Cloning and expression of a human serotonin 5-HT4 receptor cDNA.
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]GR 113808 revealed K(D) values of 0.15 +/- 0.01 nM for the human receptor and 0.3 +/- 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5-HT4 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.
Van den Wyngaert
I
I
Department of Experimental Molecular Biology, Janssen Research Foundation, Beerse, Belgium.
Gommeren
W
W
Verhasselt
P
P
Jurzak
M
M
Leysen
J
J
Luyten
W
W
Bender
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003596:Cytosine; D017931:DNA Primers; D016368:Frameshift Mutation; D006168:Guinea Pigs; D006801:Humans; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D051381:Rats; D011985:Receptors, Serotonin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D044422:Receptors, Serotonin, 5-HT4; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012702:Serotonin Antagonists / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism; D013552:Swine; D014162:Transfection
D017931:DNA Primers; D007211:Indoles; D011985:Receptors, Serotonin; D011994:Recombinant Proteins; D012702:Serotonin Antagonists; D013449:Sulfonamides; D044422:Receptors, Serotonin, 5-HT4; D003596:Cytosine; C082871:GR 113808