Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8547-8551, September 1993 Pharmacology Molecular cloning, characterization, and localization of a high-affinity serotonin receptor (5-HT7) activating cAMP formation (rat/Chinese hamster ovary celi/in situ hybridization) MARTIAL RUAT*t, ELISABETH TRAIFFORT*, ROB LEURS*, JOEL TARDIVEL-LACOMBE*, JORGE DIAzt, JEAN-MICHEL ARRANG*, AND JEAN-CHARLES SCHWARTZ* *Unite de Neurobiologic et Pharmacologie (U. 109) de l'Institut National de la Sant6 et de la Recherche Medicale, Centre Paul Broca, 2ter rue d'Alesia, 75014 Paris, France; and tLaboratoire de Physiologie, Faculte de Pharmacie, Universite Rene Descartes, 75006 Paris, France Communicated by James Black, June 7, 1993 (receivedfor review February 1, 1993) ABSTRACT By using a strategy based on nucleotide se- 5-HT2 (16) receptors, which are characterized by a coding quence homology, we have cloned a cDNA encoding a func- sequence interrupted by introns (17, 18) and a positive tional serotonin (5-HI) receptor. The deduced amino acid coupling with phospholipase C. Finally, the not yet cloned sequence ofthe 5--HT7 receptor displays limited homology with 5-HT4 receptor (19) and the recently cloned 5-HT6 receptor that ofother 5-HT receptors. In addition to the seven stretches (20, 21) display submicromolar or micromolar affinity for of hydrophobic amino acids that characterize the superfamily 5-HT and are positively coupled to adenylyl cyclase. of receptors interacting with guanine nudeotide-binding pro- There is evidence, however, for the existence ofadditional teins, the 448-aa sequence of the 5-HT7 receptor contains a 5-HT receptors. For instance, high-affinity [3H]5-HT binding hydrophobic domain located at its N-terminal end. Genomic sites, pharmacologically distinct from members of the 5-HT1 analysis indicated the presence of introns interrupting the subfamily (22, 23), and high-affinity 5-HT receptors posi- coding sequence. The 5-HT7 receptor, stably expressed in tively coupled to adenylyl cyclase, presumably distinct from transfected CHO ceils, bound [3H]5-HT with hh affnity (Kd the 5-HT4 receptor (24-26), have been described. = 1 nM), like receptors of the 5-HT1 subfamily from which, We report here the characterization of an additional 5-HT however, it was dearly distuished by its phaacology. receptor belonging to the superfamily of G-protein-coupled 5-HT in nanomolar concentrations stimulated cAMP accumu- receptors that we propose to name 5-HT7 in accordance with lation in these CHO celis by =40-fold, whereas lysergic acid recently proposed nomenclature rules (27).§ From its molec- diethylamide displayed low intrinsic agonist actiity. These ular, pharmacological, and signaling properties, it appears various properties differentiate the 5-HT7 receptor from the that the 5-HT7 receptor may define another subfamily of four other subfamilies of mammalian 5-HT receptors (i.e., the mammalian 5-HT receptors. 5-HTi-, 5-HT2-, 5-HT3-, and 5-HT4-like subfamllies) and, therefore, appear to define another receptor subfamily. North- MATERIALS AND METHODS ern blot and in situ hybridization analyses showed the 5-HT7 Cloning and Sequencing of a Rat cDNA. A rat brain cDNA transcripts to be expressed in discrete areas of the limbic brain was screened at low with a (e.g., pyramidal hippocampus cells, tenia tecta, amygdaloid, or library (28) stringency 32P-labeled nmammiary nudei), suggesting that the receptor mediates DNA fragment corresponding to the coding region of the rat serotoninergic controls in functions like mood, learning, or substance P receptor gene [nt -27 to + 1275 (29)]. One clone neuroendocrine and vegetative behaviors. (ASP45) was shown to hybridize weakly with an Afl II-Bgl II restriction fragment (nt 1193-1446) of the D2 receptor gene (30). A 32P-labeled DNA fragment (523 bp) of this clone was Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter used to screen, under high stringency, a rat hypothalamus that exerts its effects mainly in the central nervous and cDNA library constructed in AZAPII (Stratagene). A 1.5-kb gastrointestinal systems by interacting with a large variety of HindIlI-HindlIl DNA fragment ofone clone (AHPT3) among receptors. These were initially distinguished through the use six positive identical clones, purified >2 x 106 times, was of traditional pharmacological approaches with isolated or- subcloned in pGEM-4Z (Promega) and sequenced. It encodes gans (1) and, then, in binding studies (2). More recently, with an open reading frame of 359 aa starting from aa 89 (Fig. 1). the introduction of molecular biology approaches in the field A 32P-labeled DNA fragment encoding aa 90-193 was used to (3), a wealth of additional 5-HT receptor subtypes could be screen a partial Hae I-cut rat genomic library constructed in characterized. EMBL3 vector (Stratagene). Three positive clones, AHae I-a, Now, it appears that, whereas the 5-HT3 receptor is a -b, and -c, were purified >6 x 10W times. A 6.5-kb fragment ligand-gated ion channel (4), mammalian 5-HT receptors with from AHae I-a was subcloned into pGEM4Z for sequencing. seven putative transmembrane domains (TMs) and coupled It contained an open reading frame of 182 aa starting from to guanine nucleotide-binding (G) proteins can be divided into in 1 and a consensus for an intron in the three subfamilies, depending on molecular, ligand binding, Met' Fig. sequence and effector-coupling properties. Members of the 5-HT1 3' end. An identical 1.6-kb HindIII-HindIll fragment from subfamily [except, perhaps, the preliminarily characterized AHae I-b and -c was also subcloned, sequenced, and found to 5-HT5 receptor (5)-i.e., 5-HT1A (3, 6), 5-HT1B (7-9), contain an open reading frame coding for aa 184-435 (Fig. 1). 5-HTlD (10-12), 5-HT1E (13), and 5-HT1F (14) receptors-are Consensus splice sites for intronic sequences were found in encoded by intronless genes, display nanomolar affinity for 5' and 3' ends of the coding sequence (data not shown). 5-HT, and are negatively coupled to adenylyl cyclase. The 5-HT2 subfamily contains the homologous 5-HT,c (15) and Abbreviations: 5-HT, serotonin; G protein, guanine nucleotide- binding protein; LSD, lysergic acid diethylamide; TM, transmem- brane domain. The publication costs ofthis article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L19654). 8547 Downloaded by guest on September 30, 2021 8548 Pharmacology: Ruat et aL Proc. Natl. Acad. Sci. USA 90 (1993) H MMDVNSSGRPDLYGHLRSLILPEVGRGLQDLSPDGGAHPVVSSWMPHLLSGFLEVTASPA 60 synthesized by PCR, and subcloned in pGEM4Z. 35S-labeled TM 1 antisense- or sense-strand RNA probes were obtained using PTWDAPPDNVSGCGEQINYGRVEKVVIGSILTLITLLTIAGNCLVVISVCFVKKLRQPSN 120 TM 2 TM 3 a Riboprobe kit (Promega). YLIVSLALADLSVAVAVMPFVSVTDLIGGKWIFGHFFCNVFIAMDVMCCTASIMTLCVIS 180 4 TM 4 IDRYLGITRPLTYPVRQNGKCMAKMILSVWLLSASITLPPLFGWAQNVNDDKVCLISQDF 240 RESULTS TM 5 GYTIYSTAVAFYIPMSVMLFMYYQIYKAARKSAAKHKFPGFPRVQPESVISLNGVVKLQK 300 Deduced Amino Acid Sequence of the 5-HT7 Receptor. Se- TM 6 EVEECANLSRLLKHERKNISIFKREQKAATTLGIIVGAFTVCWLPFFLLSTARPFICGTS 360 quencing ofoverlapping rat hypothalamus cDNA and genomic TM 7 DNA led to the characterization of a nucleotide CSCIPLWVERTCLWLGYANSLINPFIYAFFNRDLRTTYRSLLQCQYRNINRKLSAAGMHE 420 fragments 4 sequence containing an open reading frame encoding a protein ALKLAERPERSEFVLQNSDHCGKKGHDT 448 of448 aa(Fig. 1) with an estimated molecular weight of49,836. The coding region of the gene is interrupted by at least two FIG. 1. Deduced amino acid sequence ofthe 5-HT7 receptor. The seven TMs (TM1-TM7) characteristic of G-protein-coupled recep- introns. Analysis of AHae I-a DNA insert revealed an open tors and an additional hydrophobic domain (H) are overlined. Arrows reading frame of 182 aa beginning with a consensus initiator indicate the positions of introns. Symbols *, , and >> represent methionine (GGCACGATGATG) (35), a nonsense codon at consensus sites for glycosylation and protein kinase A and C position -75, and an intron sequence starting afteraa 182 (data phosphorylation, respectively. not shown). Sequencing the AHPT3 cDNA insert revealed an open reading frame corresponding to 359 residues (aa 89-448 The full-length cDNA was obtained by PCR (31). A primer, in Fig. 1) and possessing a stop codon TGA. The full-length containing a Bgl II restriction site and located 82 nt down- nucleotide sequence was obtained by reverse transcription of stream from the first in-frame TGA stop codon (primer 1), poly(A)+ mRNAs from rat hypothalamus followed by PCR was synthesized. It was used at a concentration of 375 nM for amplification using oligonucleotides flanding the 5' and 3' the synthesis of a single-strand cDNA with avian myeloblas- coding region found in AHae I-a and AHPT3 DNA fragments. tosis virus reverse transcriptase (20 units; Boehringer Mann- Clone ASP45 was found to correspond to the C-terminal amino heim) and 2 ,ug of rat hypothalamus poly(A)+ mRNAs as acid sequence starting at residue 306. template. This template was amplified using 75 nM ofprimer Hydropathicity analysis (36) revealed eight clusters of 1 and primer 2 (containing a HindIll restriction site located 44 nt, upstream of the first in-frame ATG) for 35 identical 20-25 hydrophobic amino acids that could span the cell and for 1.5, and 3 min, membrane (Fig. 1, TM1-TM7 and H). Comparison of the cycles (94.5°C, 56°C, 72°C 1.5, amino acid sequence of 5-HT7 receptor with G-protein- respectively) with 5 units of Taq DNA polymerase (Perkin- coupled receptors indicates significant homology, particu- Elmer/Cetus). PCR products were electrophoresed and a larly with other 5-HT receptors. The highest overall homol- band of the predicted size (=1500 bp) was excised, purified ogy (38%) was found with a 5-HT receptor of Drosophila, using Geneclean II (Bio 101), digested with HindIlI and Bgl 5-HT,ko, (37), and homology was 60% when only TMs are II, ligated into pGEM-4Z, and sequenced.