Cloning and Expression of a Human Serotonin 5‐HT₄ Receptor cDNA

Abstract

Abstract:Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT4 receptor sequence, we have cloned the complete coding region for a human 5-HT4 receptor. The sequence shows extensive similarity to the published porcine 5-HT4A and rat 5-HT4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154. Moreover, we also found the same additional cytosine in the rat 5-HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT4 receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT4 receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT4 receptors in guinea pig striatal tissue. [ 3H]GR113808 revealed KD values of 0.15 ±0.01 nMforthe human receptor and 0.3 ±0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5-HT 4 antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT4-specific agonists caused an increase in cyclic AMP levels. Key Words: Serotonin-5-HT4 receptor-Cloning--Human-Rat.

Differences in Signal Transduction of Two 5-HT4Receptor Splice Variants: Compound Specificity and Dual Coupling with Gαs- and Gαi/o-Proteins

“…values that were consistent with previous findings on transiently transfected cells (Ansanay et al, 1996;Van den Wyngaert et al, 1997) and different brain tissues (Grossman et al, 1993;Waeber et al, 1994;Arranz et al, 1998). We confirm that C-terminal variations of h5-HT 4a and h5-HT 4b receptors did not modify binding K D values, consistent with the findings on EP3 and somatostatin receptors (Tsunehisa et al, 1993;Vanetti et al, 1993) and very recently also 5-HT 4 receptors (Bach et al, 2001).…”

“…However, we have shown that it is not cAMP-dependent in HEK cells and has different thresholds and pharmacology on both receptor isoforms. It is important to state that a direct comparison of tissue expression levels (guinea pig striatum ϳ200 fmol/mg; Van den Wyngaert et al, 1997) to data derived on recombinant cells is problematic, because the physiological concentration of neurotransmitter receptors in the synaptic Experiments were performed as described for Table 4. Results are means of three independent experiments performed in duplicate and are based on fitted concentration-response curves as presented in Fig.…”

Investigations into the Binding Affinities of Different Human 5-HT₄ Receptor Splice Variants

“…Drugs 5-HT creatinine sulphate complex, 5-MeOT and 3-isobutyl-1-methylxanthine (IBMX) and pargyline hydrochloride were from Sigma (Castle Hill, N.S.W., Australia); [N-methyl- 3 …”

Mapping of serotonin 5-HT4 receptor mRNA and ligand binding sites in the post-mortem human brain

“…The receptor gene was cloned from several species and shows a relatively low amino acid sequence homology (Ͻ50%) with other serotonin receptor subtypes (Gerald et al, 1995;Claeysen et al, 1996). The complete coding region for the human (h) 5-HT 4 receptor was cloned as well (Van den Wyngaert et al, 1997). The cloned h5-HT 4 receptor was expressed in COS7 cells and characterized by radioligand binding (Van den Wyngaert et al, 1997).…”

“…The complete coding region for the human (h) 5-HT 4 receptor was cloned as well (Van den Wyngaert et al, 1997). The cloned h5-HT 4 receptor was expressed in COS7 cells and characterized by radioligand binding (Van den Wyngaert et al, 1997). Recently, cloning and expression of three additional h5-HT 4 receptor splice variants have been reported (Claeysen et al, 1997;Blondel et al, 1998).…”