Characterization and functional expression of cDNAs encodingIdentification of a novel subtype of thyrotropin-releasing hormone receptor Isabelle Bidaud1, Philippe Lory2, Pierre Nicolas1,
Trang 1Characterization and functional expression of cDNAs encoding
Identification of a novel subtype of thyrotropin-releasing hormone receptor
Isabelle Bidaud1, Philippe Lory2, Pierre Nicolas1, Marc Bulant1and Ali Ladram1
1 Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, CNRS-Universite´ Paris, Paris; 2 Institut de Ge´ne´tique Humaine, CNRS-UPR 1142, Montpellier, France
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
awaiting discovery in other animal species The three Xeno-pusTRHRs 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 (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; InsP 3 , inositol
1,4,5-triphos-phate; SLIC, single-strand ligation of cDNA; TM, transmembrane
domain; TRH, releasing hormone; TRHR,
thyrotropin-releasing hormone receptor; TSH, thyroid-stimulating hormone.
Proteins and enzymes: thyrotropin-releasing hormone (THYL_PIG);
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)
Trang 2from 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
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)
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:
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,
CA, USA) The mixture was incubated for 60 min at 42C, heated for 5 min at 94C, 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 mMTris/ HCl, pH 8.3; 50 mMKCl; and 1.5 mMMgCl2), 0.5 mMof 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 94C for 45 s, 45 C for 1 min, and 72C for 3 min, followed by a final extension at 72 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
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.
Trang 3purified (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
primer (5¢-GGCCACGCGTCGACTAGTACTTTTTTTT
TTTTTT-TT-3¢; final concentration: 0.5 lM, Life
Technol-ogies) in 20 lL containing 1X reaction buffer (20 mMTris/
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 42C
for 50 min and stopped by incubation at 70C for 15 min
and quickly placing the tubes on ice The mixture was
incubated with ribonuclease H for 20 min at 37C 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
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
22C 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
GAGACCATACAGAAC-C-3¢); second set, A52
(5¢-AGAGTGAGCAGGTAGCGAGAGGAG-3¢)/TRHR-8
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 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 (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
(Fig 1D) All the PCR experiments were done as described above with hybridization temperatures of 46 and 48C 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
CGTAACTTTTGCTG-3¢)/TRHR1-4 antisense (5¢-TC TGTTAAATGTACCTAAGTAGGCA-3¢) and TRHR2-2
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 inXenopus 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 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)
Trang 4Calcium 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
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 37C 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
MERLINsoftware (LSR, Cambridge UK) to a
monochro-mator (Spectramaster) and a 12/14 bit frame transfert rate
digital camera (Astrocam).MERLINsoftware was also used
to calculate the 340/380 fluorescence ratio (Rf) The
intensity of fluorescent light emission (k¼ 510 nm) using
excitation at 340 and 380 nm was monitored for each single
fura-2 loaded cell in the field TRH (1 lMand 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
XenopusEF1a elongation factor that generates an
approxi-mately 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
3 [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 withPHYLIP
R E S U L T S Cloning of xTRHR cDNA subtypes from Xenopus laevis brain RT-PCR experiments were performed using brain Xenopus laevismRNA 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 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 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)
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
Trang 5differed 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
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) identical (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,
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
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 lMTRH (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 l ) were also tested
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.
Trang 6Since 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
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 Xenopustissues 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 XenopusEF1a 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 Xenopustissues, 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
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
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.
Trang 7color 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
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
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
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
N-term
EL1
EL2
EL3
IL1
IL2
IL3
C-term
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).
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 l M ) 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.
Trang 8TRH 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
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
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 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 rF max /
rF min , where rF max corresponds to maximum Rf 340/380 during the
drug application, and rF min corresponds to Rf 340/380 just before drug
application The change in the ratio Rf 340/380 during application of
TRH (1 and 10 l M ) and ATP (10 l M ) is shown with the corresponding
average rF max /rF min ratios for the control and the cells expressing the
different xTRHR subtypes.
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.
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.
Trang 9residue 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
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
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 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).
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