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J Biol Chem, Vol. 273, Issue 48, 32281-32287, November 27, 1998
From Astra Research Centre Montreal, 7171 Frederick-Banting, Ville
St Laurent, Quebec H4S 1Z9, Canada
A cDNA encoding a thyrotropin-releasing
hormone (TRH) receptor expressed in the pituitary was previously cloned
(De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado,
L. M., Del Camino, D., and Barros, F. (1992) J. Biol.
Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L.,
Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell
Endocrinol. 95, R11-R15). We now describe the isolation of a rat
cDNA encoding a novel subtype of TRH receptor (termed TRHR2)
displaying an overall homology of 50% to the pituitary TRH receptor.
Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression
of high affinity TRH binding with a different pharmacological profile
than the pituitary TRH receptor. De novo expressed
receptors were functional and resulted in stimulation of calcium
transient as assessed by fluorometric imaging plate reader analysis.
The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of
TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior
pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be
more restricted to the central nervous system particularly to the
sensory neurons of spinothalamic tract and spinal cord dorsal horn,
which may account for the sensory antinociceptive actions of TRH.
The thyrotropin-releasing hormone
(TRH)1 is a tripeptide
(pyroglutamic acid-histidine-proline-amide) synthesized from a
precursor polypeptide whose sequence contains 5 copies of the TRH
sequence (5-7).
Originally isolated from the hypothalamus, TRH is present in the
central nervous system (thalamus, cerebral cortex, and spinal cord,) as
well as in the periphery (pancreas, gastrointestinal tracts, and
placenta). In the hypothalamus, TRH is synthesized by peptidergic
neurons of supraoptic and paraventricular nuclei. It is then axonally
transported to be stored in the median eminence. When secreted in the
bloodstream, it reaches the pituitary where it stimulates the
production of thyroid stimulating hormone which in turn stimulates the
production of thyroxin (T4) in the thyroid gland (8).
In addition to this pivotal role in controlling the synthesis and
secretion of thyroid stimulating hormone and other hormones from the
anterior pituitary, TRH has been implicated as a neurotransmitter (9).
TRH abundantly exists in the central nervous system and exogenous
administration of TRH elicits a variety of behavioral changes (see Ref.
10 for a review).
The distribution of TRH containing cells, fibers, or receptors suggests
a potential role of TRH in the perception of noxious stimuli. TRH is
present in the periaqueductal gray, the nuclei raphe magnus and
pallidus, and the dorsal horn of the spinal cord. TRH-binding sites
have been described in the brain, the pituitary, in both the dorsal and
ventral horns of the spinal cord as well as in peripheral tissues. When
injected centrally, TRH induces a short lasting supraspinal
antinociception. The analgesia induced by intracerebroventricular TRH
injection is powerful since it is twice as great, on a molar basis, as
that of morphine (11). This TRH-induced antinociception is detected in
models of chemically and mechanically, but not thermally induced pain.
On the other hand, intrathecal TRH injections do not affect basal
antinociceptive thresholds (11). However, it is known that TRH enhances
spinal reflexes (both in vivo and in vitro) and
modulates pain transmission (4, 12, 13). Although the mode of action of
TRH at the level of the spinal cord is unclear, there is evidence
suggesting that the TRH-induced facilitation of spinal transmission
involves the activation of the
N-methyl-D-asparatate receptor (14).
TRH actions are mediated by the stimulation of specific cell surface
receptors. Studies of pituitary TRH receptors have suggested that the
TRHR1 receptor triggers the phospholipase C-protein kinase C
transduction pathway (3, 15). A cDNA sequence encoding a
G-protein-coupled TRHR was originally isolated from mouse pituitary using an expression cloning strategy (15). Subsequently, several groups
have described the cloning of rat TRH receptor cDNAs expressed in a
pituitary tumor cell line (GH3) or in the pituitary gland (1, 3). In addition, two isoforms of the rat TRHR have been shown to be
generated from a single gene by alternative splicing (2). These
isoforms are 387 and 412 amino acids in length and they differ in their
C-terminal cytoplasmic tail. Functional expression in
Xenopus laevis oocytes indicated that both
cDNAs encode functional TRHRs with indistinguishable
electrophysiological responses to TRH (16). Using an homology screening
approach, a human TRHR cDNA was also cloned (17). The predicted
amino acid sequence of the human TRHR showed high homology with the rat
and mouse TRHRs with the exception of the C-terminal region (17). The level of sequence homologies between the putative protein sequences encoded by these cDNAs, excluding the C-terminal sequence, is more
than 93%. It seems therefore that the currently available cloned TRHR
receptor cDNAs encode species variants of the same functional
subtype of TRH receptor.
Pharmacological observations, however, suggest the existence of TRH
receptor subtypes. A dissociation of endocrine from CNS effects was
observed using synthetic TRH analogues. Indeed some TRH analogues
containing the C-terminal thioamide group and norvaline or norleucine
in position 2 neither bind to pituitary nor to brain receptors and
generate no thyroid stimulating hormone release activity. Yet, both
analogues affected sleeping time and breathing frequency (18). The
differential effects evoked by TRH analogues with a modified COOH
terminus, compared with TRH analogues that have an intact COOH
terminus, further support the existence of TRH receptor subtypes (19).
Comparatively, the TRH analogues in which the integrity of the COOH
terminus is preserved (CG-3509, CG-3703, and YM-14673) have shown some
efficacy in the treatment of CNS trauma. Whereas compounds with a
modified COOH terminus have proven to be ineffective in treating
traumatic spinal cord injury despite the fact that such analogues
possess similar profile of activity with regard to their endocrine
effects (19). Biochemical experiments also suggested the existence of
TRH receptor subtypes. In particular, isoelectric focusing studies of
receptors solubilized from the brain or the pituitary revealed
respective pI values of 5.5 and 4.9. Although these differences may be
caused by tissue differences in post-translational processing, one
possible interpretation is that receptors in the pituitary and the
brain differ in amino acid sequence (10). Interestingly,
electrophysiological experiments and measurements of intracellular
calcium concentration suggested that TRH and TRH metabolites present in
the brain may act at different subtypes of TRH-binding sites (16). We
describe here the isolation and characterization of a novel rat TRH
receptor clone which we have designated TRHR2.
Cloning and Sequencing of a Rat Thyrotropin-releasing Hormone
Receptor (rTRHR2)
The templates for PCR amplification were synthesized using
GeneAmp RNA PCR kits (Perkin-Elmer) using 200 ng of spinal cord poly(A)+ RNA and were amplified using the following
degenerate primers designed against several G protein-coupled
receptors: TM3-4, 5'-GCCAT(C or T)(A or G)(C or G)(C or T)(A or
G)TIGAI(A or C)G(A or G)TA-3'; TM7-4, 5'-(A or C)(A or T)GG(C or T)(A
or G)TAGAI(C or G)AI(A or C)GG(A or G)TT-3'.
The reaction mixture contained 200 pmol of each TM3-4 and TM7-4 primers
and 2.5 units of Taq DNA polymerase in 50 mM
KCl, 1.5 mM MgCl2, 10 mM Tris-HCl,
200 µM dNTPs, pH 9.0. The reaction tubes were heated at
95 °C for 1 min and subjected to 39 cycles of denaturation
(95 °C/1 min), annealing (42 °C/1 min), and extension (72 °C/1 min).
The PCR amplification products of the sizes between 500 and 800 bp were
subcloned into the pGEM-T vector from (Promega) and recombinant clones
were sequenced using the T7 sequencing kit (Pharmacia). The clone
pGemT-1-75 was found to have certain degree of homology to rat TRHR-1,
and was used to screen a rat brain stem-spinal cord cDNA library in
Hybridization-positive phages were purified and the plasmids
(pBS/TRHR2) rescued by helper phage mediated excision (20, 21) which
were then sequenced after having generated a series of overlapping
clones using the Erase-A-Base kit (Promega). To generate a mammalian
expression vector, a 1.3-kilobase StuI-XbaI restriction fragment, from pBS/TRHR2 was isolated and subcloned between
the EcoRV and XbaI sites of pcDNA3
(InVitrogen). This expression vector was called pcDNA3-TRHR2 which
contains the entire coding sequence, 30 bp of 5'-untranslated and 220 bp of 3'-untranslated sequence, in addition to 39 bp originating from
the polylinker of pBluescript.
Stable Transfection
HEK293 cells in a 10-cm Petri dish were transfected with 20 µg
of pcDNA3-TRHR2. After 14 days of selection in culture medium containing 600 µg/ml G418, resistant colonies were pooled. Single clones were purified by 2 rounds of limited dilution in 96-well plates.
Clones of HEK293 cells expressing different levels of rTRHR2 receptor
were selected using a [3H]TRH binding assay. Transiently
transfected cells were also used for the radioligand binding and
functional assays.
Radioligand Binding Assays
The cells expressing TRH receptors were harvested, pelleted, and
resuspended in ice-cold lysis buffer (50 mM Tris, pH 7.0, 2.5 mM EDTA, with 0.1 mM phenylmethylsulfonyl
fluoride). Radioligand binding assays were performed on the crude
membrane preparation resuspended in membrane buffer (50 mM
Tris, 0.32 M sucrose, pH 7.0). For the binding assays,
membranes were diluted into 50 mM Tris, 3 mM
MgCl2, 1 mg/ml bovine serum albumin, pH 7.4. Aliquots (80 µl) of membranes were added to 96-well plates containing 1.3 nM of the radioligand
([3-methyl-3H]TRH (NET-705, 64 Ci/mmol))(70
µl) and test compounds (150 µl) at various concentrations in 300 µl total. The total (TB) and nonspecific (NS) binding were determined
in the absence and presence of 3 µM TRH, respectively.
The plates were mixed and incubated at 25 °C for 90 min then
vacuum-filtered through Packard GB/B Unifilter plates (presoaked for at
least 2 h in 0.1% polyethyleneimine), washed three times with 1 ml each, of wash buffer (50 mM Tris, pH 7.0, 3 mM MgCl2, dried, and counted in the 2.9-35 KeV
window in a TopCount (Packard) after adding 50 µl of MS-20
scintillation fluid/well.
Values of IC50 were obtained from sigmoidal fits of a
nonlinear curve-fitting program (Prism version 2.01 from GraphPad
Software, San Diego, CA). The Ki values are
determined from the equation Ki = IC50/(1+(L*/Kd)), where L* is the
radioligand concentration and Kd is 3.05 nM. The Kd was obtained from saturation
curves of TRHR2 membranes with
[3-methyl-3H]TRH (Kd = 3.05 ± 0.6 nM; Bmax = 12.03 ± 0.99 pmol/mg of protein, mean ± S.D.,
n = 3).
FLIPR Experiment
A stable HEK293 clone expressing the TRHR2 receptor and
GH4Cl neuronal cell line endogenously expressing the rat
TRH-R1 receptor were fed 24 h prior to experimentation. On the day
of the experiment, 10-12 million cells were centrifuged and gently
resuspended in loading solution (loading solution for HEK293 cells: 11 ml of Dulbecco's modified media + 10% fetal bovine serum containing 21 ml of Fluo3-AM (4 mM final) and 21 ml of 20% pluronic
acid and, for GH4Cl cells: 11 ml of F-10 media + 10% fetal
bovine serum containing 42 ml of calcium green (8 mM final)
and 42 ml of 20% pluronic acid) and incubated at 37 °C for 1 h
in a humidified chamber. Following the incubation step, cells were
washed three times in 30 ml of Hanks' + 20 mM Hepes + 0.1% bovine serum albumin (pH 7.4) and resuspended in wash buffer. The
cells were plated in 96-well poly-D-lysine-coated black
plates at a count of 100,000/well (100 ml), spun at 200 × g for 3 min, and analyzed using the FLIPR (fluorometric
imaging plate reader) system (Argon Laser: 488 nM) to study
calcium transients in response to TRH and related compounds. The data
was analyzed using the Graph Prism nonlinear regression method to
obtain maximum activation and EC50 values.
Northern Blot Analysis
A rat multiple tissue Northern blot
(CLONTECH) containing 2 mg of rat
poly(A)+ mRNA isolated from heart, brain, spleen, lung,
liver, skeletal muscle, kidney, and testes was used to study the
distribution of rTRHR2 in various tissues. The blot was pre-hybridized
at 42 °C for 3 h in a solution containing 50% formamide,
5 × SSPE, 10 × Denhardt's solution, 100 mg/ml salmon sperm
DNA, and 2% SDS. Hybridization with the full-length randomly primed
cDNA (Ready-To-Go DNA labeling kit; Pharmacia) was carried out at
42 °C for 18 h in the solution described above and the final
wash was 2 × SSC, 0.05% SDS at 60 °C. The blot was exposed at
In Situ hybridization
Animals and Tissue Preparation--
Adult male Sprague-Dawley
rats (~300 g; Charles River, St. Constant, Quebec) were sacrificed by
decapitation. Brain, pituitary, and spinal cord were promptly removed,
snap-frozen in isopentane at In Situ Hybridization--
Sections were postfixed in 4%
paraformaldehyde (BDH, Poole, United Kingdom) in 0.1 M
phosphate buffer (pH 7.4) for 10 min at room temperature and rinsed in
3 changes of 2 × SSC. Sections were then equilibrated in 0.1 M triethanolamine, treated with 0.25% acetic anhydride in
triethanolamine, rinsed in 2 × SSC and dehydrated in an ethanol
series (50-100%). Hybridization was performed in a buffer containing
75% formamide (Sigma), 600 mM NaCl, 10 mM Tris
(pH 7.5), 1 mM EDTA, 1 × Denhardt's solution
(Sigma), 50 mg/ml denatured salmon sperm DNA (Sigma), 50 mg/ml yeast
tRNA (Sigma), 10% dextran sulfate (Sigma), 10 mM
dithiothreitol, and [35S]UTP-labeled cRNA probes (10 × 106 cpm/ml) at 55 °C for 18 h in humidified
chambers. Following hybridization, slides were rinsed in 2 × SSC
at room temperature, treated with 20 mg/ml RNase IA (Pharmacia) in
RNase buffer (10 mM Tris, 500 mM NaCl, 1 mM EDTA, pH 7.5) for 45 min at room temperature, and washed
to a final stringency of 0.1 × SSC at 65 °C. Sections were then dehydrated and exposed to Biomax MR Kodak film for 10 days. Neuroanatomical structures were identified according to the Paxinos and
Watson rat brain atlas (22).
Degenerated oligonucleotides were designed to the conserved
regions of transmembrane domains 3 and 7 of several G protein-coupled receptors belonging to the adrenergic subfamily (23). In order to
isolate novel G protein-coupled receptors expressed in the spinal cord,
reverse transcriptase-PCR reactions were performed using rat spinal
cord mRNA as a template. The amplified fragments whose sizes were
between 500 and 800 bp were ligated to a cloning vector. After
screening of the subcloned fragments by DNA sequence analysis, clone
pGemT-1-75 showed marked sequence homologies with known G
protein-coupled receptors. The most homologous sequence was the human
and rat thyrotropin-releasing hormone receptors (TRHR) (3, 17, 24).
To obtain the full-length cDNA for this clone, a rat brain
stem/spinal cord cDNA library was screened using the insert of pGemT-1-75 as a probe. DNA isolated from a positive phage by helper phage-induced excision and the cDNA insert of the resulting plasmid (pBS-TRHR2) was sequenced. An open reading frame of 352 amino acids was
detected flanked by 3'-untranslated region and 5'-untranslated region
of, respectively, 220 and 361 bp. The nucleotide sequence and predicted
amino acid are displayed in Fig. 1. The
relative molecular mass of the predicted protein is 39,500 daltons.
Hydropathy analysis of the encoded protein is consistent with the
putative topography of seven transmembrane domains, indicative of the G protein-coupled receptor family (23). In addition, sequence analysis
revealed that the open reading frame of clone pBS-TRHR2 contains
several conserved structural motifs and residues found among the
members of the neuropeptide receptor family including an asparagine in
TM1 (Asn-40), a leucine (Leu-64) and an aspartic acid (Asp-68) in TM2
and a serine (Ser-109), an arginine (Arg-120) and a tyrosine residue
(Tyr-121) in TM3. Other motifs of the rTRHR2 receptor gene are the
presence of a potential site for N-glycosylation in the
amino terminus (Asn-6) and the presence of several serines and
threonines in the carboxyl terminus and the third intracellular loop,
which may serve as potential sites for phosphorylation by protein
kinases.
An alignment of the deduced amino acid sequences of known TRH receptors
is shown in Fig. 2. The overall amino
acid sequence homology between rTRHR2 and the known rat TRH receptor is
50.6%. The homologies between the known rat TRH receptor (hereafter
called rTRHR1) and rTRHR2 in TM1 to TM7 are 61, 80, 74, 58, 52, 77, and 71%.
Cloning and Characterization of a cDNA Encoding a Novel
Subtype of Rat Thyrotropin-releasing Hormone Receptor*
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ZAP II (Stratagene) in order to obtain full-length clone
(hybridization: 2 × SSC, 5 × Denhardt's solutions and
0.2% SDS, 65 °C, final wash: 5 mM EDTA, 0.2% SDS, pH
8.0, 65 °C).
80 °C for 7 days to Kodak Biomax film with intensifying screens.
40 °C for 20 s, and stored at
80 °C. Frozen tissue was sectioned at 14 mm in a Microm HM 500M
cryostat (Germany) and thaw-mounted onto ProbeOn Plus slides (Fisher
Scientific, Montreal, Quebec). Sections were stored at 80 °C prior
to in situ hybridization.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (59K):
[in a new window]
Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of a novel rat TRH receptor (TRHR2; sequence numbers 1 and
2). Nucleotides are presented in the 5' to 3' orientation. Deduced
amino acid sequence by translation of a long open reading frame is
shown, along with the 5' to 3' untranslated sequences. Numbers
above the sequence represent nucleotide (top line). The
amino acid sequence is displayed on the bottom line using
the single amino acid letter code.
View larger version (81K):
[in a new window]
Fig. 2.
Alignment of the rat TRHR2 sequence with
sequences of rat, mouse, and human TRHR1. The alignment was
prepared using the Clustal W. Amino acids identical to those in the rat
TRHR-2 sequence are boxed.
When transiently transfected into HEK293 cells, the expression plasmid pcDNA3-TRHR2 generated the expression of specific [3H]TRH-binding sites. No specific [3H]TRH binding sites were generated by the transfection of the vector itself or a control pcDNA3 expression construct encoding the delta opioid receptor. To pharmacologically characterize the rTRHR2 receptor, we generated stable HEK293 cells expressing the rTRHR2 receptor by selecting pcDNA3-TRHR2 transfected cells using G418. Binding experiments were performed on a selected cell line called HEK293/TRHR2. A single class of saturable [3H]TRH-binding site was detected displaying an estimated Kd for [3H]TRH of 3 ± 0.6 nM and a Bmax of 12 ± 1 pmol/mg of proteins. Various TRH-related peptides were used in competition experiments. These experiments using [3H]TRH as a tracer revealed Ki values of 2.3 nM for pGlu-3-methyl-His-Pro-amide, 7.3 nM for TRH, 1507 nM for pGlu-His-Pro-Gly, and >10,000 nM for pGlu-Glu-Pro-amide (Table I).
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In addition we compared the pharmacology of the novel TRHR2 receptor to that of TRHR1 receptors expressed in GH4Cl cells (25) by performing competition binding experiments. The Ki values measured using HEK293/TRHR2 or GH4Cl cells are summarized in Table I. The affinity of TRH is similar for TRHR2 and TRHR1. For both the TRHR1 receptor, present in GH4Cl cells, and for the TRHR2 receptor present in HEK293/TRHR2 cells, methyl-TRH (pGlu-3methyl-His-Pro-amide) displays a slightly higher affinity than TRH. The ligand pGlu-His-Pro-Gly binds with a better affinity to the TRHR2 receptor (Ki = 1.4 µM) than to the TRHR1 receptor (Ki = 7.2 µM). This may indicate that TRHR2 is less susceptible than TRHR1 to NH2-terminal modification of the ligand.
The rTRHR1 receptor has previously been shown to couple to
phospholipase C via a pertussis toxin-insensitive G
q/11
protein resulting in the mobilization of intracellular calcium
stores. Based on these prior observations, we investigated if rTRHR2
could also activate the phospholipase C effector system.
Using different peptidic TRH-derived compounds, the calcium signaling responses mediated by rTRHR1 endogenously expressed in GH4Cl cells and HEK293 stably transfected with rTRHR2 receptors were compared using the FLIPR system. Increasing concentrations of TRH, 3-CH3-TRH (pGlu-3-methyl-His-Pro-amide), pGlu-His-Pro-Gly, and pGlu-Glu-Pro-amide peptides were added to cells expressing either the rTRHR1 or the rTRHR2 receptors to measure calcium responses. As shown in Fig. 3, TRH and 3-CH3-TRH were equipotent at TRHR1 and TRHR2 receptors at stimulating increases in intracellular calcium, whereas pGlu-His-Pro-Gly was more potent at functionally activating the rTRHR2 receptors (EC50, 42 ± 22 nM) as compared with the rTRHR1 receptors (EC50, 1833 ± 223 nM). The pGlu-Glu-Pro-amide peptide, however, displayed no functional binding at either the TRHR1 and TRHR2 receptors. Hence, based on these observations the rTRHR2 receptor is a fully functional receptor coupled to the phospholipase C signaling pathway.
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, TRH; 
, 2-methyl-TRH; 
,
pGlu-His-Pro-Gly; 
, pGlu-Glu-Pro-NH2.
Using Northern blot analysis of RNA isolated from heart, brain, spleen, lung liver, skeletal muscle, kidney, and testes, we detected expression of TRHR2 mRNA only in brain tissue (see Fig. 4). The apparent size of the mRNA is about 8 kilobases. Other tissues contained either no or insufficient amount of message to be detected after 1 week exposure under the conditions described.
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The regional distribution of TRHR2 within the rat CNS and pituitary gland was further examined using in situ hybridization (Fig. 5, A-C). Film autoradiograms prepared from sagittal brain and spinal cord sections hybridized with 35S-labeled TRHR2 antisense riboprobe revealed a discrete and unique pattern of distribution. No signal was detected from sections hybridized with 35S-labeled TRHR2 sense probe (data not shown). In brain (Fig. 5A), the highest levels of TRHR2 mRNA were observed in the pontine nucleus, thalamus, and cerebellar cortex. Clearly, the pontine nucleus was the most intensely labeled structure in sagittal brain sections. A prominent TRHR2 hybridization signal was also present in thalamus where virtually every thalamic nucleus was either intensely or moderately labeled. Within the cerebellar cortex, intense TRHR2 labeling was detected in the lower tier of the molecular layer in all lobules. Moderate levels of TRHR2 mRNA were present in the anterior olfactory nucleus, in deeper layers of the neocortex, throughout the superior and inferior colliculi, in central gray as well as in several brainstem nuclei. Other cephalic areas such as the basal ganglia, hippocampus, and hypothalamus were generally devoid of TRHR2 labeling. In spinal cord (Fig. 5B), expression of TRHR2 mRNA was detected exclusively within the dorsal horn. The labeling was moderate and uniformly distributed throughout the dorsal gray matter, spanning approximately laminae III to VI. In contrast to the CNS, no TRHR2 hybridization signal was detected in the pituitary gland (Fig. 5C).
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For comparison, series of adjacent sections were probed in parallel
with a TRHR1 riboprobe (Fig. 5, D and E). The
level and pattern of TRHR1 mRNA expression in rat CNS and pituitary
were entirely different from that of TRHR2. Overall, CNS levels of TRHR1 mRNA were low as compared with those of TRHR2. In brain (Fig.
5D), the highest levels of TRHR1 were observed in the
accessory olfactory bulb. Moderate to weak TRHR1 labeling was present
in the olfactory bulb, nucleus of the diagonal band of Broca, various hypothalamic nuclei, dentate gyrus, superficial gray layer of the
superior colliculus, and in certain brainstem nuclei. In spinal cord
(Fig. 5E), TRHR1 mRNA signal was observed predominantly
over motoneurons of the ventral horn. In the pituitary gland (Fig. 5F), TRHR1 labeling was intense and selectively distributed
over the anterior pituitary.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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We have cloned a cDNA representing a novel rat thyrotropin-releasing hormone receptor (rTRHR2). Of all known G protein-coupled receptor sequences (EMBL/GenBank data base), the greatest homology was displayed between rTRHR2 and the human TRH receptor (3, 17, 24). In the TM regions, the sequence homology between rTRHR2 and this receptor ranges from 52 to 80%. The alignment of this rTRHR2 sequence, relative to other G protein-coupled receptors or other members of the neuropeptide receptor subfamily, indicates a unique sequence, proving rTRHR2 is a newly characterized receptor.
The transfection of HEK293 cells with a mammalian expression vector containing the rTRHR2 sequence resulted in the generation of high affinity TRH-binding sites. The Kd values for TRH binding is in the low nanomolar range. The binding of labeled TRH was displaced by TRH analogues but not by TRH unrelated ligands. Pyroglutamylglutamylproline-amide, a peptide structurally related to TRH isolated from the rabbit prostate, does not bind to the rTRHR2 nor to the TRHR1 expressing cells (Ki > 10,000 nM)(27). The rTRHR2 receptor evoked strong transient increases in [Ca2+]i concentration in a dose-dependent fashion when activated by various concentrations of TRH, 3-CH3-TRH, and pGlu-His-Pro-Gly but not by pGlu-Glu-Pro-amide. The pGlu-His-Pro-Gly peptide was the only ligand to selectively differentiate between the rTRHR1 and rTRHR2 subtypes since it functionally activated the rTRHR2 receptors more potently than the rTRHR1 receptors, and this difference was also reflected in binding affinities. These results demonstrate that the rTRHR2 gene encodes a functional receptor. Similar types of signaling responses were observed in cells expressing TRHR1 receptors (3, 28).
The rat TRHR2 receptor differs from the TRHR-1 in another aspect, i.e. the very limited distribution of its messenger RNA. The message for rat TRHR1 has been shown to be widely present both in periphery and in central nervous system as described by Northern analysis and reverse transcriptase polymerase chain reaction (29-31). The message for TRHR2, however, as assessed by Northern analysis appears to be restricted to or at least predominately expressed in brain. Under our conditions we could not detect any signal in any peripheral tissues included in our blots even after 7 days exposure. A single band between 8.0 and 8.5 kilobases was detected in the RNA from the brain. We have extended these studies of the presence of the TRHR2 in brain to include the regional distribution of the message in brain and spinal cord by in situ hybridization.
Our in situ hybridization studies indicate that distribution of TRHR2 mRNA in rat CNS is discrete and unique. A priori, the CNS pattern of TRHR2 expression suggests the involvement of at least two distinct modalities, somatosensory and motor. The discrete expression of TRHR2 mRNA in dorsal horn of the spinal cord, reticular formation, somatosensory nuclei of the thalamus (ventral posterolateral thalamic nucleus, ventral posteromedial thalamic nucleus), and neocortex is consistent with ascending pathways such as the spinothalamic and trigeminothalamic tracts (pain and crude touch) as well as the medial lemniscal system (discriminative touch). The presence of particularly high levels of TRHR2 in the pontine nucleus and the cerebellum is consistent with a role in motor control and/or proprioception, suggesting these receptors may also be the anatomical substrate for the previously described TRH effects on motor control (19).
Although both receptors subtypes are expressed in the rat brain, our in situ hybridization studies demonstrate the pattern of TRHR2 mRNA distribution to be entirely different from that of TRHR1. In fact, the CNS expression of TRHR2 and TRHR1 are virtually mutually exclusive, suggesting these receptors mediate quite different physiological roles. A clear example of this is the pituitary. Our findings as well as those of others (32) demonstrate that the anterior pituitary selectively expresses moderate levels of TRHR1 mRNA. In contrast, no detectable levels of TRHR2 expression were observed in the pituitary gland. Since TRH's neuroendocrine actions are predominantly associated with the anterior pituitary (8), these actions appear to be specific to TRHR1. Conversely, TRHR2, but not TRHR1, is selectively expressed in many key CNS regions associated with pain transmission, namely dorsal horn of the spinal cord, thalamus, central gray and rostral ventral medulla, specifically implicating a role for TRHR2 in mediating TRH's putative antinociceptive properties (11, 13).
Today, the development of TRH analogues for therapy is justified by the
potential benefit of TRH agonists in improving motor neuron functions
or other neurological functions, without triggering the endocrine
effects evoked by TRH stimulation of the pituitary. In particular, TRH
has been shown to improve the recovery following spinal cord injury
(19). Importantly, TRH has also been involved in the modulation of
spinal cord pain transmission (12, 33) and has been shown to act as an
antinociceptive agent more potent than morphine when injected centrally
(11). The regional distribution of the rTRHR2 message in the
spinal cord and in the thalamus points to a potential role for rTRHR2
in the modulation of pain perception. rTRHR2 thus represents an
important tool for the development of selective TRH-like
compounds of potential use in neurobiology.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 219979 AF091715.
To whom correspondence should be addressed. Tel.: 514-832-3206;
Fax: 514-832-3228; E-mail: philippe.walker{at}arcm.ca.astra.com.
The abbreviations used are: TRHR, thyrotropin-releasing hormone receptor; CNS, central nervous system; PCR, polymerase chain reaction; bp, base pair(s); T4, thyroxin; FLIPR, fluorometric imaging plate reader.
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Abstract
Introduction
Procedures
Results
Discussion
References
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