CLONING AND EXPRESSION OF A HUMAN SEROTONIN 5-HT4 RECEPTOR CDNA

Using a combination of library screening and nested PCR based on a par tial human serotonin 5-HT4 receptor sequence, we have cloned the compl ete coding region for a human 5-HT4 receptor, The sequence shows exten sive similarity to the published porcine 5-HT4A and rat 5-HT4L recepto r cDNA; however, in comparison with the latter, we find an open readin g frame corresponding to only 388 amino acids instead of 406 amino aci ds. This difference is due to a frame shift caused by an additional cy tosine 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 iden tified 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 recepto r, transiently expressed in COS-7 cells, showed radioligand binding pr operties similar to 5-HT4 receptors in guinea pig striatal tissue. [H- 3]GR 113808 revealed K-D values of 0.15 +/- 0.01 nM for the human rece ptor and 0.3 +/- 0.1 nM in the guinea pig tissue. Binding constants we re determined for four investigated 5-HT4 antagonists and three agonis ts, and appropriate binding inhibition constants were found in each ca se. Stimulation of transfected COS-7 cells with 5-HT4-specific agonist s caused an increase in cyclic AMP levels.