Eur J Biochem 269, 4566–4576 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03152.x Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis Identification of a novel subtype of thyrotropin-releasing hormone receptor Isabelle Bidaud1, Philippe Lory2, Pierre Nicolas1, Marc Bulant1 and Ali Ladram1 Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, CNRS-Universite´ Paris, Paris; 2Institut de Ge´ne´tique Humaine, CNRS-UPR 1142, Montpellier, France Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals where thyrotropin-releasing hormone (TRH) is known to act as a powerful stimulator of thyroid-stimulating hormone (TSH) secretion The TRH receptor of amphibians has not yet been characterized, although TRH is specifically important in the adaptation of skin color to environmental changes via the secretion of a-melanocyte-stimulating hormone (a-MSH) Using a degenerate PCR strategy, we report on the isolation of three distinct cDNA species encoding TRHR from the brain of Xenopus laevis We have designated these as xTRHR1, xTRHR2 and xTRHR3 Analysis of the predicted amino acid sequences revealed that the three Xenopus TRHRs are only 54–62% identical and contain all the highly conserved residues constituting the TRH binding pocket Amino acid sequences and phylogenetic analysis revealed that xTRHR1 is a member of TRHR subfamily and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype awaiting discovery in other animal species The three Xenopus TRHRs have distinct patterns of expression xTRHR3 was abundant in the brain and much scarcer in the peripheral tissues, whereas xTRHR1 was found mainly in the stomach and xTRHR2 in the heart The Xenopus TRHR subtype was found specifically in the intestine, lung and urinary bladder These observations suggest that the three xTRHRs each have specific functions that remain to be elucidated Expression in Xenopus oocytes and HEK-293 cells indicates that the three Xenopus TRHRs are fully functional and are coupled to the inositol phosphate/calcium pathway Interestingly, activation of xTRHR3 required larger concentrations of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation Thyrotropin-releasing hormone (TRH) was first isolated from the mammalian hypothalamus and characterized by its ability to stimulate thyroid-stimulating hormone (TSH) secretion [1,2] Most of the effects of TRH on the pituitary are mediated by activation of the phospholipase C trans- duction pathway involving a Gq-like G-protein [3] Regulation of TSH and prolactin secretions has also been reported in amphibians [4–6], but in this species, TRH is extremely important in the modulation of a-melanocytestimulating hormone (a-MSH) secretion by pituitary melanotrope cells of the pars intermedia [7,8] a-MSH, in turn, is pivotal in the adaptation of skin color to environmental changes [9] TRH causes a transient increase in inositol 1,4,5-triphosphate (InsP3) formation in the pars intermedia cells of the frogs, indicating that TRH stimulates the phospholipase C pathway in melanotrope cells [10] In these cells, TRH induces also an increase of the intracellular calcium concentration [11] Amphibians also have two TRH precursors whose amino acid sequences differ by about 16% [12,13] Both contain seven copies of the TRH progenitor sequence, whereas only five TRH units are found in the rat and mouse [14,15], and six in humans [16] The 5¢-flanking region of the amphibian TRH gene lacks the regulatory sequence CAGGGTTTCC that seems to be important for regulating the thyroid hormone gene in humans [16] and rats [17] Although TRH receptors (TRHRs) have been cloned from several species, no molecular information is presently available on the TRHR in amphibians A mouse pituitary cDNA encoding a G-protein-coupled TRH receptor (TRHR) was first isolated in 1990, using an expression cloning strategy [18] The nucleotide sequence of this receptor was subsequently used to clone TRHR cDNAs Correspondence to A Ladram, Laboratoire de Bioactivation des ´ Peptides, Institut Jacques Monod, UMR 7592, CNRS-Universite Paris 6/7, place Jussieu, 75251 Paris cedex 05, France Fax: + 33 44275994, Tel.: + 33 44276952, E-mail: ladram@ijm.jussieu.fr Abbreviations: a-MSH, a-melanocyte-stimulating hormone; EL, extracellular loop; IL, intracellular loop; InsP3, inositol 1,4,5-triphosphate; SLIC, single-strand ligation of cDNA; TM, transmembrane domain; TRH, thyrotropin-releasing hormone; TRHR, thyrotropinreleasing hormone receptor; TSH, thyroid-stimulating hormone Proteins and enzymes: thyrotropin-releasing hormone (THYL_PIG); thyrotropin-releasing hormone precursor (Q62361); thyrotropinreleasing hormone receptors (TRFR_RAT; Q9R297; TRFR_MOUSE; Q9ERT2; TRFR_BOVIN; TRFR_SHEEP; TRFR_CHICK; Q9DFB0; Q9DFA9); prolactin (PRL_HORSE); thyroid-stimulating hormone (TSHB_RAT); a-melanocyte-stimulating hormone (MLA_ANOCA) Note: cDNA sequences reported in this paper have been deposited into the EMBL database under accession numbers AJ420780, AJ420781 and AJ420782 (Received 13 March 2002, revised July 2002, accepted 30 July 2002) Keywords: thyrotropin-releasing hormone receptors; subtypes; amphibian; cloning; functional expression Ó FEBS 2002 TRH receptor subtypes from X laevis (Eur J Biochem 269) 4567 from various species, including those of rats [19–21], humans [22–24], sheep [25], oxen [26], chickens [27] and, more recently, fish [28], that all belong to the TRHR1 family Two cDNA isoforms of the TRHR1, generated by alternative splicing, have been isolated from GH3 rat anterior pituitary tumor cells These two isoforms, which differ in their C-terminal cytoplasmic tails, display no functional differences when expressed in rat-1 fibroblasts [29] A novel type TRHR subfamily (TRHR2) was discovered recently TRHR2 receptors that were 46%, 48% and 43% identical to the rat long isoform (TRHR1) have been cloned and characterized from rats [30,31], mice [32] and fish [28] Rat TRHR2 is more widely distributed in the brain than is TRHR1 [33] and they differ in their agonist-induced internalization and down-regulation/desensitization These features suggest that they differ both functionally and structurally [34] Rat TRHR2 is also basally more active than TRHR1, acting via pathways mediated by the transcription factors AP-1, Elk1 and CREB [35] To clarify the functional significance of the TRH ligand/ receptor system in amphibians, a species where TRH has been extensively studied and where it has particular functions, we have described the isolation of full-length cDNAs encoding three subtypes of the Xenopus laevis brain TRHR (xTRHR1, xTRHR2 and xTRHR3) and their functional expression in Xenopus oocytes and mammalian cells We have also determined the tissue distributions of xTRHR mRNA species by RT-PCR This study therefore represents an important molecular landmark towards the identification of the precise roles of TRH in amphibians EXPERIMENTAL PROCEDURES Cloning and sequencing of TRH receptor cDNAs Polyadenylated [poly(A)+] RNA isolation Three adult male Xenopus laevis toads (CNRS, Rennes, France) were anaesthetized by placing them on ice, killed by decapitation and their brains immediately removed poly(A)+ RNA (2–4 lg) was isolated from approximately 50 mg of brain tissue using the Micro-FastTrack mRNA isolation kit (Invitrogen) RT-PCR analysis Degenerated oligonucleotides were designed to conserved regions of the transmembrane domains (TM) of several previously cloned TRHRs A first set of primers was selected from TM1 and the end of TM6: TRHR-1 (sense), 5¢-GGKATYGTKGGKAAYATHA TGGT-3¢; TRHR-2 (antisense), 5¢-TAMGGCATCCAM A-RMARNGC-3¢ A second one for the nested PCR was chosen in the TM2-EL1 region and in the beginning of TM6: TRHR-3 (sense), 5¢-TGGGTKTAYGGKTAYGT KGGNTG-3¢; TRHR-4 (antisense), 5¢-ACMGCMARCA TYTTMGTNACYTG-3¢ All oligonucleotides were synthesized by Genset (Paris, France) The two sets of oligonucleotide primers generated a 550-bp nested PCR product from TRHR cDNA (see Fig 1A) Brain poly(A)+ RNA (1 lg) was reverse transcribed into cDNA using random hexamers (20 pmol) in a volume of 20 lL containing 1X reaction buffer (50 mM Tris/HCl, pH 8.3; 75 mM KCl; and mM MgCl2), each deoxy-NTP at 0.5 mM, ribonuclease inhibitor (0.5 U), and Moloney murine leukemia virus reverse transcriptase (200 U; Clontech, Palo Alto, Fig Diagram of the xTRHR cDNA, PCR primers and PCR products (A) Amplification of the middle region of the xTRHR cDNA by nested PCR The relative positions of the degenerated primers TRHR1, TRHR2, TRHR3, and TRHR4 are shown with the final PCR product (B) 3¢-RACE The 3¢-end amplified fragment of the xTRHR cDNA is shown The positions of the two specific oligonucleotide primers, TRHR5 and TRHR6, are indicated AUAP: abridged universal amplification primer (C) 5¢-SLIC xTRHR cDNA was ligated to the chemically 3¢-end modified oligonucleotide A5NV Three successive PCRs were performed using specific primers designed to the middle region of the receptor and to the A5NV portion The resulting 5¢-end amplified fragment is shown (D) Construction of fulllength xTRHR3 cDNA A fragment of the receptor starting from the 5¢-end and ending in the middle of the transmembrane domain was amplified using the specific primers, TRHR10 and TRHR7, and a template corresponding to a mixture of the PCR products obtained in (A) and (C) The full-length cDNA was finally obtained using this fragment in association with the 3¢-end one and the specific primers TRHR10 and TRHR11 CA, USA) The mixture was incubated for 60 at 42 °C, heated for at 94 °C, and diluted with water to 100 lL An aliquot (5 lL) of the brain cDNA mixture was amplified by PCR in 50 lL containing 1X PCR buffer (10 mM Tris/ HCl, pH 8.3; 50 mM KCl; and 1.5 mM MgCl2), 0.5 mM of each deoxy-NTP, TRHR-1 and TRHR-2 degenerated primers (0.4 lM each), and 0.2 U AmpliTaq DNA polymerase (Applied Biosystems) We used a 30-cycle program consisting of 94 °C for 45 s, 45 °C for min, and 72 °C for min, followed by a final extension at 72 °C for 10 Five microliters of this amplified mixture was then submitted to nested PCR using more internal degenerated primers, TRHR-3 and TRHR-4, under the same conditions The PCR products were analyzed by agarose gel (1%) electrophoresis The 550 bp amplified fragment was 4568 I Bidaud et al (Eur J Biochem 269) purified (Concert Rapid Gel Extraction System, Life Technologies), cloned into the pGEM-T easy vector (Promega Corp.) and sequenced with an ABI PRISM 377 automated DNA sequencer (Applied Biosystems Inc., Foster City, CA, USA) using the fluorescent dye-labeled dideoxynucleotide method, both T7 and Sp6 primers, and the Taq polymerase Three subtypes of brain Xenopus thyrotropin-releasing hormone receptor were obtained and designated xTRHR1, xTRHR2 and xTRHR3 Amplification of cDNA ends The information on the nucleotide sequence of the cloned middle region of the xTRHR allowed us to determine the 3¢-translated and -untranslated regions of the brain xTRHR cDNA in 3¢-RACE experiments Two specific sense oligonucleotide primers were designed to the TM5 and TM5-IL3 regions of the xTRHR: TRHR-5, 5¢-CCTCTACACCCCCATT TACTTC-3¢; TRHR-6, 5¢-CACGGTTCTGTATGGAC TCATAG-3¢ (Fig 1B) 500 ng of brain poly(A)+ RNA were reverse transcribed into cDNA using an adapter primer (5¢-GGCCACGCGTCGACTAGTACTTTTTTTT TTTTTT-TT-3¢; final concentration: 0.5 lM, Life Technologies) in 20 lL containing 1X reaction buffer (20 mM Tris/ HCl, pH 8.4; 50 mM KCl), 2.5 mM MgCl2, each deoxyNTP at 0.5 mM, 10 mM dithiothreitol, and SuperScript II transcriptase reverse (200 U, Life Technologies) The reaction was initiated by incubating the mixture at 42 °C for 50 and stopped by incubation at 70 °C for 15 and quickly placing the tubes on ice The mixture was incubated with ribonuclease H for 20 at 37 °C to eliminate the RNA template Two microliters of this brain cDNA mixture was then amplified by PCR under the same conditions as for RT-PCR, using the TRHR-5 sense primer (0.2 lM) and the antisense abridged universal amplification primer (AUAP: 5¢-GGCCACGCGTCGACTAGTAC-3¢; 0.2 lM; Life Technologies) Two microliters of this amplified mixture was then submitted to nested PCR using the TRHR-6 primer and the abridged universal amplification primer, under the same conditions (Fig 1B) The PCR products were analyzed by agarose gel electrophoresis, purified, and cloned into the pGEM-T easy vector for sequencing with both T7 and Sp6 primers 5¢ Single-strand ligation of cDNA [36] (5¢-SLIC) experiments were performed to obtained the 5¢-translated region of the brain xTRHR cDNA Brain Xenopus poly(A)+ RNA was extracted and reverse transcribed The cDNA was then ligated with the 3¢-end chemically modified oligonucleotide, A5NV (300 ng, 5¢-CTGCATCTATCTA ATGCTCCT-CTCGCTACCTGCTCACTCTGCGTGA CATC-NH2-3¢, Genset, Paris, France), in 11 lL containing T4 RNA ligase (50 U, Biolabs), 1X T4 RNA ligase buffer, and 23% polyethylene glycol The mixture was incubated at 22 °C for 72 h and the cDNA was purified Specific oligonucleotide primers were designed to A5NV (A51, A52 and A53 sense primers) and to the middle region of the xTRHR cDNA (TRHR-7, TRHR-8, and TRHR-9 antisense primers) Three successive PCR experiments were performed using three sets of primers: first set, A51 (5¢GATGTCACGCAGAGTGAGCAGGTAG-3¢)/TRHR-7 (5¢-GAGACCATACAGAAC-C-3¢); second set, A52 (5¢AGAGTGAGCAGGTAGCGAGAGGAG-3¢)/TRHR-8 (5¢-GGGGGTGTAGAGGTTTCTGGAGAC-3¢); third set, A53 (5¢-CGAGAGGAGCATTAGA-TAGATG Ĩ FEBS 2002 CAG-3¢)/TRHR-9 (5¢-GCCGAAATGTTGATGCCCA GATAC-3¢) (Fig 1C) The PCR products were analyzed by agarose gel electrophoresis, purified and cloned into the pGEM-T easy vector for sequencing xTRHR1 and xTRHR2 cDNA ends were obtained by a strategy similar to that described above Construction of full-length xTRHR cDNAs We used the following strategy as we were unable to amplify the fulllength cDNA directly by nested PCR, probably due to the too low expression and the large size of the receptor We used a mixture of the two partially overlapping cDNA fragments corresponding to the 5¢-region and the middle region as template for the first PCR of xTRHR3, with the oligonucleotide primers TRHR-7 and TRHR-10 (5¢-GTTTTGGGGTGGATTAAGGTAG-3¢) (Fig 1D) An 816-bp amplified fragment was purified A mixture of this cDNA fragment and the 3¢-region of the xTRHR3 cDNA was then used in a second PCR with the specific oligonucleotide primers TRHR-10 and TRHR-11 (5¢-CTACGCCACACTGTATGTTGTC-3¢) (Fig 1D) All the PCR experiments were done as described above with hybridization temperatures of 46 and 48 °C for the first and second PCR, respectively A 1400-bp fragment corresponding to the full-length xTRHR3 cDNA was finally purified, cloned into the pGEM-T easy vector, and sequenced in both directions using T7 and Sp6 primers Full-length cDNAs corresponding to the xTRHR1 and xTRHR2 subtypes were amplified as described above using partially overlapping cDNA fragments and the pair of primers TRHR1-2 sense (5¢-ATAATGGATAA CGTAACTTTTGCTG-3¢)/TRHR1-4 antisense (5¢-TC TGTTAAATGTACCTAAGTAGGCA-3¢) and TRHR2-2 sense (5¢-CAGCAAAATGGAAAATAGTAGC-3¢)/ TRHR2-4 antisense (5¢-CGACACTGTAGTAG-AGAT CACC-3¢), respectively The PCR products (xTRHR2: 1200 bp, xTRHR1: 1200 bp) corresponding to full-length cDNA were finally purified, cloned into the pGEM-T easy vector, and sequenced in both directions TRHR cDNA fragments were isolated from pGEM-T easy vector by Not1 excision and subcloned into the Not1 site of the mammalian expression vector pcDNA3.1(–) (Invitrogen) These expression vectors containing the entire coding sequence of xTRHR1, xTRHR2 and xTRHR3 were called pcDNA3.1-xTRHR1, pcDNA3.1-xTRHR2 and pcDNA3.1-xTRHR3 Voltage clamp experiments in Xenopus oocytes Xenopus oocytes were isolated, prepared and maintained using standard procedures [37], and microinjected with pcDNA3.1-xTRHR1, pcDNA3.1-xTRHR2 and pcDNA3.1-xTRHR3 (approximately 10 ng of plasmid/ oocyte) Whole cell currents were measured days later using a two-microelectrode voltage clamp technique (Genclamp, Axon Instruments) The activity of the Ca2+-activated chloride channel was recorded using a standard calcium/chloride solution containing (in mM): 96 NaCl, KCl, MgCl2, CaCl2 and Hepes (pH 7.4) The holding potential was )80 mV Data acquisition and analysis were monitored by the pCLAMP7 suite (Axon Instruments) Ó FEBS 2002 TRH receptor subtypes from X laevis (Eur J Biochem 269) 4569 Calcium imaging experiments in HEK-293 cells Human embryonic kidney (HEK-293) cells were grown to 70–90% confluence in 35 mm dishes (Nunc) in DMEM supplemented with 10% fetal bovine serum (Eurobio) and 1% penicillin streptomycin (Gibco) One day after transfection with pcDNA3.1-xTRHR, cells were trypsinized and plated onto polyornithine-coated Laboratory-Tek borosilicate chambers (Nunc) and cultured for a further 24 h For the measurement of intracellular Ca2+, cells were incubated with 2.5 lM of the acetoxymethyl ester derivative of the dual-excitation ratiometric Ca2+ sensitive indicator fura-2 (Molecular Probes) at 37 °C in the dark for 30 in Locke buffer containing (in mM): 140 NaCl, KCl, 1.2 KH2PO4, 1.2 MgSO4, CaCl2, 10 glucose and 10 Hepes (pH 7.2) Cells were then washed in Locke buffer and mounted onto the stage of an inverted microscope (Olympus IX70) equipped with epifluorescence optics and interfaced with MERLIN software (LSR, Cambridge UK) to a monochromator (Spectramaster) and a 12/14 bit frame transfert rate digital camera (Astrocam) MERLIN software was also used to calculate the 340/380 fluorescence ratio (Rf) The intensity of fluorescent light emission (k ¼ 510 nm) using excitation at 340 and 380 nm was monitored for each single fura-2 loaded cell in the field TRH (1 lM and 10 lM) and ATP (10 lM) were prepared freshly in Locke buffer and placed close to the cells studied Data are presented as mean ± SEM, and n is the number of cells used Student’s t-test was used for statistical analysis RT-PCR distribution of xTRHR mRNAs Poly(A)+ RNA was isolated from the brain, heart, liver, ventral and dorsal skin, testis, stomach, intestine, urinary bladder and lungs of adult male Xenopus laevis toads Poly(A)+ RNA extracted from rat testes and ovaries were used as positive and negative controls, respectively RTPCR experiments were performed under the same conditions described for the RT-PCR analysis (oligonucleotide primers: TRHR-1/TRHR-2 and TRHR-3/TRHR-4; length of the amplified fragment: 550 bp) The poly(A)+ RNA preparations were checked for contamination with genomic DNA by treating each mRNA sample with and without reverse transcriptase before the PCR reactions The PCR products were analyzed by agarose gel electrophoresis The purified 550 bp fragments from the positive tissues were cloned into the pGEM-T easy vector and sequenced The amounts of TRHR mRNA in these tissues were compared using a set of oligonucleotide primers corresponding to the Xenopus EF1a elongation factor that generates an approximately 280 bp product as an internal control Phylogenetic analysis The nucleotide sequence of TRH receptors from humans (GenBank accession number NM_003301), sheep (X95285), oxen (D83964), rats (NM_013047, AF091715), mice (NM_013696, AF283762), chickens (Y18244) and the teleost fish Catostomus commersoni (AF288367, AF288368) were obtained from GenBank The nucleotide sequences of the TRHR transcripts were aligned with CLUSTAL W [38] and by eye Molecular phylograms from the alignment were determined with the maximum likelihood methods in Phylip [39] Distance methods and parsimony methods were also used and gave similar results Levels of support for branches were estimated with bootstrapping methods (500 replicates) and with PHYLIP RESULTS Cloning of xTRHR cDNA subtypes from Xenopus laevis brain RT-PCR experiments were performed using brain Xenopus laevis mRNA as template and degenerated oligonucleotides designed to the conserved regions of transmembrane domains of several TRHR cloned in mammalian species Since no signal was obtained after a first PCR, a second PCR was realized with more internal oligonucleotide primers A 550-bp amplified fragment (Fig 1A) was ligated into the cloning pGEM-T easy vector Screening of 18 subclone fragments by DNA sequence analysis revealed three distinct TRHRs, xTRHR3, xTRHR2 and xTRHR1 Their relative abundances were xTRHR3  xTRHR2 > xTRHR1 The nucleotide sequence of these partial cDNAs were only 63–65% identical (xTRHR3/2: 63%; xTRHR3/1: 65%; xTRHR2/1: 64%), while their deduced amino acid sequences were 56–66% identical (xTRHR3/2: 58%; xTRHR3/1: 66%; xTRHR2/1: 56%) 5¢ and 3¢ amplification of cDNA ends (see Experimental procedures, Fig 1B,C) gave the full-length cDNAs of these TRHR subtypes (Fig 2) The sequence of xTRHR3 contained a 1215-bp open reading frame encoding a protein of 404 amino acid residues with a theoretical molecular weight of 45.5 kDa Hydropathy analysis using the Kyte and Doolittle algorithm [40], predicted seven transmembrane domains, in agreement with the topology proposed for other G protein-coupled receptors [41] The deduced amino acid sequence contained three potential sites for N-linked glycosylation (N-X-S/T) in the N-terminus at positions 3, 14 and 19 (Fig 3) Interestingly, Asn19 also represents a potential glycosylation site that is absent in mammalian and chicken TRH receptors The glycosylation site in EL2 (extracellular loop 2) of the mammalian receptor was not found in xTRHR3, as for chicken TRHR The amphibian receptor had several amino acids that are highly conserved in mammals These included all the putative residues that interact with TRH (Tyr113, Asn117, Tyr287 and Arg311), and the two Cys residues (105 and 186) that form a disulfide bond between EL1 and EL2 to maintain the receptor in a high affinity conformational state Several Ser and Thr residues were also present in the C-terminus and IL3 (intracellular loop 3) regions of the Xenopus receptor These may be sites for phosphorylation by protein kinases However, only one of the two homologous Cys residues that may be palmitoylated in the mouse receptor was found in the C-terminal tail of xTRHR3 (Cys342) The complete nucleotide sequences of xTRHR2 and xTRHR1 were obtained with the same strategy as that used for xTRHR3 The nucleotide sequences of the translated region of xTRHR2 (1206 bp) and xTRHR1 (1194 bp) cDNAs are shown in Fig These sequences encode a seven transmembrane domain protein of 401 amino acids (45.2 kDa) for xTRHR2 and 397 amino acids (45.0 kDa) for xTRHR1 Alignment of the deduced amino acid sequences with that of xTRHR3 (Fig 3) showed that xTRHR2 and xTRHR1 contained most of the amino acid residues that are conserved in other TRH receptors, but 4570 I Bidaud et al (Eur J Biochem 269) Ó FEBS 2002 Fig Nucleotide sequence of the three Xenopus TRHR cDNA subtypes The alignment (CLUSTAL W) of the complete translated sequences starting at ATG is shown Asterisks (*) indicate identical nucleotides between the three cDNA sequences differed in several respects from xTRHR3 xTRHR1 had only two potential sites for N-linked glycosylation in its N-terminus, at the conserved positions (3 and 10), while xTRHR2 had these sites at positions and 12 The glycosylation site in EL2 (Asn167 for xTRHR1 and Asn172 for xTRHR2) and the two homologous Cys residues (335 and 337 for xTRHR1, 339 and 341 for xTRHR2) in the C-terminal tail were also found The three Xenopus TRHR subtypes were found to be only 54–62% identical (62–63% for the nucleotide sequence) The N-termini, the IL3, and the C-termini of the three Xenopus subtypes contained important differences, and were only 16–30% (N-term), 25–47% (IL3) and 27– 40% (C-term) identical (Table 1) These regions also differed markedly from the known TRH receptors, especially xTRHR3 and xTRHR2 This is particularly interesting considering the functional importance of the third intracellular loop and the C-terminal tail in receptor coupling and regulation The amphibian EL1, IL1, IL2, and EL3 regions were only 53–80%, 67–100%, 62–87%, and 50–80% identical to those of mammalian TRHR1, whereas these regions of the mammalian type receptors are identical xTRHR2 was 63% identical to mouse TRHR2, 57% identical to the rat TRHR2, and 51% identical to fish TRHR2 However, if the most divergent regions of the xTRHRs (i.e N-term, IL3 and C-term) are excluded, xTRHR2 seems to belong to the TRHR subfamily because it is significantly similar to the rat, mouse and fish TRHR2 in EL1 (73–87% identity), EL2 (64–68%), EL3 (50–70%), IL1 (50–83%), and IL2 (81–94%) xTRHR1 is closer to the TRHRs subtype with 66–78% identity Our data indicate that xTRHR3 is only 58–62% identical to the TRHR1 family (including xTRHR1) and only 54%, 47%, 61% and 43% identical to the Xenopus, rat, mouse and fish TRHR2s This observation, plus the fact that the sequences most similar to xTRHR3 found in the data banks were TRHRs, suggested that xTRHR3 is a novel TRHR subtype Functional expression of xTRHR subtypes in Xenopus oocytes and HEK-293 cells The xTRH receptors were expressed in Xenopus oocytes and the mammalian HEK293 cell line (Figs and 5) Oocytes injected with xTRH receptor cDNA days previously showed a typical Ca2+dependent Cl– current when the bath contained lM TRH (Fig 4A) This inward current consists of a large, rapid and transient response that is typical of Ca2+-dependent Cl– channels activated after stimulation of PLC and the subsequent InsP3-dependent mobilization of Ca2+ from intracellular stores Control oocytes not injected with pcDNA3.1-xTRHR (data not shown) gave no response Several TRH concentrations (0.01–10 lM) were also tested Ó FEBS 2002 TRH receptor subtypes from X laevis (Eur J Biochem 269) 4571 Fig Comparison of the deduced amino acid sequences of the three Xenopus TRHRs The alignment was prepared using CLUSTAL W Asterisks (*) indicate residues identical in the three subtypes Putative transmembrane domain helixes (bold letters) were assigned based on those of the previously cloned TRH receptors Arrows indicate the residues (Y106, N110, Y282 and R306) that are highly conserved in the other TRHRs and that interact directly with TRH The additional potential glycosylation site (Asn19) in the N-terminus and the absence of the homologous Cys335 (Arg340) in the C-tail of xTRHR3 are indicated in gray background The non conventional putative phosphorylation sites of xTRHR1 (cAMP/cGMP-dependent protein kinase) and xTRHR2 (tyrosine kinase) are indicated with dashed and solid lines Since TRH desensitized the receptor (data not shown), one dose of TRH was tested and the maximum current amplitude of each recording was measured and reported as a function of the TRH concentration (Fig 4B) The average dose–response profiles showed differences between the three xTRHR subtypes, with oocytes expressing xTRHR3 cDNA giving a particularly poor response to 0.1 lM and lM TRH Similar studies in mammalian HEK-293 cells confirmed that Xenopus TRH receptors acted via the phosphoinositide-calcium transduction pathway (Fig 5) TRH (1 and 10 lM) did not activate a Ca2+ transient in control cells not transfected with xTRHR cDNA, while ATP (10 lM), which activates P2Y receptors [42], produced Ca2+ transients in living cells The responses of HEK-293 cells transfected with the three xTRHR subtypes differed in the same way as the transfected oocytes One micromolar TRH did not trigger Ca2+ transient in cells transfected with pcDNA3.1-xTRHR3, whereas the same TRH dose produced a Ca2+ response in cells expressing xTRHR2 and xTRHR1 cDNAs Distribution of xTRHRs The distributions of xTRHRs in the brain, liver, testis, urinary bladder, stomach, ventral and dorsal skin, lung, heart and intestine were also examined No signal was obtained by Northern blotting, probably because there was too little of the Xenopus receptors, so we used RT-PCR (Fig 6) The cDNA from each organ was amplified using the two sets of degenerated primers (TRHR-1/TRHR-2 and TRHR-3/TRHR-4) that gave us the middle portion of the xTRHRs (Fig 1A) The expected fragment was found in the rat testis (positive control) but not in the rat ovary (negative control) (Fig 6A) No signal was detected in the absence of the cDNA template (data not shown) A 550-bp amplified product was observed in all the Xenopus tissues tested except the liver and the ventral skin The amount of the xTRH receptor mRNAs in these tissues was assayed using a set of primers corresponding to the Xenopus EF1a elongation factor cDNA as internal control (Fig 6B) The highest concentration of xTRHR mRNA was detected in the Xenopus brain, with a considerable amount in the intestine (Fig 6C) Similarly strong signals were obtained in the lung and heart, with a smaller signal in the testis There was much less TRHR mRNA in the urinary bladder and stomach The xTRHR subtypes were identified by purifying the 550-bp PCR product from all the Xenopus tissues, cloning them in the pGEM-T easy vector, and sequencing Sequence analysis of numerous clones indicated that the three xTRHR subtypes were present in the brain (18 clones tested: four xTRHR1, five xTRHR2 and nine xTRHR3), heart (22 clones: one xTRHR1, 17 xTRHR2 and four xTRHR3), and stomach (14 clones: nine xTRHR1, three xTRHR2 and two xTRHR3) Only xTRHR1 was present in the lung (11 clones tested), the intestine (three clones) and the urinary bladder (four clones) However, the two other subtypes could be present in these tissues We have also detected xTRHR1 and xTRHR2 in the testis and xTRHR1 in the dorsal skin DISCUSSION TRH is a powerful stimulator of TSH secretion by the anterior pituitary cells of mammals, but this function is less clear in amphibians, where TRH seems to be implicated in regulating a-MSH, thus controlling the adaptation of skin Ó FEBS 2002 4572 I Bidaud et al (Eur J Biochem 269) Table Amino acid identities in the various portions of the three TRHRs subtypes and comparison with a lower vertebrate (fish) and a mammal (mouse) containing both TRHR type and type Percentage identities were calculated by CLUSTAL W % identitya X3 N-term X3 X2 X1 EL1 X3 X2 X1 EL2 X3 X2 X1 EL3 X3 X2 X1 IL1 X3 X2 X1 IL2 X3 X2 X1 IL3 X3 X2 X1 C-term X3 X2 X1 X2 X1 M1 100 – – 16 100 – 30 22 100 40 19 55 100 – – 47 100 – 67 53 100 100 – – 61 100 – 100 – – F1 M2 F2 30 41 31 22 34 33 18 57 17 67 60 80 53 67 73 47 87 53 53 73 60 50 46 100 43 46 68 36 43 36 50 68 50 57 64 43 80 100 – 70 60 100 60 50 80 60 50 70 40 50 50 60 70 60 100 – – 50 100 – 83 67 100 83 67 100 83 67 100 33 67 33 67 83 83 100 – – 69 100 – 75 62 100 81 62 87 81 62 87 62 87 56 69 94 62 100 – – 25 100 – 47 33 100 33 31 67 41 25 60 22 35 22 30 33 27 100 – – 27 100 – 40 27 100 40 25 72 36 21 51 24 28 27 12 10 a X1, X2, X3, Xenopus TRHR subtype 1, and 3; M1, mouse TRHR1 (NM_013696); M2, mouse TRHR2 (AF283762); F1, fish TRHR1 (AF288367); F2, fish TRHR2 (AF288368) color to changes in the environment To obtain further information on the way TRH acts in this species, characterization of TRH receptors is necessary Therefore, in this study, we provide the first molecular characterization of several TRH receptors from Xenopus laevis (xTRHRs) We have cloned and functionally expressed three distinct xTRHR subtypes The specific functional properties of the recombinant xTRHRs have been analyzed in Xenopus oocytes and HEK-293 cells We also report on the distribution profiles of the xTRHR mRNAs We used a degenerate PCR cloning strategy to isolate three distinct subtypes of TRHR cDNA (xTRHR1, xTRHR2 and xTRHR3) from Xenopus brain These encode the entire sequences of the proteins The amino acid sequence of xTRHR1 is very similar (74–78% identity) to that of its mammalian subtype counterparts, indicating that it is a member of the type TRHR subfamily The dissimilarity between xTRHR2 and the two other Xenopus Fig Functional expression of xTRH receptors in Xenopus oocytes (Upper) Typical Ca2+-activated Cl– current traces obtained in xTRHR3 (upper trace), xTRHR2 (middle trace) and xTRHR1 (bottom trace) cDNA injected oocytes Xenopus oocytes were constantly perfused with ND96 solution and TRH (1 lM) was applied to oocytes for 30 s Note the fast desensitization of the responses (Lower) Responses of the three xTRHR subtypes to different concentrations of TRH The maximum current amplitude of each recording was measured and reported as a function of TRH concentration The white star indicates the average value and n represents the number of oocytes tested for each condition TRHRs and its similarity to most of the regions of the mouse, rat and fish TRHR2 indicate that xTRHR2 is a member of the recently described TRHR subfamily xTRHR3 corresponds to a novel TRHR subtype that is only 58–62% identical to the TRHR1 family, including xTRHR1, and only 54%, 47%, 61% and 43% identical to the Xenopus, rat, mouse and fish TRHR2s We analyzed the molecular evolution of TRHR transcripts from various animal species to identify the origins of the TRH receptor subtypes The molecular phylogram of TRHR sequences is not completely resolved, but two distinct clades are apparent (Fig 7) Sequences from human, sheep, ox, rat, mouse, chicken and Xenopus type Ó FEBS 2002 TRH receptor subtypes from X laevis (Eur J Biochem 269) 4573 Fig Ca2+ imaging experiments on HEK-293 cells expressing xTRHR subtypes We measured the change in Ca2+ concentration was examined in HEK-293 cells loaded with fura-2 and evaluated from the ratio of fluorescence at 340 nm and 380 mm (Rf 340/380) The average amplitude of the response of each cell was estimated by the ratio rFmax/ rFmin, where rFmax corresponds to maximum Rf 340/380 during the drug application, and rFmin corresponds to Rf 340/380 just before drug application The change in the ratio Rf 340/380 during application of TRH (1 and 10 lM) and ATP (10 lM) is shown with the corresponding average rFmax/rFmin ratios for the control and the cells expressing the different xTRHR subtypes TRH receptor cluster tightly together, suggesting that they represent orthologous loci in these species A second clade of orthologous sequences consists of type TRH receptors from rat, mouse, fish and Xenopus As shown in the phylogram, the TRHR sequences not cluster according to animal species This pattern implies that type and type TRH receptors loci originated in a common ancestor prior to the divergence of the species sampled and that concerted evolution has played a very small role in the evolution of this gene family The relationships of type and type TRHRs from Xenopus in the second clade suggest that these two loci are not the result of duplication of a Xenopus gene, but that the type receptor originated in the common ancestor of fish and amphibian Although this particular locus may now be extinct in fishes and mammals, it is more likely that the type receptor is awaiting discovery in these species The putative binding pocket identified in the transmembrane domains of the mouse receptor is completely conserved in the three Xenopus TRHR subtypes (Fig 3) The candidate residues interacting directly with TRH are Tyr106 and Asn110 in TM3, Tyr282 in TM6, and Arg306 in Fig RT-PCR distribution of Xenopus TRHR in various tissues (A) Amplification of the middle portion of xTRHR cDNA (550 bp) using the two sets of degenerated oligonucleotide primers, TRHR-1/ TRHR-2 and TRHR-3/TRHR-4 (see Fig 1A) PCR products were analyzed by agarose gel (1%) electrophoresis (B) Amplification of cDNA templates with a set of primers corresponding to the Xenopus EF1a elongation factor cDNA (280 bp) as internal control of the poly(A) + RNA (C) Tissue comparison of the level of expression of xTRHRs with samples containing the same total quantity of mRNA The cDNA templates used were from: Xenopus liver (3), brain (lane 4), testis (lane 5), urinary bladder (lane 6), stomach (lane 7), lung (lane 8), heart (lane 9), intestine (lane 10) and dorsal skin (lane 11) Rat testis (lane 1) and ovary (lane 2) were used as positive and negative controls, respectively TM7 (in Xenopus and mouse TRHR1) [41] Tyr106 and Asn110 have been reported to form hydrogen bonds with the pyroGlu residue of TRH and Arg306 with the ProNH2 Fig Molecular phylogram of nucleotide sequences of TRH receptor transcripts reconstructed by maximum likelihood methods Type TRH receptor from the teleost fish Catostomus commersoni was the most basal sequence and was used to root the tree Bootstrap values from 500 replicates greater than 50% are indicated at nodes Ó FEBS 2002 4574 I Bidaud et al (Eur J Biochem 269) residue Tyr282 was reported to interact hydrophobically with the imidazole ring of TRH Other residues are highly conserved in the three Xenopus TRH receptors These include the two Cys residues 98 and 79 (in Xenopus and mouse TRHR1), said to form a disulfide bond between EL1 and EL2 to maintain the TRH receptor in a high-affinity conformational state [43] The residues Asp71 and Arg283 that are necessary for receptor activation [44,45] are also present These residues are thought to form ionic or hydrogen bonds with other TM residues to keep the receptor in the active conformation after TRH binds Altogether these data indicate that these novel G proteincoupled receptors are clearly TRH receptors An important finding of this study is the description of a novel TRH receptor subtype that does not belong to the subtypes and of TRHR This xTRHR3 subtype has several distinctive features This is the only TRH receptor that contains an additional potential glycosylation site in the N-terminus (Asn19) xTRHR3 lacks the glycosylation site in EL2, as the chicken, fish (type and 2), rat (type 2) and mouse (type 2) TRH receptors Glycosylation may play a role in the receptor expression or stability [46] Another feature of TRHRs is the presence of two Cys residues in their C-terminal tails that are observed in xTRHR1 (Cys335 and 337) and xTRHR2 (Cys339 and 341) By contrast, only one of these residues (Cys342) corresponding to the homologous Cys337 is present in xTRHR3 (also in fish TRHR2) Since palmitoylation of homologous Cys may be necessary for optimal interaction with the internalization machinery [47], it is tempting to suggest that xTRHR3 might be differently processed in the cell machinery The C-terminal region of the chicken and mammalian TRHR1 contains another residue, Phe363 (in mouse TRHR1), which may be important in signaling endocytosis [3] This residue is present at position 369 in xTRHR1 but is not found in the two other Xenopus TRHR subtypes; it is also absent from fish TRHR1 and rat, mouse and fish TRHR2 There are unconventional putative phosphorylation sites in the Xenopus TRH receptors The C-terminal tail of xTRHR1 contains a putative phosphorylation site for cAMP/cGMP-dependent protein kinase (R/K-R/K-X-S/T) at position 339 (KKRS); this is also found in fish TRHR1, but in IL3 (KKDS at position 235) xTRHR2 contains a putative tyrosine kinase phosphorylation site (R/K-XX or XXX-D/E-XX or XXX-Y) in the C-tail (KAGPEGDLY at position 389) xTRHR2 also has two putative casein kinase II phosphorylation sites that are not found in IL3 of the TRH receptor (also one in fish TRHR2) Altogether these data greatly contribute to the understanding of the molecular blueprint of the Xenopus TRH receptors and further indicate that differential regulations of the xTRHR subtypes may participate to their physiological functions RT-PCR analyses showed that the TRH receptors are present in the central and peripheral tissues of Xenopus laevis An in situ hybridization study is in progress to accurately determine the anatomical distribution of the three xTRHR subtype mRNAs in the Xenopus brain Previous studies revealed that mammalian TRHR2 is more widely distributed in the central nervous system than is TRHR1 [30,33,34], suggesting that TRHR2 mediates many of the known functions in the brain that are not transduced by TRHR1 In the Xenopus peripheral tissues, the intestine contains the highest concentration of xTRHR mRNA The heart and the stomach contain the three xTRHRs, but xTRHR2 is most abundant in the heart and xTRHR1 in the stomach We also found xTRHR1 and xTRHR2 in the testis and xTRHR1 in the dorsal skin Interestingly, xTRHR3 is weakly expressed in the peripheral tissues, while xTRHR1 seems to be specific to the intestine, lung, and urinary bladder The physiological functions mediated by the three Xenopus TRHR subtypes in the central nervous system and in the peripheral tissues remain to be elucidated Using functional expression strategies, we finally demonstrate that the three xTRHRs are fully functional when expressed either in Xenopus oocytes or in mammalian HEK-293 cells Typical Ca2+-dependent Cl– currents were recorded when TRH was added Xenopus oocytes expressing xTRHRs Similarly, in transfected HEK-293 cells, a TRHinduced intracellular Ca2+ response was also observed, indicating that the Xenopus TRH receptors are coupled to the PLC/ InsP3 pathway All three receptors produced a rapidly desensitizing response following TRH application Interestingly, activation of xTRHR3 in both Xenopus oocytes and mammalian cells required larger concentrations of TRH to produce Ca2+-dependent responses comparable to those produced by xTRHR1 and xTRHR2 This lower response is probably not due to the vector itself since the response of the two other subtypes would also be affected, suggesting rather for xTRHR3 a lower stability or affinity for TRH Although our results indicate that xTRHR3 contains all the structural characteristics of the TRHR receptors, we effectively cannot exclude that xTRHR3 is an orphan receptor Pharmacological experiments will be necessary to assess if the weak effect of TRH observed for xTRHR3 corresponds to a low expression (Bmax) or affinity (Kd) Current work is in 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(5¢-GGGGGTGTAGAGGTTTCTGGAGAC-3¢); third set, A5 3 (5¢-CGAGAGGAGCATTAGA-TAGATG Ĩ FEBS 2002 CAG-3¢)/TRHR-9 (5¢-GCCGAAATGTTGATGCCCA GATAC-3¢) (Fig... CGTAACTTTTGCTG-3¢)/TRHR1-4 antisense (5¢-TC TGTTAAATGTACCTAAGTAGGCA-3¢) and TRHR2-2 sense (5¢-CAGCAAAATGGAAAATAGTAGC-3¢)/ TRHR2-4 antisense (5¢-CGACACTGTAGTAG-AGAT CACC-3¢), respectively The PCR... 31 Itadani, H., Nakamura, T., Itoh, J., Iwaasa, H., Kanatani, A. , Borkowski, J., Ihara, M & Ohta, M (1998) Cloning and characterization of a new subtype of thyrotropin-releasing hormone receptors