Báo cáo khoa học: Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopus laevis Identification of a novel subtype of thyrotropin-releasing hormone receptor

doi:10.1046/j.1432-1033.2002.03152.x

Eur. J. Biochem. 269, 4566–4576 (2002) (cid:2) FEBS 2002

Characterization and functional expression of cDNAs encoding thyrotropin-releasing hormone receptor from Xenopuslaevis Identification of a novel subtype of thyrotropin-releasing hormone receptor

Isabelle Bidaud1, Philippe Lory2, Pierre Nicolas1, Marc Bulant1 and Ali Ladram1 1Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, CNRS-Universite´ Paris, Paris; 2Institut de Ge´ne´tique Humaine, CNRS-UPR 1142, Montpellier, France

awaiting discovery in other animal species. The three Xeno- pus 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 1 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. Inter- estingly, activation of xTRHR3 required larger concentra- tions of TRH compared with the other two receptors, suggesting marked differences in receptor binding, coupling or regulation.

Keywords: thyrotropin-releasing hormone receptors; sub- types; amphibian; cloning; functional expression.

Thyrotropin-releasing hormone receptor (TRHR) has already been cloned in mammals where thyrotropin-releas- ing 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 dege- nerate 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 1 and xTRHR2 belongs to subfamily 2, while xTRHR3 is a new TRHR subtype

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]. Regu- lation 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-melanocyte- stimulating hormone (a-MSH) secretion by pituitary mel- anotrope 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, 2 place Jussieu, 75251 Paris cedex 05, France. Fax: + 33 1 44275994, Tel.: + 33 1 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-triphos- phate; SLIC, single-strand ligation of cDNA; TM, transmembrane domain; TRH, thyrotropin-releasing hormone; TRHR, thyrotropin- releasing hormone receptor; TSH, thyroid-stimulating hormone. Proteins and enzymes: thyrotropin-releasing hormone (THYL_PIG); thyrotropin-releasing hormone precursor (Q62361); thyrotropin- releasing 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-stimula- ting 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 8 July 2002, accepted 30 July 2002)

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1

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 anter- ior 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 2 TRHR subfamily (TRHR2) was discov- ered 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.

E X P E R I M E N T A L P R O C E D U R E S

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).

CA, USA). The mixture was incubated for 60 min at 42 (cid:3)C, heated for 5 min at 94 (cid:3)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 (cid:3)C for 45 s, 45 (cid:3)C for 1 min, and 72 (cid:3)C for 3 min, followed by a final extension at 72 (cid:3)C for 10 min. Five microliters of this amplified mixture was then submitted to nested PCR using more internal degen- erated 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

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 syn- thesized 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 contain- ing 1X reaction buffer (50 mM Tris/HCl, pH 8.3; 75 mM KCl; and 3 mM MgCl2), each deoxy-NTP at 0.5 mM, ribonuclease inhibitor (0.5 U), and Moloney murine leuke- mia virus reverse transcriptase (200 U; Clontech, Palo Alto,

Fig. 1. Diagram of the xTRHR cDNA, PCR primers and PCR prod- ucts. (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 oligonu- cleotide 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 full- length xTRHR3 cDNA. A fragment of the receptor starting from the 5¢-end and ending in the middle of the transmembrane domain 6 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.

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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.

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.

2

corresponding to the

Construction of full-length xTRHR cDNAs. We used the following strategy as we were unable to amplify the full- length 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 (Fig. 1D). (5¢-GTTTTGGGGTGGATTAAGGTAG-3¢) An 816-bp amplified fragment was purified. A mixture of the this cDNA fragment and the 3¢-region of xTRHR3 cDNA was then used in a second PCR with specific oligonucleotide primers TRHR-10 and the (5¢-CTACGCCACACTGTATGTTGTC-3¢) TRHR-11 (Fig. 1D). All the PCR experiments were done as described above with hybridization temperatures of 46 and 48 (cid:3)C for the first and second PCR, respectively. A full-length 1400-bp fragment xTRHR3 cDNA was finally purified, cloned into the pGEM-T easy vector, and sequenced in both directions using T7 and Sp6 primers.

TRHR1-2

antisense

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 Technol- ogies) in 20 lL containing 1X reaction buffer (20 mM Tris/ HCl, pH 8.4; 50 mM KCl), 2.5 mM MgCl2, each deoxy- NTP 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 (cid:3)C for 50 min and stopped by incubation at 70 (cid:3)C for 15 min and quickly placing the tubes on ice. The mixture was incubated with ribonuclease H for 20 min at 37 (cid:3)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 ampli- fied 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.

Full-length cDNAs corresponding to the xTRHR1 and xTRHR2 subtypes were amplified as described above using partially overlapping cDNA fragments and the pair of (5¢-ATAATGGATAA primers sense CGTAACTTTTGCTG-3¢)/TRHR1-4 (5¢-TC TGTTAAATGTACCTAAGTAGGCA-3¢) and TRHR2-2 (5¢-CAGCAAAATGGAAAATAGTAGC-3¢)/ sense 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

[37],

standard procedures

Xenopus oocytes were isolated, prepared and maintained and microinjected using and with pcDNA3.1-xTRHR1, pcDNA3.1-xTRHR2 pcDNA3.1-xTRHR3 (approximately 10 ng of plasmid/ oocyte). Whole cell currents were measured 2 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, 2 KCl, 1 MgCl2, 2 CaCl2 and 5 Hepes (pH 7.4). The holding potential was )80 mV. Data acquisition and analysis were monitored by the pCLAMP7 suite (Axon Instruments).

5¢ Single-strand ligation of cDNA [36] (5¢-SLIC) experi- ments 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 (cid:3)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 anti- sense 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 (5¢-CGAGAGGAGCATTAGA-TAGATG set, A53

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Calcium imaging experiments in HEK-293 cells

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.

R E S U L T S

Cloning of xTRHR cDNA subtypes from Xenopus laevis brain. RT-PCR experiments were performed using brain Xenopus laevis mRNA as template and degenerated oligo- nucleotides designed to the conserved regions of transmem- brane 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 (cid:2) 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%).

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 trans- fection with pcDNA3.1-xTRHR, cells were trypsinized and plated onto polyornithine-coated Laboratory-Tek borosili- cate 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 (cid:3)C in the dark for 30 min in Locke buffer containing (in mM): 140 NaCl, 5 KCl, 1.2 KH2PO4, 1.2 MgSO4, 2 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 monochro- mator (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. RT- PCR experiments were performed under the same condi- tions 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 approxi- mately 280 bp product as an internal control.

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 transmem- brane 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).

Phylogenetic analysis

rats

commersoni

fish Catostomus

3

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. 2. 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

The nucleotide sequence of TRH receptors from humans (GenBank accession number NM_003301), sheep (X95285), oxen (D83964), (NM_013047, AF091715), mice (NM_013696, AF283762), chickens (Y18244) and the teleost (AF288367, AF288368) were obtained from GenBank. The nucleotide sequences of the TRHR transcripts were aligned with [38] and by eye. Molecular phylograms from the CLUSTAL W alignment were determined with the maximum likelihood

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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 3 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.

xTRHR2 seems to belong to the TRHR subfamily 2 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 1 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.

identical

The three Xenopus TRHR subtypes were found to be only 54–62% identical (62–63% for the nucleotide se- quence). 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) (Table 1). These regions also differed markedly from the known TRH receptors, especi- ally xTRHR3 and xTRHR2. This is particularly interesting considering the functional importance of the third intracel- lular 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 1 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,

Functional expression of xTRHR subtypes in Xenopus oocytes and HEK-293 cells. The xTRH receptors were expressed in Xenopus oocytes and the mammalian HEK- 293 cell line (Figs 4 and 5). Oocytes injected with xTRH receptor cDNA 2 days previously showed a typical Ca2+- dependent Cl– current when the bath contained 1 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.

4 of the complete translated sequences starting at Fig. 2. Nucleotide sequence of the three Xenopus TRHR cDNA subtypes. The alignment (CLUSTAL W) ATG is shown. Asterisks (*) indicate identical nucleotides between the three cDNA sequences.

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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 1 lM TRH. Similar studies in mammalian HEK-293 cells confirmed that Xenopus TRH receptors acted via the phosphoinositide-calcium transduction path- way (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.

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.

Fig. 3. 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 con- served 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 indi- cated in gray background. The non conven- tional putative phosphorylation sites of xTRHR1 (cAMP/cGMP-dependent protein kinase) and xTRHR2 (tyrosine kinase) are indicated with dashed and solid lines.

D I S C U S S I O N

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

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

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6 Table 1. 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 1 and type 2. Percentage identities were calculated by CLUSTAL W .

% identitya

X3 X2 X1 M1 F1 M2 F2

100 – – 16 100 – 30 22 100 40 19 55 30 41 31 22 34 33 18 57 17

100 – – 47 100 – 67 53 100 67 60 80 53 67 73 47 87 53 53 73 60

100 – – 61 100 – 50 46 100 43 46 68 36 43 36 50 68 50 57 64 43

N-term X3 X2 X1 EL1 X3 X2 X1 EL2 X3 X2 X1 EL3 X3 X2 X1 100 – – 80 100 – 70 60 100 60 50 80 60 50 70 40 50 50 60 70 60 IL1

X3 X2 X1 100 – – 50 100 – 83 67 100 33 67 33 67 83 83 83 67 100 83 67 100 IL2

X3 X2 X1 100 – – 69 100 – 75 62 100 81 62 87 81 62 87 62 87 56 69 94 62 IL3

100 – – 25 100 – 47 33 100 33 31 67 41 25 60 22 35 22 30 33 27

a X1, X2, X3, Xenopus TRHR subtype 1, 2 and 3; M1, mouse TRHR1 (NM_013696); M2, mouse TRHR2 (AF283762); F1, fish TRHR1 (AF288367); F2, fish TRHR2 (AF288368).

X3 X2 X1 C-term X3 X2 X1 100 – – 27 100 – 40 27 100 40 25 72 36 21 51 24 28 27 8 12 10

color to changes in the environment. To obtain further information on the way TRH acts in this species, charac- terization 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.

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 2. 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 tran- scripts 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 1

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 1 counterparts, indicating that it is a member of the type 1 TRHR subfamily. The dissimilarity between xTRHR2 and the two other Xenopus

Fig. 4. 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 (bot- tom 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 meas- ured 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.

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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. 6. 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.

TRH receptor cluster tightly together, suggesting that they represent orthologous loci in these species. A second clade of orthologous sequences consists of type 2 TRH receptors from rat, mouse, fish and Xenopus. As shown in the phylogram, the TRHR sequences do not cluster according to animal species. This pattern implies that type 1 and type 2 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 3 and type 2 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 3 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 3 receptor is awaiting discovery in these species.

Fig. 5. Ca2+ imaging experiments on HEK-293 cells expressing xTRHR subtypes. We measured the change in Ca2+ concentration was exam- ined 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.

The putative binding pocket identified in the transmem- brane 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. 7. Molecular phylogram of nucleotide sequences of TRH receptor transcripts reconstructed by maximum likelihood methods. Type 1 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.

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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 protein- coupled receptors are clearly TRH receptors.

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 demon- strate 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 TRH- induced 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 progress to elucidate these issues. Overall, this study demonstrates that expression of distinct TRH receptors can account for the specific features of the TRH signaling in Xenopus oocytes and further suggests the existence of a third TRHR subtype that has yet to be identified in other species.

A C K N O W L E D G M E N T S

The authors thank Drs J. Moreau and T. Foulon for their expert assistance, and Dr M.C. Gershengorn for a critical reading of this manuscript. This work was funded entirely by the Centre National de la Recherche Scientifique (CNRS).

R E F E R E N C E S

An important finding of this study is the description of a novel TRH receptor subtype that does not belong to the subtypes 1 and 2 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 do the chicken, fish (type 1 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 the data greatly contribute to the understanding of molecular blueprint of the Xenopus TRH receptors and further indicate that differential regulations of the xTRHR subtypes may participate to their physiological functions.

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