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J. Biol. Chem., Vol. 276, Issue 3, 2153-2158, January 19, 2001
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From the Oregon Regional Primate Research Center and Department of
Physiology and Pharmacology, Oregon Health Sciences University,
Beaverton, Oregon 97006
Received for publication, August 28, 2000, and in revised form, October 3, 2000
Gonadotropin-releasing hormone (GnRH) regulates
pituitary gonadotropin release and is a therapeutic target for human
and animal reproductive diseases. In the present study we have utilized
the technique of fluorescence resonance energy transfer to
monitor the rate of GnRH receptor-receptor interactions. This technique relies on the observation that the degree of physical intimacy of
molecules can be assessed by the tendency of proximal fluorophores to
exchange energy. Our data indicate that GnRH agonist, but not antagonist, occupancy of the GnRH receptor promotes physical intimacy (microaggregation) between receptors. The time course indicates that
this occurs promptly (<1 min) after occupancy and persists for at
least 80 min and within the physiologically relevant range of the
releasing hormone. The process measured is not inhibited by 0.1 mM vinblastin, 2 µM cytochalasin D, or
3 mM EGTA, an observation that distinguishes it from
macroaggregation (patching, capping, and internalization).
These observations, along with reports from other laboratories, are
consonant with a growing body of evidence that indicates that
microaggregation is an early event following agonist occupancy of the
receptor and part of the mechanism by which effector regulation occurs.
Even before the gonadotropin-releasing hormone
(GnRH)1 receptor was cloned
and sequenced or known to be a member of the G-protein-coupled receptor
(GPCR) superfamily, evidence from antibody cross-linking studies
suggested that promoting small scale receptor-receptor interactions
(such as dimerization) stimulated multiple gonadotrope end points
including gonadotropin release, regulation of receptor numbers, and
target cell sensitivity (1-6). Subsequently it was shown that
treatment of gonadotropes with releasing hormone agonists caused
receptors to move sufficiently close to one another that a radioiodine
molecule could be transferred from a lactoperoxidase molecule
covalently linked to a receptor-bound agonist onto an adjacent receptor
(7). These two types of observations suggested that agonist occupancy
of the receptor promotes microaggregation (physical association within
100-120 Å), the occurrence of which is sufficient to stimulate
cellular responses. For these reasons, microaggregation has been
suspected to be a component of the mechanism leading to hormonal
activation of the gonadotrope (1). This process appears to occur as
quickly as it can be measured (<1 min) (7) and is distinguished from
macroaggregation (patching, capping, internalization), an
event that occurs later (>20 min), appears sensitive to
vinblastin, cytochalasin D, and EGTA, and is associated with extinction
of responses (8, 9).
In the intervening years since microaggregation was proposed,
receptor-receptor interactions have been suggested to occur for a range
of GPCRs and for other receptors (10-14). Recently it was suggested
(15, 16) that heterodimers among GPCRs may provide a significant level
of physiological regulation. The GnRH receptor is an intriguing member
of the GPCR superfamily because it is the smallest known at this time
with a short extracellular N-terminal tail and virtually no
C-terminal intracellular tail, a region associated with modifications
that regulate the loss of other receptors.
Unfortunately there has not been a reliable or convenient method
adequate to measure interactions between GnRH receptors, let alone
develop mathematic descriptions for this process (17, 18). Fluorescence
resonance energy transfer (FRET) is useful for assessment of
protein-protein interactions. To occur, it is necessary that two
different fluorophores are within 100 Å of each other and that the
emission spectrum of the donor overlaps the absorption spectrum of the
acceptor. We (19, 20) and others (21) have reported the usefulness of
chimeras of GnRHR-spacer-GFP (green fluorescent protein) for tracking
receptor trafficking. The recent availability of red fluorescent
proteins, excellent spectral partners of GFP for FRET at distances of
100 Å (22), allows the simultaneous visualization of both fluorescent
proteins in a confocal microscope, the observation of receptor
aggregation in real time, and the measurement of aggregation rates. We
show that occupancy by GnRH agonists promotes receptor microaggregation at doses, and with a time course, appropriate for a role of this event
in GnRH receptor signaling in vivo.
Rat GnRHR complementary DNA (cDNA) in pcDNA1 was
generously provided by Dr. W. W. Chin. The expression vector
pcDNA3.1 was purchased from Invitrogen (San Diego, CA). pEGFP-N1
vector, which encodes a GFP variant (F64L, S65T-GFP; REF) for human
codon usage preferences was purchased from
CLONTECH Laboratories, Inc. pDsRed vector, a human
codon-optimized variant that uses the strong cytomegalovirus IE
promoter and allows fusions to the N terminus of RFP1 (referred to as
RFP below), was also purchased from CLONTECH. DMEM,
Opti-MEM, LipofectAMINE, and polymerase chain reaction (PCR) reagents
were purchased from Life Technologies, Inc. Restriction enzymes
and competent cells for cloning were purchased from Promega Corp. (Madison, WI). The GnRH agonist, buserelin
(D-t-butyl-D-Ser6-Pro9des
Gly10 EA GnRH, Hoechst), and antagonist,
"Nal-Arg"
(Ac-D-Nal1-D-4-chlorophenylalanine2-D-3-pyridylalanine3,
Arg5-D-Arg6-D-Ala10-GnRH,
Contraceptive Development Branch, NIH) were obtained as indicated. All
GnRH analog solutions were prepared 10-fold concentrated, and 50 µl
of this solution was added to 450 µl of medium so that the final
concentration was achieved. Other reagents were of the highest degree
of purity available from commercial sources.
Generation of Chimeras of rGnRHR and Fluorescent Proteins
Wild-type rGnRHR cDNA in pcDNA1 was subcloned into
pcDNA3.1 at BamHI and XhoI restriction enzyme
sites. The rGnRHR-C tail-GFP, the fusion of the N terminus of the GFP
to the C terminus of the rat GnRHR with the catfish GnRHR intracellular
C tail, was constructed as described previously (18).
The chimera of rGnRHR-C tail and RFP was constructed by overlap
extension PCR, a procedure used to join DNA fragments that contain an
overlap region. The first PCR reaction included the T7 primer and a
30-mer chimeric reverse primer (CTTGGAGGAGCGCACCAT/CTGTCCACTGGG) encoding for the RFP1-N1 and rGnRHR-C tail without the stop codon. The
sequence for RFP including the full coding region and 3'-untranslated region was amplified from pRFP1-N1 vector using a 30-mer chimeric forward primer (GACCAACCCAGTGGACAG/ATGGTGCGCTCC) and a pRFP1-N1 reverse
primer (RED-N1-Rev). The DNA fragments from the two PCR reactions for
rGnRHR-C tail and RFP were used as templates in a third PCR reaction
with primer sets of T7 and RED-N1-Rev. The third PCR reaction produced
a full-length chimeric cDNA for rGnRHR-C tail-RFP. The chimeric
cDNA was flanked by the restriction sites present in the polylinker
of pcDNA3.1 vector. The cDNA was digested with BamHI
and XbaI and cloned into the same sites of pcDNA3.1 vector. The identity of the chimeric constructs and the correctness of
the PCR-derived coding sequence were verified by Dye Terminator Cycle
Sequencing (PerkinElmer Life Sciences). Large scale plasmid DNA
for transfection was prepared using a Qiagen EndoFree Plasmid Maxi Prep
kit (Qiagen Inc., Valencia, CA). The purity and identity of the plasmid
DNA were further verified by restriction enzyme analysis.
Transient Transfection of GH3 Cells
For confocal microscopy of live cells, glass coverslips (24 × 40 mm) were immersed in 12 N HCl for 1-2 h, rinsed
three times with sterile distilled water, and then secured in a sterile
100-mm Petri dish with dental wax. The GH3 cells were
maintained in growth medium (DMEM (Irvine Scientific, Santa Ana, CA)
containing 10% fetal calf serum (FCS; Life Technologies, Inc.) and 20 µg/ml gentamicin (Gemini Bioproducts, Calabasas, CA)) in a humidified
atmosphere (37 °C) containing 5% CO2. The cells were
plated at a density of 9 × 105 cells/dish.
Twenty-four h later, the cells were washed once with 3 ml of Opti-MEM
(Life Technologies, Inc.) and then were transiently transfected with
0.71 µg of cDNA for chimeric rGnRHR-C tail-RFP plus 0.45 µg of
cDNA for chimeric rGnRHR-C tail-GFP using 10 µl of LipofectAMINE
(Life Technologies, Inc.) in 1.5 ml of Opti-MEM/dish. These cDNA
concentrations resulted in an optimally detectable increase of FRET in
response to cell activation by buserelin. Increasing or decreasing the
ratio of RFP:GFP receptor chimeras resulted in loss of the FRET signal.
Five h later, 1.5 ml of DMEM, 20% FCS was added to each dish.
Twenty four h after the start of the transfection, the medium was
replaced with fresh DMEM, 10% FCS, gentamicin, and the cells were
allowed to incubate for 24 h. Forty eight h after the start of the
transfection, the cells were washed two times with warm medium (DMEM,
0.1% bovine serum albumin, gentamicin) and were then incubated with 10 µM cycloheximide for 24 h. The cells were washed
twice with 26 °C DMEM, 0.1% bovine serum albumin, gentamicin; the
coverslips containing the cells were then transferred to a cell
chamber, and room temperature medium was added to the chamber and
incubated with the live cells during confocal imaging. Where indicated,
50 µl of buserelin solution (10 Inositol Phosphate Bioassay
An inositol phosphate bioassay was used to check the biological
activity of the chimeric constructs. Briefly, GH3 cells
were plated in a 24-well Costar (Cambridge, MA) plate at
105 cells/well. Twenty four h later, the cells were washed
once with 0.5 ml of Opti-MEM and then transiently transfected with 0.8 µg of plasmid chimeric receptor DNA/well or wt rGnRHR cDNA using 2 µl of LipofectAMINE in 0.250 ml of Opti-MEM. Five h later, 0.25 ml
of DMEM, 20% FCS was added per well. Twenty four h after the start of
the transfection, the medium was replaced with 0.5 ml/well DMEM, 10%
FCS, gentamicin, and the cells were allowed to grow. Fifty four h after
transfecting, the cells were washed with DMEM, 0.1% bovine serum
albumin, 20 µg/ml gentamicin and then preloaded with 0.5 ml of DMEM
(inositol free) containing 4 µCi of [3H]inositol
(PerkinElmer Life Sciences) for 18 h at 37 °C. After preloading, the cells were washed with DMEM (inositol free) containing 5 mM LiCl and stimulated with buserelin for 2 h. The
medium was removed, and 1 ml of 0.1 M formic acid was added
to each well. The cells were frozen and thawed to disrupt the cell
membranes. Inositol phosphate accumulation was determined by
Dowex anion exchange chromatography and liquid scintillation
spectroscopy as described previously (23).
Confocal Imaging
General--
Cells expressing both GnRHR-C tail-GFP and GnRHR-C
tail-RFP were imaged in an open chamber in DMEM, 0.1% bovine serum
albumin, gentamicin medium at room temperature maintained at
26-27 °C in a Leica TCS-SP confocal microscope using a
63 × 1.25 numerical aperture water immersion objective,
pinhole 2.5 Airy disc units. GFP was excited with the 488-nm
line of an Ar laser, and RFP was excited with the 568-nm line of a Kr
laser. Emission was measured simultaneously in the green channel from
500-550 nm and in the red channel from 610-670 nm.
FRET was measured in both fixed and live cells, with and without
activation by buserelin, by imaging GFP before and after the red
fluorescent protein was photobleached with a large dose of irradiation
with the 568-nm line of a Kr laser. The increase of donor fluorescence
(green) after receptor (red) bleaching was interpreted as evidence of
FRET occurring from GFP to RFP.
Time Lapse--
For all time-lapse experiments, GFP was excited
with 488-nm blue light with the minimum intensity necessary to obtain
an image, to minimize photobleaching and cell damage due to absorption
of light energy. Emission channels were the same as described above; the image recorded in the red channel was due partly to GFP
contribution in the overlapping red spectral range, partly to
excitation of RFP fluorescence at 488 nm, and partly to the resonance
energy transfer from GFP to RFP. Images were acquired every 5, 10, or 30 s for the first 10 min, every 2 min for up to 30 min,
and every 5 min for up to 2 h. Five sections were acquired for
each time point to compensate possible focus shifts during the
time-lapse experiment. Four-dimensional image stacks were open and
organized in MetaMorph (Universal Imaging, West Chester, PA). For each
time point, only one section was chosen of the five acquired, so that it imaged the same plane within the cell throughout time, compensating for focus shift. Regions of interest were selected on membranes so that
they did not include any of the intracellular red fluorescent signal,
areas of intense membrane blebbing or motion. The average intensity of
the red and green fluorescence was measured in each area of interest at
all time points using MetaMorph. The red to green ratio was calculated
and then normalized to unity for the value at time 0 and graphed as a
function of time.
The GnRHR-C tail-RFP chimera is expressed on the plasma membrane,
as was reported for the GnRHR-C tail-GFP construct (19). In addition,
RFP can be found in cytoplasmic compartments (Fig. 1, A and B); this
material may be misfolded copies or molecules otherwise targeted for
destruction. The GnRHR-C tail-RFP is functional and elicits
inositol phosphate production in response to the GnRH agonist
buserelin, similar to that of the wild-type receptor (Fig. 1C) and to the GnRHR-C tail-GFP construct (19).
In cells expressing both receptor constructs, the red and green
fluorescent proteins are colocalized (interdispersed) on the plasma
membrane. If receptors are present as individual monomers, their
fluorescence will not be affected by each other. If molecules of GFP
and RFP come into appropriate physical proximity, energy absorbed by
illumination of the green fluorophore is transmitted by FRET to the red
fluorophore, which will then emit a red photon. In this case, a
reduction of green fluorescence is associated with the increase of red
fluorescence. FRET only occurs if three conditions are simultaneously
satisfied. First, the emission spectrum of the donor must
overlap with the absorption spectrum of the acceptor. Second, the two
fluorophores must be within 100 Å of each other. Third, the transition
dipoles of the donor and acceptor must be favorably oriented. The
spectra of GFP and RFP are well suited for FRET. If they are part of
dimerized GnRHR-C tail molecules, the distance and orientation will be
favorable for FRET at least part of the time, because each fluorophore
is attached to a flexible 51-amino acid-long linker that confers
sufficient flexibility to allow the fluorophore to attain the
correct dipole orientation. This flexibility will likely prevent FRET
from occurring every time receptors come into intimate association, however.
We show evidence that FRET is increased in GH3 cells
activated by the GnRH agonist buserelin but is not increased in
response to medium or to a GnRH antagonist. These observations are
consonant with GnRH receptor microaggregation as a component of
signal transduction.
FRET can occur only in the presence of an acceptor molecule. If RFP
(the acceptor) is removed or destroyed, it would be predicted that the
fluorescence of GFP (the donor) would increase upon illumination. Fig.
2 shows examples of cells imaged before
(A and B) and after (C and
D) photobleaching of RFP on an area including the plasma membrane. The integrated intensity of green (Fig. 2, A and
C) and red (B and D)
fluorescence is measured for the same rectangular area that
includes the membrane. For Fig. 2, panel I, cells were fixed
after 30 min of treatment with buserelin. For the cell shown, the green
fluorescence intensity increased from 68,217 to 69,313 (arbitrary
units) when the red fluorescence intensity decreased from 41,844 to
17,542 after specific photobleaching of RFP by 568-nm radiation.
Changes in green fluorescence measured for other cells in the same
sample ranged from 5 to 15%. In Fig. 2, panel II, control
cells were fixed without buserelin treatment. In the example shown,
green fluorescence was 43,903 before and 43,406 after photobleaching of
RFP from 13,880 to 5,857. Changes for other cells in the same
population ranged from
Gonadotropin-releasing Hormone Receptor Microaggregation
RATE MONITORED BY FLUORESCENCE RESONANCE ENERGY TRANSFER*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
6
M) was added to 450 µl of medium in the imaging chamber,
for a final concentration of 107
M, unless otherwise specified. When vinblastin or
cytochalasin D were used, cells were incubated for 60 min in 0.1 mM vinblastin or 30 min in 2 µM cytochalasin
D in medium at 37 °C and then imaged at room temperature as
described above. In these experiments, vinblastin or cytochalasin D
were continuously present during the imaging period. Chelation of
calcium with 3 mM EGTA in the imaging medium caused cells
to detach from the coverslips. To retain their orientation for
time-lapse imaging, 0.5% low melting point agarose (Bio-Rad) was added
both to the EGTA-containing medium and to the control medium.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
GnRHR-C tail-GFP (A)
and GnRHR-C tail-RFP (B) are expressed on the plasma
membrane. Both fluorophores are expressed on the plasma membrane,
and significant amounts of RFP can be found inside the cytoplasm.
C, the GnRHR-C tail-RFP construct ( 
) expressed on the
membrane elicits a strong inositol 1,4,5-trisphosphate response
to buserelin, similar to wild-type receptor (
), as was shown
previously for GnRH-C tail-GFP. IPs, inositol
phosphates.
5 to 5%. Fig. 2, panel III shows a
live cell imaged 16 min after addition of buserelin. In this case, the
intensity of green fluorescence changed from 43,040 to 49,736 when the
intensity of RFP was reduced from 20,565 to 7,875. The increase in
donor fluorescence observed after acceptor photobleaching was small but
consistent with values expected for FRET probability, considering the
factors mentioned above.
View larger version (84K):
[in a new window]
Fig. 2.
Photobleaching of RFP in small areas on the
membrane causes a small but consistent increase of GFP fluorescence in
cells that have been exposed to buserelin but has no effect in control
cells treated with medium alone. Panel I, cell fixed after
30 min of exposure to buserelin before (A and B)
and after (C and D) photobleaching. Integrated
fluorescence intensities in the rectangular area were as follows: GFP,
68217 before (A) and 69313 after (C)
photobleaching of RFP from 41844 (B) to 17542 (D). Panel II, control cell fixed without
exposure to buserelin. Integrated intensities for green fluorescence
were as follows: 43,903 before (A) and 43,406 after
(C) photobleaching of RFP from 13,880 (B) to
5,857 (D). Panel III, live cell imaged 16 min
after addition of buserelin to the culture medium. Green integrated
intensities were 43,040 (A) before and 49,736 (C)
after photobleaching of RFP from 20,565 (B) to 7,875 (D).
If GnRH agonists provoke receptor microaggregation, it is predictable
that treatment with buserelin in cells expressing GnRHR-C tail-GFP and
GnRHR-C tail-RFP will cause the increase of red fluorescence at the
expense of green fluorescence. We imaged live cells temporally after
addition of 107 M buserelin to
the culture and measured the average intensity of green and red
fluorescence in the same small region on the plasma membrane at all
time points. Fig. 3 is an example of cell regions analyzed over 20 min after activation by buserelin. Green and
red fluorescence intensities were normalized to their values at time 0. The graph shows a small but constant increase of red fluorescence,
increasing ~10% after 20 min. The intensity of green fluorescence
drops robustly, partly due to energy transfer to RFP and partly due to
photobleaching. The photobleaching rate of GFP alone is not affected by
the presence of buserelin, suggesting that the increase in the ratio of
red to green fluorescence intensity measured over time, observed only
in the presence of buserelin, is not caused by the faster bleaching of
GFP.
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When several small regions were arbitrarily chosen along the membrane of the same cell, not all displayed an increase in the rate of red/green fluorescence after addition of buserelin. We conclude that receptor aggregation, as monitored by FRET, is not a uniform process throughout the cell membrane but rather occurs in "islands" of microaggregation. Results presented are averages over larger regions. For the same cell, regions that show internalization of the receptor tend to have red:green ratios >30% higher than regions that do not. When internalization is not visible, low and high FRET areas are visually indistinguishable.
Even though we are aware of other possible contributing factors, for
simplicity we used the increase of the ratio of red:green fluorescence
as a measure of FRET and consequently of receptor aggregation. Use of
this simplification is supported by the observation that, in the
absence of activation, the red:green fluorescence ratio is largely
constant in time for a small region of a cell membrane. Fig.
4A shows the fluorescence
ratio for a cell for 10 min before (
) and after exposure to
buserelin () from 0 to 20 min. The ratio fluctuates very
little around the initial value, but, as buserelin is added, the ratio
increases significantly and largely linearly. To test whether the
change of medium may play an artifactual role in our observation, we
changed from the initial medium to fresh medium and then to
107 M buserelin (Fig. 4B). The
ratio of red:green fluorescence intensity did not change with the
medium exchange and remained constant for the 24 min imaged in these
conditions. As soon as medium was replaced with fresh medium containing
107 M buserelin, the FRET signal,
as measured by the red:green intensity ratio, increased constantly for
the next 20 min (Fig. 4B).
|
) in the same medium
for 10 min and then by a solution containing
10When cells were exposed to GnRH antagonist, Nal-Arg (107
M), the red:green ratio remained constant for the duration
of the experiment, through two changes of medium alone and one addition of medium containing Nal-Arg for 1 h (Fig. 4D). When
Nal-Arg was replaced by a solution containing a 100-fold molar excess
of buserelin, the ratio of red:green fluorescence increased less and
reached a plateau after ~20 min (Fig. 4C).
The experiments were repeated (three or more times) with different
batches of cells, transfected and imaged at different times with
similar results. Linear fit of the red/green intensity over the first
20 min gives rate constants with an average over seven experiments of
0.054 ± 0.019 min1 for buserelin,
0.005 ± 0.010 min1 for medium, and
0.003 ± 0.014 min1 for Nal-Arg.
After the initial fast rise in FRET signal lasting about 20 min, the
signal remains nearly constant for at least 1 h and decreases thereafter. Motion of the cell membrane in this time frame makes persistent observation of specific regions difficult and adds to the
noise level (Fig. 5A).
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Lowering the concentration of buserelin did not abolish the increase of
FRET, but in most cases it reduced the rate of FRET increase (data not
shown). Even 1010 M buserelin,
for which we did not observe receptor internalization during the times
examined, produced an increase of the red to green fluorescence ratio
(Fig. 5B).
We then examined the action of 0.1 mM vinblastin, an alkaloid drug that inhibits patching and capping (7) but has no action on microaggregation. Vinblastin did not affect the increase in the red to green fluorescence ratio after addition of buserelin (Fig. 5C). Because the effect of vinblastin is mediated by the disruption of the actin cytoskeleton, we tested the effect of cytochalasin D, a microfilament-destabilizing agent. As observed for vinblastin, treatment with cytochalasin did not block the FRET signal (Fig. 5D). No internalization was observed in the presence of either vinblastin or cytochalasin D at the concentrations used.
Absence of calcium in the culture medium causes a dramatic reduction in cluster formation and internalization (i.e. macroaggregation (8)). We added 3 mM EGTA, together with 0.5% low melting point agarose, to the imaging medium to examine whether the FRET signal was dependent on the presence of calcium. The FRET response was robust, similar to that of the control in the presence of agarose (Fig. 5, E and F), suggesting that microaggregation rather than macroaggregation was being observed by this measurement.
The present study utilized the FRET technique to examine GnRH receptor-receptor interactions. Our findings suggest that agonist occupancy of the receptor enhances energy transfer, and we infer that a decreased receptor-receptor distance has occurred. Occupancy of the receptor by an antagonist (or no occupancy at all) is not accompanied by increased energy transfer and suggests, therefore, that intimate receptor-receptor interactions do not occur in this circumstance. This interaction can be measured within minutes and appears linear for 20-30 min. The time course is consistent with our previous estimate (7), although the prior method did not allow a detailed rate determination. Based on other interactions (24-26) assessed by the FRET technique, it is reasonable to view a receptor-receptor distance, upon microaggregation, of <120 Å consistent with prior estimates for the distance between agonist binding sites on adjacent receptors (1). We show that energy transfer in response to an agonist is independent of vinblastin treatment, which is a convenient means of distinguishing microaggregation from large scale patching, capping, and internalization.
It is somewhat surprising that GFP and RFP allow apparently normal receptor function and are useful markers for receptors (24), given their large size and shape; however, recent observations suggest that estimates of issues of specific orientation may not be as significant as once believed (22). In the case of the GnRH receptor, inclusion of a spacer between the receptor and fluorophore appears to be essential, because without this, the chimeric protein is not properly routed (19). The specific spacer used, however, does not appear to be critical. Although useful for rate calculations, the FRET technique does not allow quantification of numbers of receptor microaggregates either before or after hormone treatment, and interpretation is further complicated because the energy transfer only occurs when GFP and RFP are proximal (<100 Å) and the geometry permits a productive event. It would not be unreasonable to imagine that <20% of all actual receptor-receptor collisions are measured, because even under optimal conditions only 50% of the collisions would be GFP·RFP.
The present study provides data that are consonant with a role of
microaggregation in GnRH receptor signaling. These data provide
estimates of rates of this process in real time. In addition, our
findings suggest that microaggregation is restricted to particular areas of the membrane. Furthermore, these data indicate that the long
C-terminal tails characteristic of most GPCRs, but absent in the GnRH
receptor, are not required for microaggregation. Evidence for
receptor-receptor interactions have now been observed for a number of
receptors (10-14, 27-29), and it has been suggested that these
interactions are important for explanation of independent mediation of responses (30) and, more recently, for heterogeneous receptor regulation (15, 16).
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FOOTNOTES |
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* This work was supported by Grants HD19899, HD18185, and RR00163 from the National Institutes of Health.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.
To whom correspondence and requests for reprints should be
addressed: 505 NW 185th Ave., Beaverton, OR 97006. Tel.: 503-690-5297; Fax: 503-690-5569; E-mail: connm@ohsu.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007850200
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ABBREVIATIONS |
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The abbreviations used are: GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; FRET, fluorescence resonance energy transfer; GnRHR, GnRH receptor; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; RFP, red fluorescent protein; Nal, 2-naphthylalanine; Nal-Arg, Ac-D-Nal1-D-4-chlorophenylalanine2-D-3-pyridylalanine3, Arg5-D-Arg6-D-Ala10-GnRH; rGnRHR, rat GnRHR; FCS, fetal calf serum.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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