Tài liệu 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 ppt

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 Bidaud
1
, Philippe Lory
2
, Pierre Nicolas
1
, Marc Bulant
1
and Ali Ladram
1
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-
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
andxTRHR2intheheart.TheXenopus 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 (InsP
3
) 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:
Abbreviations: a-MSH, a-melanocyte-stimulating hormone; EL,
extracellular loop; IL, intracellular loop; InsP
3
,inositol1,4,5-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)
Eur. J. Biochem. 269, 4566–4576 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03152.x
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 GH
3
1
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.
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 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 m
M
Tris/HCl, pH 8.3; 75 m
M
KCl; and 3 m
M
MgCl
2
), each deoxy-NTP at 0.5 m
M
,
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 42 °C,
heated for 5 min at 94 °C,anddilutedwithwaterto100 lL.
An aliquot (5 lL) of the brain cDNA mixture was amplified
by PCR in 50 lL containing 1X PCR buffer (10 m
M
Tris/
HCl, pH 8.3; 50 m
M
KCl; and 1.5 m
M
MgCl
2
), 0.5 m
M
of
each deoxy-NTP, TRHR-1 and TRHR-2 degenerated
primers (0.4 l

M
each), and 0.2 U AmpliTaq DNA
polymerase (Applied Biosystems). We used a 30-cycle
program consisting of 94 °C for 45 s, 45 °Cfor1min,
and 72 °C 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
fragmentinassociationwiththe3¢-end one and the specific primers
TRHR10 and TRHR11.
Ó FEBS 2002 TRH receptor subtypes from X. laevis (Eur. J. Biochem. 269) 4567

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
2
adapter
primer (5¢-GGCCACGCGTCGACTAGTACTTTTTTTT
TTTTTT-TT-3¢; final concentration: 0.5 l
M
, Life Technol-
ogies) in 20 lL containing 1X reaction buffer (20 m
M
Tris/

HCl, pH 8.4; 50 m
M
KCl), 2.5 m
M
MgCl
2
, each deoxy-
NTP at 0.5 m
M
,10m
M
dithiothreitol, and SuperScript II
transcriptase reverse (200 U, Life Technologies). The
reaction was initiated by incubating the mixture at 42 °C
for 50 min and stopped by incubation at 70 °Cfor15min
and quickly placing the tubes on ice. The mixture was
incubated with ribonuclease H for 20 min at 37 °Cto
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 l
M
) and the antisense abridged universal amplification
primer (AUAP: 5¢-GGCCACGCGTCGACTAGTAC-3¢;
0.2 l
M
; 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-NH
2
-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 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
set, A53 (5¢-CGAGAGGAGCATTAGA-TAGATG
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 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
xTRHR3cDNAwasthenusedinasecondPCRwith
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¢)andTRHR2-2

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 2 days later
using a two-microelectrode voltage clamp technique
(Genclamp, Axon Instruments). The activity of the
Ca
2+
-activated chloride channel was recorded using a
standard calcium/chloride solution containing (in m
M
): 96
NaCl, 2 KCl, 1 MgCl

2
, 2 CaCl
2
and 5 Hepes (pH 7.4).
The holding potential was )80 mV. Data acquisition and
analysis were monitored by the pCLAMP7 suite (Axon
Instruments).
4568 I. Bidaud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 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 Ca
2+
, cells were incubated
with 2.5 l
M
of the acetoxymethyl ester derivative of the
dual-excitation ratiometric Ca
2+
sensitive indicator fura-2
(Molecular Probes) at 37 °C in the dark for 30 min in Locke
buffer containing (in m
M
): 140 NaCl, 5 KCl, 1.2 KH
2

PO
4
,
1.2 MgSO
4
, 2 CaCl
2
, 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 l
M
and 10 l
M
)and
ATP (10 l
M
) 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.
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 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 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
Ó FEBS 2002 TRH receptor subtypes from X. laevis (Eur. J. Biochem. 269) 4569
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.
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%
identicaltotheratTRHR2,and51%identicaltofish
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 Ca
2+
-
dependent Cl
–

current when the bath contained 1 l
M
TRH
(Fig. 4A). This inward current consists of a large, rapid and
transient response that is typical of Ca
2+
-dependent Cl
–
channels activated after stimulation of PLC and the
subsequent InsP
3
-dependent mobilization of Ca
2+
from
intracellular stores. Control oocytes not injected with
pcDNA3.1-xTRHR (data not shown) gave no response.
Several TRH concentrations (0.01–10 l
M
)werealsotested.
Fig. 2. Nucleotide sequence of the three Xenopus TRHR cDNA subtypes. The alignment (
CLUSTAL W
)
4
of the complete translated sequences starting at
ATG is shown. Asterisks (*) indicate identical nucleotides between the three cDNA sequences.
4570 I. Bidaud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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 l
M
and 1 l
M
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 l
M
)didnotactivateaCa
2+
transient in control cells not transfected with xTRHR
cDNA, while ATP (10 l
M
), which activates P2Y receptors
[42], produced Ca
2+
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 Ca
2+
transient in cells
transfected with pcDNA3.1-xTRHR3, whereas the same
TRH dose produced a Ca
2+
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
Fig. 3. Comparison of the deduced amino acid
sequences of the three Xenopus TRHRs. The
alignment was prepared using
CLUSTAL W
5
.
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.
Ó FEBS 2002 TRH receptor subtypes from X. laevis (Eur. J. Biochem. 269) 4571
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.
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
6
.
% identity
a
X3 X2 X1 M1 F1 M2 F2
N-term
X3 100 16 30 40 30 22 18
X2 – 100 22 19 41 34 57
X1 – – 100 55 31 33 17
EL1
X3 100 47 67 67 53 47 53
X2 – 100 53 60 67 87 73
X1 – – 100 80 73 53 60
EL2
X3 100 61 50 43 36 50 57
X2 – 100 46 46 43 68 64
X1 – – 100 68 36 50 43
EL3
X3 100 80 70 60 60 40 60

X2 – 100 60 50 50 50 70
X1 – – 100 80 70 50 60
IL1
X3 100 50 83 83 83 33 67
X2 – 100 67 67 67 67 83
X1 – – 100 100 100 33 83
IL2
X3 100 69 75 81 81 62 69
X2 – 100 62 62 62 87 94
X1 – – 100 87 87 56 62
IL3
X3 100 25 47 33 41 22 30
X2 – 100 33 31 25 35 33
X1 – – 100 67 60 22 27
C-term
X3 100 27 40 40 36 24 8
X2 – 100 27 25 21 28 12
X1 – – 100 72 51 27 10
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 Ca
2+
-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.
4572 I. Bidaud et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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.
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 ProNH
2
Fig. 5. Ca
2+
imaging experiments on HEK-293 cells expressing xTRHR
subtypes. We measured the change in Ca
2+
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
,whererF
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
)andATP(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.
Ó FEBS 2002 TRH receptor subtypes from X. laevis (Eur. J. Biochem. 269) 4573
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.
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.Anin situ hybridization study is in progress to
accurately determine the anatomical distribution of the
threexTRHRsubtypemRNAsintheXenopus 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 Ca
2+
-dependent Cl
–
currents were
recorded when TRH was added Xenopus oocytes expressing
xTRHRs. Similarly, in transfected HEK-293 cells, a TRH-
induced intracellular Ca
2+
response was also observed,
indicating that the Xenopus TRH receptors are coupled to
the PLC/ InsP
3
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 Ca
2+

-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 (B
max
) or affinity
(K
d
). 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.
ACKNOWLEDGMENTS
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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