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

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

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

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

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/

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

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

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.

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:

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-

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

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

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.

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

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

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

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+

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