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INTRODUCTION |
Thyrotropin-releasing hormone
(TRH)1 is involved in
controlling the production of thyroid-stimulating hormone and prolactin from the anterior pituitary gland. TRH functions via binding to its
receptor subtype that belongs to the large family of G-protein-coupled receptors (GPCRs), the first of which identified (1-4) is now known as
TRH receptor 1 (TRHR). As with many other GPCRs, there has been great
interest in the mechanisms of regulation of TRHRs. Although the events
underlying TRHR intracellular signaling and trafficking have been
studied (5-11), the potential for TRHRs to undergo receptor-receptor
interactions has not been previously addressed. Traditionally, GPCRs
were thought to function as monomeric units, coupling to their cognate
G-proteins in a 1:1 stoichiometry upon agonist activation. However, a
growing body of biochemical and functional evidence supports the
existence of homo- and heterodimers and oligomers and thus a critical
role for GPCR-GPCR interactions in receptor function.
Early functional evidence for GPCR dimerization came from the
observation that the co-expression of two defective receptors can
restore receptor activity by trans-complementation between mutant or
chimeric receptors (12, 13). Additional functional evidence has come
from analyzing the effect of dominant receptor mutants on wild-type
receptor function (14-18), and co-immunoprecipitation studies of
epitope-tagged receptors have been used to demonstrate that many GPCRs
can exist as homodimers or oligomers (14, 19-26). It has been
suggested that hetero-oligomerization may represent a general
phenomenon among GPCRs, resulting in a greater diversity of GPCR
function (27-35).
In order to assess whether homo- and hetero-oligomerization exists
among GPCRs in living cells, newly developed biophysical methods have
been utilized to provide convincing evidence for the formation of
receptor complexes. Fluorescence resonance energy transfer (FRET) has
been used to show homodimerization/homo-oligomerization of somatostatin
receptors (26) and GnRHRs (36) and heterodimerization between
somatostatin receptor, somatostatin receptor 5, and the dopamine D2
receptor (29). Bioluminescence resonance energy transfer (BRET)
represents a novel derivation of the FRET technique (37). This approach
involves the transfer of energy resulting from the degradation of
coelenterazine by Renilla luciferase (Rluc) to green
fluorescent protein or a red-shifted variant, enhanced yellow
fluorescent protein (EYFP), which in turn emits fluorescence. BRET is
strictly dependent on the molecular proximity between the energy donor
(Rluc) and acceptor (EYFP), making it ideal for studying
protein-protein interactions. Furthermore, it has advantages over FRET
in that it avoids the need for fluorescence excitation and thus
possible cell damage and photobleaching (37). BRET was initially used
to detect interactions between the cyanobacteria clock protein KaiB in
Escherichia coli (37). More recently, BRET has
been used to demonstrate that the
2-adrenergic receptor exists as functional dimers in living cells and also the
agonist-induced interaction between the receptor and adaptor protein
-arrestin 2 (38). Here we have taken advantage of this newly
developed biophysical technique to investigate whether the TRHR could
exist as oligomers in living mammalian cells. We have shown that in the
unbound state, the TRHR exists as preformed homo-oligomeric complexes
and that this interaction is modulated by agonist activation of the
receptor. BRET was also used to examine the agonist-promoted interaction between TRHR and GnRHRs with the intracellular adaptor protein
-arrestin to demonstrate a direct real time interaction in
intact cells.
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EXPERIMENTAL PROCEDURES |
Eukaryotic Expression Constructs--
The TRHR/Rluc construct
was generated by amplification of the rat TRHR coding sequence (39)
without its stop codon using sense and antisense primers containing
BamHI and NotI sites, respectively. The fragment
was then cloned in frame into the pRluc vector constructed by insertion
of the Rluc coding region into pcDNA3 (Invitrogen). Similarly, the
TRHR/EYFP fusion construct was created by insertion of the
BamHI/NotI fragment in frame into the EYFP vector
constructed by insertion of the EYFP coding region into pcDNA3.
The GnRHR/Rluc and GnRHR/EYFP constructs were generated by
amplification of the rat GnRHR +1 stop codon mutant coding region (40)
without its stop codon using sense and antisense primers containing
EcoRV and NotI sites, respectively. This
generated a 10-amino acid C-terminal extension to the GnRHR onto which
the Rluc or EYFP coding sequence could be added. This GnRHR
EcoRV/NotI fragment was then cloned into the
pcDNA3 vector containing either Rluc or EYFP as described above.
The Rluc/EYFP fusion construct was generated by amplification of the
Rluc coding region without its stop codon containing HindIII
and EcoRV sites and insertion of this
HindIII/EcoRV fragment in frame into the
HindIII/EcoRV sites of the EYFP vector.
The rat GnRHR and rat GnRHR/TRHR carboxyl-terminal tail chimera
(GnRHR/TRHR tail) have been previously described (40). The cDNA for
human protease-activated receptor 1 was provided by L. Brass
(University of Pennsylvania, Philadelphia, PA). The dynamin dominant
negative mutant (K44A) construct was provided by S. Schmid (University
of California, San Francisco, CA). The
2-adrenergic/Rluc (
2-AR/Rluc) and
2-adrenergic/EYFP
(
2-AR/EYFP) fusion constructs were provided by M. Bouvier (University of Montreal) and were previously described
(38).
Cell Culture and Transfection--
HEK 293 and COS 1 cells
(ATCC) maintained in Dulbecco's modified Eagle's medium containing
10% fetal calf serum, glutamine (0.3 mg/ml), and
penicillin/streptomycin (100 units/ml) (Life Technologies, Inc.) at
37 °C in 5% CO2 were seeded at a density of 5 × 105 or 1 × 106 cells/ml per 60- or 100-mm
dish, respectively. Transient transfections were performed the
following day with 5 or 10 µg of total DNA using Superfect (Qiagen),
and cells were used 48 h post-transfection.
Iodination of GnRH Agonist--
Iodinated
[D-Trp6,Pro9,N-Et]GnRH
(Sigma) was prepared using the lactoperoxidase method and purified by
chromatography on a Sephadex G-25 column in 0.01 M acetic
acid/0.1% bovine serum albumin. The specific activity was 42 µCi/µg and was calculated as described previously (41).
Confocal Imaging--
Transfected HEK 293 cells were plated onto
poly-L-lysine-coated eight-well chamber slides 24 h
after transfection. Treatments were carried out 48 h
post-transfection, and then cells were fixed in 4% paraformaldehyde,
mounted in Fluoroguard (Bio-Rad), and sealed with a coverslip. Cells
were excited with a 488-nm laser light and examined using a Bio-Rad
confocal laser microscope under an oil immersion × 60 objective
with light filtered in the green channel from 500 to 550 nm.
Whole Cell Receptor Binding Assay--
Dose displacement
receptor binding assays were performed on COS 1 cells transiently
transfected with untagged and tagged TRHR or GnRHR expression
constructs in 24-well plates. Briefly, cells were incubated for 120 min
at 4 °C in assay buffer (HEPES-buffered Dulbecco's modified
Eagle's medium containing 0.1% bovine serum albumin) with
125I-labeled
[D-Trp6,Pro9,N-Et]GnRH
or [3H][Me-His2]TRH (PerkinElmer Life
Sciences) with or without unlabeled agonist. The concentration of
unlabeled agonist used ranged from 10
6 to
10
11 M. The cells were then
washed and solubilized in 0.2 M NaOH, 1% SDS, and the
radioactivity was counted, with receptor binding expressed as a
percentage of maximum specific binding.
Receptor Internalization Assay--
Receptor internalization
assays were performed in HEK 293 cells as previously described (42).
Briefly, transiently transfected cells in 24-well plates were incubated
with 125I-labeled
[D-Trp6,Pro9,N-Et]GnRH
or [3H][Me-His2]TRH at 37 °C for 5-120
min. Surface bound radioactivity was then removed by washing with acid
solution for 12 min. Internalized radioactivity was determined after
solubilizing cells in 0.2 M NaOH, 1% SDS. Nonspecific
binding for each time point was determined under the same conditions in
the presence of 1 µM unlabeled GnRH agonist (leuprolide)
or TRH. After subtraction of nonspecific radioactivity, internalized
radioactivity was expressed as a percentage of the total binding. All
time points were performed in duplicate in at least three separate experiments.
BRET Assay--
Forty-eight hours post-transfection, COS 1 cells
were detached with phosphate-buffered saline plus 0.05% trypsin and
washed twice in phosphate-buffered saline. Approximately 50,000 cells/well were distributed in a 96-well plate and incubated in the
presence or absence of TRH (Sigma), GnRH agonist, leuprolide (Abbott
Australasia), or GnRH antagonist, Antide (Sigma) (all
10
6 M final concentration) for
the specified time period at 37 °C. The coelenterazine (h form)
(Molecular Probes, Inc., Eugene, OR) was added to a final concentration
of 5 µM, and readings were collected immediately
following this addition. Repeated readings were taken for at least
5-10 min using a custom designed BRET instrument (Berthold, Australia)
which allows sequential integration of the signals detected in the
440-500 and 510-590 nm windows. Data are represented as a normalized
BRET ratio, which is defined as the BRET ratio for the co-expression of
the Rluc and EYFP constructs normalized against the BRET ratio for the
Rluc expression construct alone (38). The BRET ratio is defined as
((emission at 510-590 nm)
(emission at 440-500 nm) × cf)/(emission at 440-500 nm), where cf
corresponds to (emission at 510-590 nm/emission at 440-500 nm) for
the Rluc construct expressed alone in the same experiment.
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RESULTS |
Characterization of Receptor Rluc and EYFP Fusion
Constructs--
TRHR fusion proteins with either Rluc or EYFP added to
the C terminus were validated to ensure they displayed functional
characteristics similar to untagged, wild-type receptors. Confocal
microscopy was used to demonstrate that the TRHR/EYFP fusion protein
was expressed on the plasma membrane (Fig.
1A, I). Radioligand
binding and internalization assays were used as an additional means to measure receptor expression at the cell surface as well as receptor function compared with wild type. Both TRHR/Rluc and TRHR/EYFP were
shown to express at the cell surface, and dose displacement binding
assays revealed similar IC50 values compared with wild-type (Table I). Furthermore, the rate of
TRHR/Rluc fusion receptor internalization was not significantly altered
in comparison with untagged receptor in response to TRH (Fig.
1B). The TRHR/EYFP fusion receptor, however, showed 10%
less ligand-induced internalization when compared with wild-type
TRHRs.

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Fig. 1.
Functional characterization of tagged TRH and
GnRH receptor constructs. A, visualization of receptor at
the cell surface was carried out using confocal microscopy on HEK 293 cells transiently transfected with TRHR/EYFP (I) or
GnRHR/EYFP (II). B, comparison of internalization
rates of tagged TRH and GnRH receptors compared with untagged receptor.
HEK 293 cells were transfected with receptor constructs, and
internalization assays were carried out after ligand treatment (5 min
to 2 h). Assays were carried out at least three times, and results
shown are from a representative experiment.
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Table I
IC50 values for wild-type and BRET fusion receptors
Whole cell binding assays were carried out in COS 1 cells expressing
either wild-type or receptor fusion proteins with increasing amounts of
unlabeled agonist (10 11 to 10 6 M). TRHR
and GnRHR displacement binding assays were performed using
[3H][Me-His2]TRH and
[125I][D-Trp6,Pro9,N-Et]GnRH,
respectively. Results shown are the mean ± S.D. of three
independent experiments.
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In order to determine specificity of the interaction observed between
TRHRs, EYFP and Rluc fusion constructs of another GPCR, the GnRHR, were
generated and validated as described above. A stop codon mutant
encoding a GnRHR with a 10-amino acid C-terminal linker described
elsewhere (40) was used. Similar to the TRHR/EYFP, the GnRHR/EYFP was
expressed on the plasma membrane, as demonstrated by confocal
microscopy, although some cytoplasmic fluorescence was observed,
suggesting the presence of an intracellular pool of receptors (Fig.
1A, II). Receptor binding assays and
internalization assays in cells expressing each of the GnRHR fusion
constructs further illustrated the expression of both GnRHR/EYFP and
GnRHR/Rluc at the cell surface. The GnRHR has previously been shown to
internalize at a slower rate compared with the TRHR in both HEK 293 and
COS cells (42). Neither the binding (Table I) nor the internalization (Fig. 1B) characteristics of either of the Rluc- and
EYFP-tagged forms of the GnRHR were affected compared with wild-type receptor.
Agonist-independent (Constitutive) TRHR Interaction--
To
determine whether constitutive interaction occurred between TRHRs in
living cells, BRET was measured in COS 1 cells co-expressing either
TRHR/Rluc and pcDNA3 vector or TRHR/Rluc and TRHR/EYFP fusion
proteins. The ratio of TRHR/Rluc:TRHR/EYFP plasmid concentration used
for transfection was 1:4. An excess of fluorescent acceptor molecule
expression over donor is thought to favor more efficient energy
transfer, and initial experiments using a range of ratios of TRHR/Rluc
to TRHR/EYFP DNA showed that the 1:4 ratio resulted in an optimal BRET
ratio (data not shown). Following the addition of the cell-permeable
Renilla luciferase substrate, coelenterazine, a
bioluminescent signal was emitted in both the 440-500- and 510-590-nm filter windows in cells co-expressing TRHR/Rluc and vector. To quantitate the BRET signal, the ratio of light emitted in the 510-590-nm window over that emitted in the 440-510-nm window was determined as described under "Experimental Procedures." Cells expressing both TRHR/Rluc and TRHR/EYFP showed an increase in the
relative amount of light emitted in the 510-590-nm window (corresponding to the emission peak wavelength of EYFP) and thus an
increase in the BRET ratio of 0.12 relative to the BRET ratio obtained
for TRHR/Rluc only (Fig. 2). To rule out
the possibility that BRET was occurring between different transfected
cells that were expressing either one of the TRHR fusion constructs,
but not both in the same cell, monotransfectants expressing either TRHR/Rluc or TRHR/EYFP were combined. BRET ratios from these combined cells (data not shown) were compared with cells expressing either TRHR/Rluc alone or co-expressing both tagged constructs.
Co-transfection of both TRHR/Rluc and TRHR/EYFP in the same cells was
necessary for BRET to occur (Fig. 2), thus providing strong evidence
that an actual interaction was occurring in the same cell.

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Fig. 2.
Constitutive and
agonist-dependent oligomerization of the TRH receptor.
BRET was measured in COS 1 cells transfected with either TRHR/Rluc
alone or with both TRHR/Rluc and TRHR/EYFP. Cells were incubated with 5 µM coelenterazine, and BRET readings were taken
immediately. TRH or GnRH at 10 6 M
final concentration was incubated for 20 min before the addition of
substrate. Data shown are represented as the normalized BRET ratio
against readings obtained with TRHR/Rluc alone. Assays were carried out
at least three times, and results shown are mean ± S.D.
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Agonist Modulation of TRHR Interaction--
Given that the
evidence indicated the existence of TRHR complexes, we next wanted to
investigate whether agonist activation affected the nature or extent of
TRHR aggregation. Following agonist treatment of COS 1 cells
co-expressing TRHR/Rluc and TRHR/EYFP, an additional increase (~65%)
in the BRET signal was observed in comparison with untreated
co-transfectants (Fig. 2). No increase in the BRET signal was observed
when either vehicle or GnRH agonist was used.
The effect of receptor activation on TRHR/Rluc and TRHR/EYFP
energy transfer increased with time of exposure to agonist, reaching a
maximum following a 20-min incubation (Fig.
3A). In addition, a
dose-dependent increase in agonist promoted the BRET ratio
(Fig. 3B), with an ED50 of 8.75 ± 1.76 nM, in a similar range to that observed for ligand binding
(Table I). To examine the relationship between receptor expression and
BRET levels, increasing concentrations of TRHR/Rluc and TRHR/EYFP at a
1:4 ratio were transfected into COS 1 cells. BRET was shown to increase
with increasing concentrations of plasmid DNA, reaching a plateau at
the concentration of 0.5 µg of TRHR/Rluc with 2.0 µg of TRHR/EYFP
(Fig. 4). Levels of receptor expression
measured by binding correlated with BRET.

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Fig. 3.
Effect of time and agonist concentration on
TRH receptor oligomerization. A, BRET ratios in COS 1 cells expressing TRHR/Rluc and TRHR/EYFP were measured immediately
after the addition of coelenterazine, and repeated measures were taken
for up to 30 min. Assays were performed at least three times, and the
results shown are single reads for each time point from a
representative experiment. B, BRET ratio measurements were
performed in response to increasing concentrations of TRH
(10 11 to 10 6
M). Data represent the mean ± S.D. generated from
four readings taken at each dose from a representative
experiment.
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Fig. 4.
Effects of TRH receptor expression levels on
BRET ratios. COS 1 cells were co-transfected with increasing
amounts of plasmid DNA for TRHR/Rluc and TRHR/EYFP, as shown. The BRET
ratio (black bars) was measured immediately
following the addition of coelenterazine. The same batches of
transfected cells were also used in whole cell binding assays
(white bars). Results are expressed as the
percentage of maximum binding/BRET ratio for each plasmid
concentration. Assays were carried out at least three times, and
results shown are mean ± S.D.
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Co-expression of Tagged Receptors--
The BRET observed between
the TRHR/Rluc and TRHR/EYFP did not result from nonspecific interaction
between the Rluc and EYFP moieties of the fusion proteins. No increase
in the signal was detected when Rluc and EYFP were co-expressed as
nonfusion proteins, compared with Rluc expressed alone (data not
shown). This is in contrast to the BRET signal generated by a fusion
construct of Rluc covalently linked to EYFP used as a positive control,
which produced an increased BRET ratio over that of Rluc alone (data not shown). The small increase in the basal levels of the BRET ratio
(~0.03) observed upon co-expression of any Rluc and EYFP fusions or
nonfusions is possibly due to a small degree of nonspecific interaction/proximity of Rluc and EYFP moieties as a result of overexpression.
GnRHR/Rluc, GnRHR/EYFP, and
2-AR/EYFP fusion constructs
were co-expressed with the TRHR fusion proteins, in order to control for the specificity of the TRHR/Rluc and TRHR/EYFP interaction and to
rule out the possibility that it is not due to receptor overexpression
at the membrane. Co-expression of the TRHR/Rluc and GnRHR/EYFP did not
lead to an energy transfer in either untreated cells or in cells
treated with either TRH or GnRH agonist alone or both together (Fig.
5A). A similar finding was
observed for cells co-expressing the combination of either the
TRHR/EYFP with the GnRHR/Rluc or TRHR/Rluc with the
2-AR/EYFP (Fig. 5A).

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Fig. 5.
Specificity of TRHR/Rluc and TRHR/EYFP
interactions. A, effect of Rluc- and EYFP-tagged receptor
constructs. COS 1 cells were transfected with TRHR/Rluc and either
pcDNA3, TRHR/EYFP, GnRHR/EYFP, or 2-AR/EYFP. The
GnRHR/Rluc was co-transfected with pcDNA3, TRHR/EYFP, or
GnRHR/EYFP. The BRET ratio was measured with or without agonist (TRH,
GnRH, or both as indicated at 10 6
M). B, effect of wild-type receptor on TRHR BRET
ratio. COS 1 cells transfected with 0.5 µg of TRHR/Rluc and 1.5 µg
of TRHR/EYFP were also transfected with 3 µg of either pcDNA3,
untagged TRHR, GnRHR, GnRHR/TRHR C-tail, or protease-activated receptor
1. The BRET ratio was taken immediately after the addition of
coelenterazine. Assays were carried out at least three times, and
results shown are mean ± S.D.
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The low level of BRET observed in COS 1 cells co-expressing both the
GnRHR/Rluc and GnRHR/EYFP compared with either GnRHR/Rluc with
TRHR/EYFP, GnRHR/EYFP with TRHR/Rluc, or GnRHR/Rluc alone (Fig.
5A) suggests that the GnRHR may not form oligomers in the absence of ligand. However, following the addition of GnRH agonist, an
increase in the BRET ratio was observed (Fig. 5A),
indicating that an interaction between GnRHR/Rluc and GnRHR/EYFP does
occur. The addition of a GnRH antagonist (Antide;
10
6 M), had no effect on BRET
(results not shown).
Co-expression of Untagged Receptors--
To further demonstrate
the specificity of the TRHR interaction, untagged TRHR was transfected
into COS 1 cells in addition to TRHR/Rluc and TRHR/EYFP. Expression of
untagged TRHR was found to markedly reduce the BRET signal generated
between the TRHR/Rluc and TRHR/EYFP fusions (Fig. 5B). In
contrast, expression of other untagged GPCRs including the GnRHR,
GnRHR/TRHR chimera and protease-activated receptor 1, did not
significantly inhibit the BRET signal observed between the two tagged
TRH receptors (Fig. 5B). Untagged receptor expression was
confirmed by ligand binding assays (data not shown). Interestingly, no
reduction in BRET was observed when the GnRHR/TRHR tail chimera was
used, indicating that the TRHR C-terminal tail is not involved in the
complex formation of this receptor. This suggests that the
ligand-independent BRET observed between TRHR/Rluc and TRHR/EYFP is due
to a specific interaction between at least two TRHR monomers and gives
evidence for the existence of preformed or constitutive receptor oligomers.
Inhibition of Receptor Internalization--
Agonist stimulation is
known to cause clustering and internalization of the TRHR (10, 42-44).
Thus, it is possible that the increase in BRET observed following TRH
stimulation could result from an increase in the number of Rluc- and
EYFP-tagged receptors in close proximity in coated pits. To establish
that the BRET signal was not due to receptor clustering, we employed
the dominant negative dynamin mutant (Dyn K44A) known to efficiently
block TRHR internalization by inhibiting clathrin-mediated endocytosis (45, 46). Dyn K44A impaired internalization of tagged TRHRs regardless
of whether they were expressed individually or together in the same
cells (Fig. 6A). However, the
presence of Dyn K44A had no effect on the agonist-dependent
increase in BRET (Fig. 6B). This supports the hypothesis
that the agonist-induced BRET signal is due to microaggregation or
oligomerization of the TRHRs and not to clustering of receptors or
macroaggregation as a result of receptor internalization.

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Fig. 6.
Effects of inhibition of TRH receptor
internalization on oligomerization. COS 1 cells expressing TRH
receptor constructs either with pcDNA3 (black
bars) or dynamin K44A (shaded bars)
were measured for levels of receptor internalization (A) and
BRET ratio in the presence or absence of TRH (B). Assays
were carried out at least three times, and results shown are
representative of a single experiment.
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Measurement of Receptor Interaction with
-Arrestin by
BRET--
To determine whether other protein-protein interactions
involving the TRHR and GnRHR fusion proteins could be measured by BRET
and to assess if other ligand-dependent receptor
interactions could be detected, we investigated the known
-arrestin
dependence of the TRHR (8, 42, 46) and the
-arrestin
independence of the GnRHR (42, 46).
The
-arrestin fusion protein was validated by co-expression of
-arrestin/EYFP with either TRHR/Rluc or GnRHR/Rluc in HEK 293 cells
in order to visualize an agonist-dependent redistribution of
-arrestin using confocal microscopy. The
-arrestin/EYFP- and
TRHR/Rluc-expressing cells showed a visible redistribution from the
cytoplasm to the cell surface after 90 s of agonist stimulation (Fig. 7A, II), as
confirmed in previous studies using
-arrestin/green fluorescent
protein (8, 42, 46). In contrast, in cells co-expressing GnRHR/Rluc,
there was no redistribution of
-arrestin/EYFP after the addition of
GnRH (Fig. 7A, IV), confirming that the addition
of Rluc or EYFP to these proteins does not affect the
-arrestin
independence of the tagged GnRHR. In order to measure the BRET ratio
between TRHR and
-arrestin, COS 1 cells were co-transfected with
TRHR/Rluc and pcDNA3 or TRHR/Rluc and
-arrestin/EYFP. There was
a very small increase in the BRET ratio upon co-expression of
-arrestin/EYFP compared with TRHR/Rluc alone, although this increase
is also seen when co-expressing TRHR/Rluc with EYFP only (data not
shown) due to a small degree of nonspecific interaction as a result of
overexpression. However, the addition of TRH caused a dramatic increase
in the BRET ratio (Fig. 7B), confirming that
-arrestin
only associates with the agonist-activated receptor (47). COS 1 cells
co-expressing GnRHR/Rluc and
-arrestin showed no increase in the
BRET ratio following the addition of GnRH agonist (Fig. 7B),
supporting our previous data indicating that GnRHR undergoes
-arrestin-independent internalization (42, 46).

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Fig. 7.
Measurement of the interaction of
-arrestin 1 and TRHR using BRET. A,
visualization of -arrestin 1/EYFP redistribution was assessed using
confocal microscopy in HEK 293 cells co-expressing either TRHR/Rluc or
GnRHR/Rluc with -arrestin 1/EYFP. I and III,
untreated; II, TRH (10 6
M, 90 s); IV, GnRH agonist
(10 6 M, 10 min). B,
BRET ratio was measured in COS 1 cells expressing TRHR/Rluc and
pcDNA3; TRHR/Rluc and -arrestin 1/EYFP; GnRHR/Rluc and
pcDNA3; and GnRHR/Rluc and -arrestin 1/EYFP. Measurements were
taken in the absence or presence of the appropriate ligand
(10 6 M). Assays were carried out
at least three times, and results shown are representative of a single
experiment.
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DISCUSSION |
There has been a recent surge of interest in the field of
GPCR-GPCR interactions, with an increasing number of reports describing the existence and functional importance of GPCR homo- and
hetero-oligomerization. A variety of functional and biochemical
techniques have been utilized to demonstrate the existence of higher
order complexes among GPCRs. We have utilized a newly developed
biophysical method, BRET, which has several advantages over previously
used methods to demonstrate that the TRHR can undergo
homo-oligomerization in living cells. Although FRET, a
proximity-dependent assay related to BRET, has also
been used to show dimerization of GPCRs in living cells (26, 29, 36),
the requirement for excitation, which results in photobleaching and
acceptor autofluorescence, can limit the usefulness of FRET (37). The
reduction in background light levels in BRET due to circumvention of
excitation should allow detection of interacting proteins at much lower
concentrations than are required for FRET and thus possibly detect
basal interactions undetectable using FRET.
In order to assess whether monomeric TRHRs were able to interact, TRHR
fusion proteins were generated with either the bioluminescent donor,
Rluc, or fluorescent acceptor, EYFP. The Rluc- and EYFP-tagged TRHR
constructs all showed cell surface expression, ligand binding, and
internalization, similar to wild-type receptor. The addition of green
fluorescent protein to the TRHR does not prevent interaction with
-arrestin (8), although TRHR/EYFP did internalize slightly slower
than the untagged receptor, as has been previously reported for green
fluorescent protein-tagged
1 and
2-adrenoreceptors (48).
Co-expression of TRHR/Rluc and TRHR/EYFP resulted in an increase in the
ratio of light emitted in the 440-500- and 510-590-nm filter windows
used to detect an energy transfer over that emitted by TRHR/Rluc alone
and thus an increase in the defined BRET ratio. The detection of BRET
between TRHR fusion proteins under basal conditions demonstrates a
close proximity between the TRHR/Rluc and TRHR/EYFP, since the maximum
distance allowing energy transfer between the Rluc and EYFP BRET pair
is ~50 Å (37). Our observation can be best explained by the
existence or formation of constitutive receptor dimers or oligomers.
This supports our previous data obtained from immunoprecipitation
studies of the hemagglutinin-tagged TRHR, where the formation of higher
molecular weight protein species was observed, most likely reflecting
dimer or oligomer formation (49). In this study, we have used the term
oligomer to refer to the interaction between the TRH receptors detected
by BRET, with the understanding that the BRET signal could result from the formation of dimeric and/or oligomeric complexes.
The observation that constitutive receptor dimers exist has been
reported for several GPCRs (21, 23, 38). It is still presently unclear
whether the receptors form dimers or oligomers at the cell surface or
intracellularly prior to trafficking to the cell surface. However,
evidence from the GABAB receptor and other GPCRs indicates
that constitutive dimers may preform intracellularly before trafficking
to the cell surface (16-18, 32, 50). If the dimer or oligomer is the
functional receptor unit, one could speculate that the constitutive
signaling observed for the TRHR (9) may relate to the preexisting TRHR
homomeric complexes.
TRHR dimer formation was found to be modulated by agonist activation,
with a further increase in the BRET signal observed for cells
co-expressing TRHR/Rluc and TRHR/EYFP following treatment with TRH.
This occurred in a time- and dose-dependent manner with an
EC50 value comparable with those obtained for the ligand
binding and signaling of the wild-type TRHR (6). Titration of the
quantity of co-transfected TRHR/Rluc and TRHR/EYFP resulted in a
correlation between receptor binding and BRET ratio, indicating that
the degree of receptor oligomerization detected is influenced by the
level of cell surface receptor expression.
The agonist promotion of the BRET ratio could be indicative of an
increase in the number of oligomers formed and/or a change in the
conformation of preexisting complexes that results in closer proximity
or more favorable orientation of donor and acceptor molecules (38). A
recent study has shown that for the metabotropic glutamate receptor,
glutamate binding stabilizes the preexisting dimer (51), whereas other
GPCRs including somatostatin receptor 5 (26) and GnRHR (36) undergo
ligand-dependent dimerization.
The constitutive and agonist-dependent BRET observed
between the TRHRs was shown to be a specific interaction in several
ways. First, no interaction was detected between co-expressed Rluc and EYFP moieties or between the tagged TRHR and either of the
complementary tagged GnRHR or
2-adrenergic receptors,
indicating that BRET was not a result of a nonspecific EYFP and Rluc
interaction. These results also suggest that heteromeric complexes do
not form between the TRHR and GnRHR or between the TRHR and
2-adrenergic receptor. Overexpression of untagged
receptors was employed to demonstrate that the constitutive BRET signal
detected was specific for the TRHR interaction, since a reduction in
the BRET signal was only observed upon co-expression of the wild-type
TRHR with TRHR/Rluc and TRHR/EYFP. Interestingly, expression of the
GnRHR/TRHR tail chimera did not interfere with BRET-detected
oligomerization, indicating that the C-terminal tail of TRHR is not
required for this process. It is proposed that several regions of a
GPCR may be involved in dimer formation, including the N terminus and
extracellular domains (24, 25, 52), transmembrane domains (14, 15, 20),
intracellular loops (12), and the C terminus (22, 32) and may involve
the formation of disulfide bonds as wells as noncovalent interactions.
Agonist stimulation of the TRHR is known to promote clustering and
internalization through clathrin-coated pits (42, 44, 45). Therefore,
we also wanted to determine whether the BRET observed following TRH
induction was due to agonist modulation of receptor oligomers and not
just due to the close proximity between Rluc- and EYFP-tagged receptors
clustering within clathrin-coated pits. BRET did not occur in cells
co-expressing TRHR and either
2-adrenergic or GnRH
receptors, all of which internalize via clathrin-coated pits (42, 53)
following TRHR agonist stimulation. Furthermore, co-expression of the
dynamin dominant negative mutant (K44A), known to efficiently block
receptor internalization (54), including TRHR internalization (45, 46),
did not affect the agonist-induced rise in the BRET signal. These
findings demonstrated that clustering of receptors within the pits is
not sufficient to allow energy transfer between noninteracting receptors.
Interestingly, the GnRHR was shown to interact following agonist
stimulation, suggesting the formation of oligomers, analogous to
findings reported recently (36) and strengthening the use of BRET to
detect GPCR oligomerization. We were unable to detect any preexisting
GnRHR complexes; however, an earlier study showed that co-expression of
the wild-type receptor and a truncated splice variant impaired GnRHR
cell surface expression and signaling (17), which may suggest the
existence of preformed GnRHR dimers. Growing evidence suggests that
heterodimerization/oligomerization may be a general phenomenon among
GPCRs (26, 29, 34, 35) and represent a means of increasing the
diversity of cellular responses to a wide range of extracellular
signals. However, the TRHR did not appear to form constitutive or
agonist-stimulated hetero-oligomers with the GnRHR, at least not under
the conditions used to demonstrate TRHR and GnRHR homo-oligomerization.
This does not rule out the possibility that the co-expression of both
receptors in the correct cell type in vivo may result in
formation of a heteromeric complex.
The BRET technique and the TRHR and GnRHR fusion constructs were
further validated by measurement of their interaction with
-arrestin
1. Our earlier studies have shown that while the TRHR undergoes
-arrestin-dependent internalization, the GnRHR does not
(42, 46). BRET measurements revealed an agonist-dependent interaction between TRHR and
-arrestin, while there was no
-arrestin interaction observed for the GnRHR. The GnRHR is a unique
GPCR in that it lacks the functionally important intracellular
C-terminal domain, which may be responsible for its inability to
interact with
-arrestin and its consequent slow internalization
kinetics (42). The interaction between the
2-AR and
-arrestin has also been demonstrated using BRET (38).
The role for both receptor homo- and hetero-oligomerization is rapidly
growing. Using the BRET technique, we were able to demonstrate that the
TRHR and GnRHRs both form homomeric complexes and that, in the case of
the TRHR, constitutive and agonist-promoted oligomerization is present.
We were also able to confirm using BRET the differential ability of the
TRHR and GnRHR to interact with
-arrestin. The development of
biophysical techniques to study receptor-protein interactions in living
cells provides an effective means to obtain direct evidence for the
role of these events in GPCR function.