TRH-R2 Exhibits Similar Binding and Acute Signaling but Distinct Regulation and Anatomic Distribution Compared with TRH-R1
Brian F. ODowd,
Dennis K. Lee,
Wei Huang,
Tuan Nguyen,
Regina Cheng,
Yang Liu,
Bing Wang,
Marvin C. Gershengorn and
Susan R. George
Departments of Pharmacology (B.F.O., D.K.L., Y.L., S.R.G.)
and Medicine (S.R.G.) University of Toronto Toronto, Ontario
M5S 1A8, Canada Centre for Addiction and Mental Health
(B.F.O., T.N., R.C., S.R.G.) Toronto, Ontario, M5S 2S1, Canada
Division of Molecular Medicine (W.H., B.W., M.C.G.)
Department of Medicine Weill Medical College of Cornell
University New York, New York 10021
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ABSTRACT
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TRH (thyroliberin) is a tripeptide
(pGlu-His-ProNH2) that signals via G
protein-coupled receptors. Until recently, only a single receptor for
TRH was known (TRH-R1), but two groups identified a second receptor,
TRH-R2. We independently discovered TRH-R2. Using an extensive set of
TRH analogs, we found no differences in TRH-R1 and TRH-R2 binding or in
acute stimulation of signaling. TRH-R2 was more rapidly internalized
upon binding TRH and exhibited a greater level of TRH-induced
down-regulation than TRH-R1. During prolonged exposure to TRH, cells
expressing TRH-R2 exhibited a lower level of gene induction than cells
expressing TRH-R1. TRH-R2 receptor mRNA was present in very discrete
nuclei and regions of rat brain. A major mRNA transcript for TRH-R2 was
seen in the cerebral cortex, pons, thalamus, hypothalamus, and midbrain
with faint bands found in the striatum and pituitary. The extensive
distribution of TRH-R2 in the brain suggests that it mediates many of
the known functions of TRH that are not transduced by TRH-R1. The
variations in agonist-induced internalization and
down-regulation/desensitization, and anatomic distribution of
TRH-R2 compared with TRH-R1, suggest important functional differences
between the two receptors.
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INTRODUCTION
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TRH (thyroliberin) is a tripeptide
(pGlu-His-ProNH2) that functions as a hormone, a
paracrine regulatory factor, and a neurotransmitter/neuromodulator. A
review of the extensive actions of TRH was published in 1989 (1 ). It is
known that TRH initiates some, if not all, of these effects by
interacting with receptors on cell surfaces and that these receptors
couple to G proteins (2 3 ). Until recently, only a single G
protein-coupled receptor (GPCR) for TRH was known (TRH-R1) (4 ). Two
groups independently identified a second gene encoding another TRH
receptor, TRH-R2, from a rat brain cDNA library (5 ) and a rat brain
stem-spinal cord cDNA library (6 ). In a limited in situ
hybridization study, Cao and colleagues (6 ) found that distribution of
TRH-R2 mRNA in the central nervous system was distinct from that of
TRH-R1 mRNA. We found that TRH-R2 exhibits higher basal signaling
activity than TRH-R1 (7 ).
A preliminary pharmacological characterization of TRH-R2 was
performed and the findings compared with TRH-R1. Both Itadani et
al. (5 ) and Cao et al. (6 ) agreed that TRH-R1 and
TRH-R2 bound TRH with equal affinity (5 6 ). Cao and colleagues (6 )
presented data that rat TRH-R2 and TRH-R1 bound the TRH analog
MeTRH1 with equal affinity
but that TRH-R2 bound another TRH analog, pGlu-His-Pro-Gly, with higher
affinity than rat TRH-R1. They concluded that these receptors exhibited
different pharmacological characteristics. These data are too
preliminary to justify such an important conclusion. It is noteworthy
that, of four amino acid residues within the transmembrane helices and
two within the extracellular loops of mouse TRH-R1 (8 ) that we
identified as sites of direct interaction with TRH (4 9 ), all six
residues are conserved in rat (10 11 ), human (12 ), and chicken (13 )
TRH-R1 and rat TRH-R2 (5 6 ). This suggests that there is likely to be
significant similarity between the binding of TRH analogs by these
receptors although differences may be found.
In this paper, we present the results of our independent discovery,
extensive anatomic localization in rat brain, pharmacological
characterization, and comparison of the cellular biology of rat TRH-R2
compared with TRH-R1. Our studies, using an extensive set of TRH
analogs, revealed no differences in TRH-R1 and TRH-R2 binding or
signaling. We have investigated the potential functional differences
between TRH-R1 and TRH-R2 and have shown differences in agonist-induced
internalization and desensitization/down-regulation of TRH-R2 compared
with TRH-R1.
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RESULTS
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Cloning of a cDNA Encoding TRH-R2
A rat brain 5'-stretch cDNA library was amplified by PCR
with a transmembrane helix 7 (TM7)-based degenerate primer (P1)
paired with two primers specific for the 5' (P2) and 3' (P3) regions
flanking the cDNA library inserts. One PCR product (~400 bp) was
found to encode a novel GPCR from TM4 to TM7, sharing the greatest
translated sequence identity of 44% to TRH-R1. This cDNA was labeled
with [32P]dCTP-
and used to probe the same
rat brain cDNA library, which resulted in the isolation of two cDNA
clones. These clones were amplified by PCR using primers P2 and P3 and
the products subcloned into the pcDNA3 vector. Both cDNAs revealed
identical sequences encoding the full-length receptor, which we named
TRH-R2. TRH-R2 encoded a protein of 352 amino acids, which shared the
greatest sequence identity of 68% in the TM domains with TRH-R1.
TRH-Binding Analysis
The affinities of mTRH-R1, rTRH-R1, and rTRH-R2 receptors for
MeTRH were indistinguishable: dissociation constant
(Kd ) = 1.9 ± 0.10 nM for
mTRH-R1, Kd = 3.8 ± 0.70 nM for
rTRH-R1, and Kd = 4.6 ± 0.40 nM
for rTRH-R2 (Fig. 1
). In competition
binding experiments, the calculated affinities of the three receptors
for TRH were indistinguishable also: inhibition constant
(Ki ) = 13 ± 1.3 nM for
mTRH-R1, Ki = 12 ± 1.9 nM for
rTRH-R1, and Ki = 16 ± 1.8 nM
for rTRH-R2. The binding affinities of a series of TRH analogs were
tested. Because substitution of the side chain of each of the three
residues of TRH has been shown to affect TRH binding to TRH-R1 (14 ),
the analogs chosen were substituted in the first position
(Pro-His-ProNH2 and
DesazapGlu-His-ProNH2) (9 ), the second position
(pGlu-Val- ProNH2) (15 ), and the third position
(pGlu-His-pyrrolidine) (15 ). Because we had shown that the conformation
of analogs restricted in their rotation by creating a methylene bridge
between residues at the 2- and 3-positions of a TRH analog exhibited
stereospecific differences in binding to mTRH-R1 (16 ), we tested the
binding of the two restricted analogs,
CH-TRH and ßCH-TRH, and the
freely rotatable parent analog, CH-TRH
(pGlu-cyclohexylAla-ProNH2). Lastly, because Cao
and colleagues (6 ) reported that pGlu-His-Pro-Gly exhibited different
affinities when binding to rTRH-R1 and rTRH-R2, we tested
pGlu-His-Pro-Gly and pGlu-His-Pro-Gly-NH2. Table 1
shows that all analogs tested bound to
mTRH-R1 and rTRH-R2 with similar affinities.

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Figure 1. Association Binding of [3H]MeTRH to
Control or TRH-Treated Cells Expressing mTRH-R1, rTRH-R1, or rTRH-R2
The experiments were performed as described in Materials and
Methods. Control COS-1 cells were incubated in growth medium
alone (Con) whereas TRH-treated cells were incubated in growth medium
containing 1 µM TRH for 1620 h (TRH). The points
represent replicate data from two experiments.
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Acute TRH-R Signaling
The potencies of TRH at mTRH-R1, rTRH-R1 and rTRH-R2 for acute
stimulation of phosphoinositide hydrolysis were indistinguishable
(Table 2
), as were the maximal levels of
TRH-stimulated second messenger formation (data not shown). Similar
potencies with the three TRH-Rs were found for
DesazapGlu-His-ProNH2
([DesazapGlu1]TRH),
pGlu-Val-ProNH2
([Val2]TRH), pGlu-His-pyrrolidine
([Pyr3]TRH),
pGlu-His-Pro-Gly-NH2, and pGlu-His-Pro-Gly.
Agonist-Induced Receptor Internalization And Down-Regulation
The rate of internalization of mTRH-R1 is affected by
alterations in its carboxyl terminus (17 18 ). mTRH-R1 (19 ) and rTRH-R1
(20 ) exhibit two alternative splice forms that affect their carboxyl
termini, but no differences in internalization rates of these variants
have been reported. A major difference between rodent TRH-R1s and
rTRH-R2 is in the carboxyl termini (Fig. 2
). The carboxyl terminus of TRH-R2 is
shorter than both alternative splice variants of rat and mouse TRH-R1
and of the 42 amino acid residues in the carboxyl terminus of TRH-R2,
only 15 are identical to those in TRH-R1. Figure 3
illustrates that there is a marked
difference in the MeTRH-stimulated rate of internalization of rTRH-R2
compared with mTRH-R1 or rTRH-R1. Internalization stimulated by MeTRH
was more rapid with rTRH-R2 (t1/2 = 0.20 min)
than with mTRH-R1 (t1/2 = 1.6 min) or rTRH- R1
(t1/2 = 1.2 min). Because agonist-stimulated
receptor internalization may lead to receptor degradation, it was
likely that prolonged exposure to TRH would cause a greater decrease in
the levels of rTRH-R2 on the cell surface than of the TRH-R1 receptors.
Figure 1
illustrates the effects of exposure to 1 µM TRH
for 1620 h on TRH-R levels in cells expressing each of the three
receptors. In these cells, TRH did not significantly decrease the
levels of mTRH-R1 (88 ±7% of control) or of rTRH-R1 (100 ± 17%
of control) but caused an approximately 40% decrease in rTRH-R2
(63 ± 16% of control). Thus, rTRH-R2 exhibited more rapid
agonist-stimulated internalization kinetics and a greater degree of
down-regulation with prolonged agonist exposure than either rodent
TRH-R1.

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Figure 2. Comparison of the Amino Acid Sequences of the
Carboxyl Termini of mTRH-R1, rTRH-R1, and rTRH-R2.
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Figure 3. TRH-Stimulated Internalization of MTRH-R1, rTRH-R1,
and rTRH-R2
The experiments were performed as described in Materials and
Methods. COS-1 cells were incubated in buffer containing 2
nM [3H]MeTRH for the times indicated. The
points represent replicate data from three experiments.
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Chronic Stimulation By TRH
As shown previously (7 ), TRH-R2 causes a greater induction
of reporter gene transcription than TRH-R1 in the absence of agonist
(Fig. 4A
); that is, TRH-R2 exhibits
higher basal signaling activity than TRH-R1. In contrast, 24 h
exposure to 1 µM TRH caused lesser induction of reporter
gene transcription in cells expressing TRH-R2 than in cells expressing
TRH-R1 (Fig. 4A
). The fold stimulation for gene induction by TRH-R2 was
between 2- and 3-fold, whereas that for TRH-R1 was between
10- and 22-fold (Fig. 4B
). Thus, TRH-R2 was less effective in
stimulating gene transcription than TRH-R1 during prolonged stimulation
by TRH.

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Figure 4. Signaling Activity of TRH-R1 and TRH-R2 during
Chronic Stimulation by TRH
Chronic signaling by 1 µM TRH was measured as activation
of CREB-mediated luciferase activity (relative light units) in HEK 293
cells after 24 h. A, Absolute levels of basal and TRH-stimulated
reporter gene induction in a representative sample of three
experiments. B, Fold stimulation by TRH in the three experiments.
Bars represent mean ± SD of triplicate
samples.
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Tissue Distribution
Tissue distribution of TRH-R2 mRNA transcripts was obtained by
Northern blot analysis using a cDNA fragment encoding TRH-R2 from TM4
to TM7 and poly(A)+ RNA isolated from various rat
tissues. In the brain, a major transcript of 9.4 kb (and a faint band
of 3.8 kb) was seen in the pons, hypothalamus, and midbrain (Fig. 5
). Faint bands of 9.4 kb were also found
in the striatum and pituitary (data not shown).

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Figure 5. Northern Blot Analysis of the Tissue Distribution
of TRH-R2 in Rat Brain
Each lane contains 10 µg of poly(A)+ RNA isolated from
various rat tissues. The molecular size is indicated on the
right.
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TRH-R2 receptor mRNA distribution visualized by in
situ hybridization histochemistry revealed abundant expression in
very discrete nuclei and regions of rat brain (Figs. 6
and 7
).
There was extremely dense expression in frontoparietal cortex,
particularly in the primary somatosensory and motor areas, and also in
the primary visual area and primary olfactory cortex. Strong signals
were also present in other areas of cortex, such as the anterior
cingulate area, concentrated in the deeper rather than in the
superficial layers of cortex. Further caudally, TRH-R2 mRNA expresssion
was moderately dense also in the posterior cingulate area,
retrosplenium, striate areas, and throughout the subiculum, in both
dorsal and ventral portions.

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Figure 6. In Situ Hybridization Studies of
TRH-R2 mRNA
In situ hybridization studies were performed in coronal
sections of rat brain using a 35S-labeled cDNA probe.
Representative coronal sections from a total of three separate
experiments are shown. The sections are designated by the series of
stereotactic coordinates derived from the rat brain atlas of Paxinos
and Watson (34 ). A, Section -0.7 mm from bregma showing dense
labeling in paraventricular nucleus of thalamus
(PVT). Signal is also seen in the deeper layers of frontoparietal
cortex (FrP), in the primary somatosensory area, and in the anterior
cingulate area (ACg). Some labeling is also visualized in the
endopiriform nucleus (En) and the medial preoptic area of hypothalamus
(MPO). B, Section -0.8 mm from bregma showing very dense signal in the
deeper layers of the frontoparietal cortex (FrP) and the anterior
cingulate area (ACg). Labeling is also seen in the bed nucleus of the
stria terminalis (BST), the medial preoptic area (MPO), the nucleus of
the diagonal band (HDB), the paraventricular nucleus of hypothalamus
(PVH), and the primary olfactory cortex (PO). C, Section -1.3 mm from
bregma showing dense expression in the frontoparietal cortex in the
primary somatosensory (FrPss) and primary motor areas (FrPm), and in
the anterior cingulate area (ACg). Very dense labeling is also seen in
the anteroventral nucleus (AV) and paraventricular nucleus of thalamus
(PVT) and in the nucleus reuniens, medial part (REm). Signal is also
evident in the anterior hypothalamic nucleus (AHy) and in the lateral
hypothalamic area (LH). D, Section -3.3 mm from bregma showing
abundant expression in several thalamic nuclei, the venteroposterior
nucleus of thalamus (VP) in the medial and lateral divisions, the
central medial nucleus of thalamus (CM), the paraventricular nucleus of
thalamus (PVT), and the medial habenular nucleus (MHb). Lesser signals
were detected in the laterodorsal (LD) and ventromedial (VM) nuclei of
thalamus and in the posterior cingulate area (PCg) and frontoparietal
cortex (FrP). Labeling is also visualized in the zona incerta (ZI),
dorsal premammillary nucleus (PMd), the amygdalohippocampal area (AHi),
and the medial nucleus of amygdala (MeA). E, Section -3.9 mm from
bregma showing labeling of the frontoparietal cortex (FrP), posterior
cingulate area (PCg), and the ventral posterolateral and posteromedial
nuclei of thalamus (VP), including the parvicellular portions of these
nuclei. Signal is visualized also in the lateral geniculate nuclear
complex, dorsal part (LGd), the medial habenular nucleus (MHb),
paraventricular nucleus of thalamus (PVT), zona incerta (ZI),
subthalamic nucleus (STh), posterior hypothalamic nucleus (PH), and
ventral premammillary nucleus (PMv). F, Section -5.3 mm from bregma
showing localization of mRNA in the deeper layers of cerebral cortex,
in the striate areas (Str) of the primary visual cortex, the
retrosplenium, and the subiculum, in both dorsal (Sd) and ventral (Sv)
parts. Dense labeling was evident in the medial geniculate nuclear
complex, in the dorsal (MGd) and ventral (MGv) parts. Labeling is also
present in the superior colliculus (SC), lateral posterior nucleus of
thalamus (LP), periaqueductal gray (PAG), mesencephalic reticular
nucleus (MR), rostral linear raphe nucleus (RL), and the ventral
tegmental area (VTA). G, Section -6.8 mm from bregma showing labeling
of the striate areas of primary visual cortex (Str), the retrosplenial
area (RSp), the subiculum, pyramidal layer of the ventral part (Sp),
and the dentate gyrus crest (DGgr). Signal was also detected in
superior colliculus (SC), periaqueductal gray (PAG), mesencephalic
reticular nucleus (MRN), pontine nucleus (PN), and interpeduncular
nucleus (IP). H, Section -7.9 mm from bregma showing signal in
inferior colliculus (IC), nucleus sagulum (SAG), mesencephalic
reticular nucleus (MRN), pontine reticular nucleus (PRN), central
nucleus raphe (CR), and pontine nucleus (PN).
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Figure 7. In Situ Hybridization Studies of
TRH-R2 mRNA
In situ hybridization studies were performed in
sections of rat brain and pituitary gland, using a
35S-labeled cDNA probe. Representative sections from a
total of three separate experiments are shown. A, Sagittal section of
rat brain 2.4 mm from midline showing signal in frontoparietal areas of
cerebral cortex (FrP), and in the striate areas (Str), retrosplenial
area (RSp), subiculum (S), and the primary olfactory area (PO).
Labeling is also evident in the ventrolateral (VL) and laterodorsal
(LD) nuclei of thalamus, the zona incerta (ZI), subthalamic nucleus
(STh), olfactory tubercle (OT), and the medial amygdaloid nucleus
(MeA). Expression of mRNA is also observed in the superior (SC) and
inferior (IC) colliculi, mesencephalic reticular nucleus (MR),
parabrachial nucleus (PB), pontine nucleus (PN), and pontine reticular
nucleus (PRt). B, Section through pituitary gland showing faint
expression of TRH-R2 mRNA in anterior lobe of pituitary (AP) and lack
of signal in the neurointermediate lobe (NIL).
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Several thalamic nuclei displayed extremely dense labeling, such as the
paraventricular, centromedial, anteroventral, and ventroposterior
thalamic nuclei and the medial habenular nucleus. TRH-R2 mRNA was
present less abundantly in other thalamic nuclei, such as the
laterodorsal, lateroposterior, and ventromedial nuclei and the medial
reuniens nucleus. In hypothalamus, TRH-R2 mRNA was most abundant in the
anterior hypothalamic area and was also present in the medial preoptic
and lateral hypothalamic areas, the paraventricular nucleus, and some
of the mammillary nuclei. Moderate labeling was also seen in the bed
nucleus of the stria terminalis, the nucleus of the diagonal band, some
of the amygdaloid nuclei , and in the subthalamic nucleus. The
geniculate nuclear complex contained very dense expression in the
medial geniculate, in both the dorsal and ventral divisions of the
nucleus, whereas in the lateral geniculate, moderate expression was
observed largely in the dorsal division.
In midbrain, a punctate pattern of TRH-R2 mRNA expression was observed
in the superior colliculus, periaqueductal gray, and the mesencephalic
reticular nucleus. Small amounts of mRNA were present in the ventral
tegmental area. The pontine gray expressed TRH-R2 mRNA very abundantly,
and the central and rostral linear raphe nuclei showed moderately dense
labeling as well. Lesser amounts were evident in the inferior
colliculus, the nucleus sagulum, the pontine reticular nucleus, and the
parabrachial nucleus. TRH-R2 receptor mRNA was detected in pituitary
gland, as sections revealed a very small amount of labeling in the
anterior lobe, whereas the neurointermediate lobe was devoid of any
signal (Fig. 7
).
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DISCUSSION
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A series of 11 TRH analogs were used to study the binding
characteristics of TRH-R2 compared with TRH-R1. We found no significant
differences in the affinities of binding by TRH-R1 and TRH-R2 of any
analog. This may have been expected because the six residues in TRH-R1
(4 9 ) that have been shown to directly interact with TRH are identical
in TRH-R2. Six TRH analogs were used in studies of acute signaling
potencies at TRH-Rs. There were no significant differences in the
potencies of these analogs at the two TRH-Rs. We cannot explain the
discrepancy between our data and that of Cao and colleagues (6 ), who
reported that pGlu-His-Pro-Gly bound with higher affinity to and
exhibited higher potency at TRH-R2 than at TRH-R1.
TRH stimulates TRH-R1 and TRH-R2 internalization and down-regulation
(4 ) (Figs. 1
and 3
). TRH-R1 internalization proceeds via
clathrin-coated pits (21 22 ) and likely leads to increased receptor
degradation because receptors are targeted to lysosomes. This mechanism
of TRH-induced TRH-R1 down-regulation is complemented by effects of TRH
to decrease TRH-R1 gene transcription (23 ) and to increase TRH-R1 mRNA
degradation (24 ). The rate of receptor internalization and the
distribution within cells vary in different cell types (17 25 ). In the
series of experiments described herein, both mouse and rat TRH-R1
exhibited slower rates of internalization than TRH-R2. In concordance
with the differences in their rates of internalization, TRH caused a
much greater down-regulation of TRH-R2 than of TRH-R1. Because TRH-R
internalization and down-regulation affect TRH signaling (4 18 26 ),
it was likely that these differences would lead to differences in TRH
signaling mediated by TRH-R1 and TRH-R2 during prolonged TRH exposure.
We measured the effect of prolonged stimulation by TRH on induction of
transcription of a reporter gene and found that down-regulation of
TRH-R2 was associated with a lesser degree of TRH-stimulated gene
induction in cells expressing TRH-R2 than in cells expressing TRH-R1.
Thus, it appears that TRH-R2 can be stimulated to signal acutely like
TRH-R1 but exhibits greater down-regulation/desensitization when
exposed to TRH chronically.
During the course of this work, the discovery of rat TRH-R2 was
reported independently by two other groups (5 6 ). Within the
open-reading frame of TRH-R2, the published sequences varied by
two single nucleotide differences located in the regions encoding the
N-terminal portion of TM4 and the carboxy terminus. Specifically,
Itadani et al. (5 ) reported an isoleucine and valine at
amino acid positions 143 and 347, respectively, while Cao et
al. (6 ) reported a methionine and glutamic acid at these
respective positions. By comparison, our TRH-R2 sequence agreed with
Ile143 in accordance with the report by Itadani
et al. (5 ) but with Glu347 as reported
by Cao et al. (6 ).
In situ hybridization analysis revealed that TRH-R2
mRNA exhibited a distinct brain distribution with especially abundant
levels of expression in areas of the cortex, thalamus, and the pontine
nucleus (Figs. 6
and 7
). In addition, other areas of the midbrain
including the medial and lateral geniculate nuclei, superior
colliculus, periaqueductal gray, mesencephalic reticular nucleus, and
central raphe nucleus displayed discrete levels of TRH-R2 mRNA
expression. Together with strong distinct signals from various sensory
and motor control areas in the cortex and thalamus (e.g. the
striate areas of the primary visual cortex, the paraventricular,
centromedial, anteroventral, and ventroposterior thalamic nuclei),
TRH-R2 may play roles in nociception, motor control, and regulation of
somatosensory transmission. Unlike the previous report (6 ), we found
expression of small amounts of TRH-R2 mRNA in the anterior lobe of the
pituitary (as seen clearly against the absence of signal in the
neurointermediate lobe of the pituitary), suggesting possible roles for
TRH-R2 in hormone regulation. Furthermore, dense labeling was seen in
the hypothalamus in the anterior and lateral hypothalamic nuclei, as
well as moderate levels of expression in the medial preoptic area,
paraventricular nucleus, posterior hypothalamic nucleus, and the
ventral and dorsal premammillary nuclei, suggesting that TRH-R2 may
play a role in appetite regulation, motivation, or other hypothalamic
functions. A comparison of the distributions of TRH-R1 and TRH-R2 mRNAs
in rat brain (Table 3
) shows that there
are distinct distributions for the two TRH-Rs although there are some
areas in which both receptors are expressed.
In summary, we have described a second GPCR for TRH. The
extensive distribution of this receptor in the brain suggests that it
mediates many of the known functions of TRH that are not transduced by
TRH-R1. In addition, the variations in agonist-induced internalization
and down-regulation/desensitization of TRH-R2 compared with TRH-R1
suggest important functional and structural differences between the two
receptors.
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MATERIALS AND METHODS
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Cloning of a cDNA Encoding TRH-R2
A rat brain 5'-Stretch cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA) was amplified by the PCR
using Pfu polymerase (Stratagene, LaJolla, CA)
and a degenerate oligonucleotide based upon the conserved TM 7 (P1:
5'-GAAGGCGTAGADBAEFGGHTT-3'; B = C or G, D = C or G or T,
E = A or C or G or T, F = A or C or G, H = A or G)
paired with two primers specific for the 5' (P2:
5'-GGTGGCGACGACTCCTGGAGC-3') and 3' (P3: 5'-GACACCAGACCAACTGGTAAT-3')
regions flanking the cDNA library inserts. PCR conditions were as
follows: denaturation at 94 C for 30 sec, annealing at 58 C for 40 sec,
and extension at 72 C for 1 min, for 30 cycles, followed by a 7-min
extension at 72 C. The PCR products were extracted with
phenol/chloroform, precipitated with ethanol and electrophoresed on a
low melting point agarose gel. PCR product bands were excised from the
gel, ligated into the EcoRV site of pBluescript SK(-)
(Stratagene), and sequenced. One insert appeared to encode
a novel GPCR and was labeled with [32P]dCTP-
(NEN Life Science Products, Boston, MA) by nick
translation (Amersham Pharmacia Biotech, Arlington
Heights, IL) and used to screen the same library amplified above as
previously described (27 ). Positive phage clones were plaque purified
and their inserts amplified by PCR using Pfu polymerase and
primers P2 and P3. The PCR products were blunt-end ligated into the
EcoRV site of pcDNA3 vector (Stratagene) and
sequenced on both strands.
Cell Culture and Transfection
COS-1 cells were maintained and transiently transfected using
the diethylaminoethyl-dextran method as described previously
(28 ). In brief, cells were seeded 1 or 2 days before transfection at
0.7 to 1.5 x106 cells per 100-mm dish. After
transfection, COS-1 cells were maintained in DMEM with 10% FCS for 1
day at which time cells were harvested and seeded into 24-well plates
at 50,000 cells per well in DMEM with 5% FCS.
HEK 293 cells were grown in DMEM containing 10% FBS. On the day before
transfection, the cells were seeded in 24-well dishes (30,000 cells per
well). After 16 h, the medium was aspirated and the cells were
transfected using calcium phosphate. The transfection cocktail
contained 1 µg/ml receptor-encoding plasmid DNA, 1 µg/ml pFR-Luc,
and 1 µg/ml pFA2-CREB (PathDetect In Vivo Signal
Transduction Pathway trans-Reporting System,
Stratagene)(7 ). [TRH-Rs signal via
Ca2+/calmodulin-dependent protein kinase (29 )
that may induce gene transcription via the transcription factor CREB
(30 ).] Total DNA was kept constant by adding "empty" plasmid.
"Mock" transfections were performed without receptor-encoding
plasmid. The cells were exposed to the transfection cocktail for 6
h and then were incubated in DMEM containing 1% FBS for 1624 h in
the absence (basal) or presence of 1 µM
TRH.
Receptor Binding
One day after reseeding into 24-well plates, association
binding experiments with [3H]MeTRH (0.110
nM) or competition binding experiments were carried out in
HBSS, pH 7.4, using 2 nM [3H]MeTRH
and various concentrations of unlabeled analogs as described previously
(31 ) with cells in monolayer for 2 h at room temperature.
Equilibrium Kd values were derived from
association experiments and equilibrium Ki values
were derived from competition binding experiments for which curves were
fitted by nonlinear regression analysis and drawn with the PRISM
program (GraphPad Software, Inc.).
Internalization and Down-regulation
Internalization of TRH-Rs was measured as specifically
bound [3H]MeTRH that was resistant to acid wash
(17 ). At the end of the incubation with 2 nM
[3H]MeTRH for the times shown, free ligand was
removed by aspirating the binding buffer and washing the cells with 2
ml ice-cold buffer. Cells were then exposed to 1 ml 50 mM
glycine, pH 3.5, 0.5 M NaCl for 1 min at 4 C, and then
washed with 1 ml binding buffer. Acid-resistant
[3H]MeTRH was counted and compared with total
specific binding. Internalized receptors are presented as the fraction
of acid-resistant [3H]MeTRH-bound receptors
divided by total [3H]MeTRH-bound receptors.
Down-regulation of TRH-Rs was measured as follows (24 ). Cells
expressing TRH-Rs were incubated in growth medium containing 1
µM TRH for 1620 h. Thereafter, the medium was aspirated
and the cells were washed once with binding buffer, and then with 1 ml
50 mM glycine, pH 3.5, 0.5 M NaCl for 1 min (to
remove TRH bound to receptors on the cell surface) and then again with
1 ml binding buffer. Total receptor number was then determined in
association binding experiments with [3H]MeTRH
(0.110 nM).
Acute Signaling
TRH-R-mediated, stimulated phosphoinositide hydrolysis was
measured in myo-[3H]inositol-labeled
cells as described (15 ).
Chronic Stimulation By TRH
Prolonged signaling by TRH was assayed using a firefly
luciferase reporter gene under the control of CREB-responsive promoter
in cells exposed to TRH for 24 h. Cells were transfected with
plasmids encoding TRH-Rs and pFR-Luc and pFA2-CREB. After 24 h,
cells in 24-well plates were washed with PBS and lysed with 0.5 ml of
lysis buffer (25 mM GlyGly, pH 7.8, 15 mM
MgSO4.6H2O, 4
mM EGTA, 1 mM dithiothreitol, 1% Triton
X-100). Cell lysates (0.025 ml) were combined automatically with 0.125
ml reaction buffer (25 mM GlyGly, pH 7.8, 15 mM
MgSO4.6H2O, 4
mM EGTA, 1 mM dithiothreitol, 15 mM
KH2PO4, 2 mM
ATP) and 0.025 ml luciferin (0.4 mM) in reaction buffer and
the luminescence measured for 10 sec in a TR717 Microplate Luminometer
(Tropix, Bedford, MA).
Northern Blot Analysis
mRNAs from several rat tissues were extracted as described
previously (27 ). Briefly, total RNA was extracted by the method of
Chomczynski and Sacchi (32 ) and poly (A)+ RNA
isolated using oligo(dT) cellulose spin columns (Pharmacia Biotech, Piscataway, NJ). RNA was denatured and size
fractionated on a 1% formaldehyde agarose gel, transferred onto nylon
membrane, and immobilized by UV irradiation. The blots were hybridized
with a 32P-labeled DNA fragment encoding TRH-R2
from TM4 to TM7, washed with 2x SSPE and 0.1% SDS at 50 C for 20 min
and again with 0.1x SSPE and 0.1% SDS at 50 C for 2 h, and
exposed to x-ray film at -70 C in the presence of an intensifying
screen.
In Situ Hybridization Analysis
Preparation of rat brain sections and in situ
hybridization procedures were done as previously described (33 ).
Briefly, brains were removed from male Sprague Dawley rats (The Jackson Laboratory, Bar Harbor, ME) within 30 sec of
decapitation, frozen, sectioned at 14 µm thickness using a microtome
cryostat, thaw-mounted onto microscope slides and stored at -70 C. A
cDNA fragment encoding the full length TRH-R2 was used as a probe for
in situ hybridization and was labeled with
[35S]dCTP-
(NEN Life Science Products). The rat brain slices were incubated for 2 h in
prehybridization solution, hybridized with the labeled probe
(106 dpm/slice) for 16 h, and washed in
conditions of increasing temperature and decreasing salt
concentrations. The hybridized sections were dehydrated in a graded
alcohol series and exposed to X-ray film (Dupont MRF-34) for 46 weeks
at -70 C and developed. For use as controls, adjacent sections were
hybridized after treatment with RNase, to confirm the specificity of
hybridization.
Statistical Analysis
Statistical analysis was performed by t test.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Marvin C. Gershengorn, Weill Medical College of Cornell University, 1300 York Avenue, Room A328, New York, New York 10021-4896.
This research was supported by grants from the Medical Research Council
of Canada, the National Institute on Drug Abuse, the Smokeless Tobacco
Research Council, Inc., and the US Public Health Service
(DK-43036).
1 Abbreviations used: MeTRH = N-t-[methylHis]-TRH; CH-TRH = pGlu-cyclohexylAla-ProNH2;
CH-TRH = (6S, 9S, 12S)-1-Aza-3-aminopyroglutamyl-4-cyclohexyl-9-carboxamide-2-oxo-bicyclo[4.3.0]non-2-ene; ßCH-TRH = (6R, 9S, 12S)-1-Aza-3-aminopyroglutamyl-4-cyclohexyl-9-carboxamide-2-oxo-bicyclo[4.3.0]non-2-ene. 
Received for publication May 4, 1999.
Revision received August 31, 1999.
Accepted for publication September 22, 1999.
 |
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