nomenclature given to the variants obtained by cDNA cloning. Thus, for all the different C-terminal variants reported so far, the corresponding exonic sequences have been found in the gene (Bender et al., 2000). In addition, an internal splice variant has been detected that results in the insertion of 14 amino acids into the second extracellular loop of the receptor. The corresponding exon has been named exon h, and when combined with the C-terminal exon b results in a variant termed h5-HT4(hb) (Bender et al., 2000). In the light of the known gene structure, the previously reported h5-HT4(e) variant, which was thus named because it was similar (although not identical) to the rat 5-HT4(e) receptor (Claeysen et al., 1999, Mialet et al., 2000a) has been re-named h5-HT4(g) (Bender et al., 2000). This new name seems more appropriate in view of the existence, at least in human, of the so-called exon ‘gef’, which can account for three different variants: the re-named human variant 4(g) and the putative human variants 4(e) and 4(f), which would be the true human counterparts of rat and mouse variants 4(e) and 4(f) (Claeysen et al., 1999).

The aims of the present work were to characterize further the variability in the human 5-HT4 receptor family. We report the cloning from human hippocampal cDNA of a novel 5-HT4 receptor variant that lacks the C-terminal exon which constitutes the varying domain in the other variants. We also report the distribution in monkey brain and human brain and periphery of this novel and other existing C-terminal variants.

Section snippets

5′ and 3′ RACE amplifications

A partial nucleotide sequence encoding a fragment encompassing most of the third cytoplasmic loop of a human 5-HT4 receptor (Ullmer et al., 1995) was used to design and synthesize PCR primers to be used in RACE reactions (Rapid Amplification of cDNA Ends) to amplify the 5′-half and the 3′-half of human 5-HT4 receptors. Primers and oligonucleotides were custom synthesized by Amersham Pharmacia Biotech (Little Chalfont, UK). Two antisense primers, p1 (5′ AGAATTCGGTCTCTGTCCTCATGCGATGAGTG) and p2

5′ RACE

Marathon-Ready cDNA from human hippocampus was subjected to amplification with the anchor primer AP1 and gene specific antisense primer p1, followed by a nested PCR with AP1 and gene specific primer p2 (see Fig. 1). Upon Southern blot analysis of the reaction products and hybridization with oligonucleotide probe on1, a hybridizing DNA fragment of approximately 800 bp was observed in both PCR reactions. The products of the nested PCR were subcloned and screened with probe on1. Sequencing of

Discussion

We report the cloning of a novel human 5-HT4 receptor C-terminal splice variant. It is of interest to note that three other isoforms already described in the literature were also cloned from the same human hippocampus cDNA library (not shown), indicating the complexity of 5-HT4 receptor variant expression in certain brain regions.

In our initial RACE experiments, two 3′RACE fragments were obtained, one of them corresponding to the already described h5-HT4(b) variant (Van den Wyngaert et al., 1997

Acknowledgements

This work was supported by grants from CICYT, SAF 96-0336 and SAF 97-0117. The authors wish to thank Dr F. Artigas and Ms L. Campa for their expert HPLC measurements of 5-HT in cell culture medium. In the very initial phases of this project, M.T.V. was recipient of a Long Term EMBO fellowship (ALTF 448-1992). Support from the CIRIT (Generalitat de Catalunya) to the Department of Neurochemistry (IIBB/CSIC) as Grup de Recerca Consolidat (1999 SGR-00210) is acknowledged.

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