Binding of Agonist but Not Antagonist Leads to Fluorescence Resonance Energy Transfer between Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptors
Regina D. Horvat,
Deborah A. Roess,
Scott E. Nelson,
B. George Barisas and
Colin M. Clay
Cell and Molecular Biology Program (R.D.H.) Animal Reproduction
and Biotechnology Laboratory Department of Physiology (D.A.R.,
S.E.N., C.M.C.) and Department of Chemistry (B.G.B.) Colorado
State University Fort Collins, Colorado 80523
 |
ABSTRACT
|
---|
We have used spot fluorescence photobleaching
recovery methods to measure the lateral diffusion of GnRH receptor
(GnRHR) fused at its C terminus to green fluorescent protein (GFP)
after binding of either GnRH agonists or antagonist. Before ligand
binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D =
18 ± 2.8 x
10-10cm2sec-1)
and high values for fractional fluorescence recovery (%R) after
photobleaching (73 ± 1%). Increasing concentrations of agonists,
GnRH or D-Ala6-GnRH,
caused a dose-dependent slowing of receptor lateral diffusion as well
as a decreased fraction of mobile receptors. Increasing concentrations
of the GnRH antagonist Antide slowed the rate of receptor diffusion but
had no effect on the fraction of mobile receptors, which remained high.
To determine whether the decrease in %R caused by GnRH agonists was
due, in part, to increased receptor self-association, we measured the
fluorescence resonance energy transfer efficiency between GnRHR-GFP and
yellow fluorescent protein-GnRHR. There was no energy transfer
between GnRHR on untreated cells. Treatment of cells with GnRH agonists
led to a concentration-dependent increase in the energy transfer
between GnRH receptors to a maximum value of 16 ± 1%. There was
no significant energy transfer between GnRH receptors on cells treated
with Antide, even at a concentration of 100 nM.
These data provide direct evidence that, before binding of ligand,
GnRHR exists as an isolated receptor and that binding of GnRH agonists,
but not antagonist, leads to formation of large complexes that exhibit
slow diffusion and contain receptors that are self-associated.
 |
INTRODUCTION
|
---|
The binding of GnRH to specific, high-affinity receptors located
on gonadotrope cells of the anterior pituitary gland represents a
central point for regulation of reproductive function (1, 2). In the
absence of GnRH input, synthesis and secretion of LH and, consequently,
reproductive function ceases (1, 3). Thus, the GnRH receptor (GnRHR) is
the site that receives and mediates the primary stimulatory input to
gonadotropes. Accordingly, much effort has been expended toward
understanding the biology of the GnRHR at both the genetic and protein
level. The extensive utilization of potent agonists and antagonists of
GnRH in the regulation of reproductive function and the treatment of a
number of pathologies further underscores the importance of a full
understanding of the events underlying physiological and
pharmacological activation of the GnRHR (4, 5, 6). In this regard, perhaps
the single greatest breakthrough was the initial isolation of the cDNA
encoding the murine GnRHR (7, 8). Hydrophobicity analysis of the
predicted polypeptide revealed the presence of seven hydrophobic amino
acid domains consistent with membership of the GnRHR in the superfamily
of G protein-coupled receptors. The mammalian GnRHR is, however,
unusual in that it lacks an intracellular carboxyl terminus, a region
that has been implicated in deactivation and internalization of other G
protein-coupled receptors.
Although the availability of cDNAs encoding the GnRHR has allowed
much progress in elucidating structure-function relationships that
exist in this protein, significant gaps in our understanding of this
molecule remain. In particular, the lack of antibodies capable of
recognizing the GnRHR in situ has precluded analyses of the
GnRHR in the unbound state and thus any changes that may be induced by
hormone binding. For this reason, it has been difficult to monitor
GnRHR biosynthesis, its trafficking through intracellular compartments,
and its behavior in the plasma membrane. As an alternative to
immunological detection, we have constructed a functional GnRHR in
which green fluorescent protein (GFP) is fused to the carboxyl terminus
of the murine GnRHR (9). This fusion receptor is appropriately
trafficked to the plasma membrane, binds hormone with an affinity
similar to that of wild-type receptor, and is capable of signal
transduction (9). Given the ability of the GnRHR fused to GFP
(GnRHR-GFP) to recapitulate these central characteristics of native
GnRHRs, this approach represents a powerful tool to address a number of
basic questions regarding the biology of the GnRHR. Toward this
end, we have used fluorescence photobleach recovery (FPR) to examine
lateral dynamics of both the unbound and bound forms of the GnRHR-GFP
in the plasma membrane (9). We found that binding of agonist to the
GnRHR slowed the rate of lateral movement of the receptor in the plasma
membrane and led to an "anchoring" event such that a significant
percentage of the receptors became laterally immobile. The binding of
the GnRH antagonist Antide also led to a reduction in the rate of
lateral diffusion but, in contrast to GnRH, did not affect the fraction
of mobile receptors. This striking difference suggests that these two
distinct postreceptor binding events reflect a fundamental difference
in the behavior of agonist- vs. antagonist-occupied
receptor. Since the reduction in the rate of lateral diffusion of the
GnRHR-GFP fusion protein was similar with GnRH and Antide, it would
appear that this event may reflect ligand binding irrespective of
agonist or antagonist. In contrast, the reduction in the mobile
fraction observed only with GnRH may reflect additional interactions
unique to the fully activated receptor.
There are several potential explanations for the differential
effects of agonist and antagonist on the lateral diffusion of the
GnRHR. For example, the reduction in the mobile fraction of GnRHRs may
simply reflect association of the agonist-occupied receptor with
membrane signaling components. Alternatively, the reduced mobile
fraction associated with agonist binding may reflect self-association
of receptors into microaggregates that allow for G protein coupling.
Indeed, receptor dimerization and/or oligomerization has been suggested
as a critical event preceding signal transduction by other G
protein-coupled receptors including the
ß2-adrenergic (10, 11) and
M3 muscarinic receptors (12). Similarly, others
have suggested that aggregation of GnRHRs occurs as an early and
essential step in GnRH signaling (13, 14, 15, 16, 17); however, the inability to
examine the aggregated state of the unbound GnRHR has precluded a
direct test of this hypothesis. Herein, we examine the relationship
between signal transduction and lateral mobility of the GnRHR-GFP
fusion protein bound to increasing doses of either the natural ligand
GnRH, the super-agonist
des-Gly10-D-Ala6-GnRH
N-ethyl amide
(D-Ala6-GnRH), and Antide,
a GnRH antagonist. Additionally, to directly test the hypothesis that
binding of agonist but not antagonist leads to self-association of
GnRHRs in the plasma membrane, we examine the efficiency of
fluorescence energy transfer between the GnRHR-GFP fusion protein and
the red-shifted variant of GFP, termed yellow fluorescent protein
(YFP).
 |
RESULTS
|
---|
A Dose-Dependent Decrease in the Fraction of Mobile Receptors
Correlates with Agonist-Induced Signaling
Previously, we have found that the GnRHR expressed as a fusion
protein with GFP binds D-Ala6-GnRH
with a dissociation constant (Kd) similar to that
of the wild-type murine GnRHR expressed in the gonadotrope-derived
T31 cell line (9). Furthermore, Chinese hamster ovary (CHO) cells
stably expressing the GnRHR-GFP fusion protein exhibited a 4.5-fold
increase in intracellular concentrations of cAMP after treatment with
10 nM GnRH (9). To more directly assess the relationship
between signaling and lateral mobility of the GnRHR in the plasma
membrane, we tested whether the effects of increasing concentrations of
GnRH, D-Ala6-GnRH, and Antide on
generation of cAMP were correlated with changes in either the rate of
lateral diffusion of the receptor or the fraction of laterally mobile
receptors. Specifically, we sought to determine whether lateral
mobility of the GnRHR-GFP fusion protein is, like signaling, dependent
on the concentration of available ligand. A dose-dependent increase in
intracellular cAMP was evident for CHO cells stably expressing
GnRHR-GFP and treated for 1 h with increasing concentrations of
either natural ligand (GnRH) or the superagonist
D-Ala6-GnRH (Fig. 1A
). Consistent with higher affinity
binding to the GnRHR, the dose-response curve for
D-Ala6-GnRH was shifted to the left
as compared with GnRH. As expected, treatment of CHO cells with the
GnRH antagonist Antide did not affect levels of cAMP, which remained
low. Background cAMP levels from nontransfected cells and from CHO
cells expressing GnRHR-GFP were 35 ± 10 and 36 ± 5
pM/106 cells, respectively.
Consistent with our earlier report of GnRHR lateral dynamics
(9), the unoccupied GnRHR-GFP fusion receptor exhibited a fast
diffusion coefficient of 18 x 10-
10cm2sec-1
(Fig. 1B
). A significant reduction in the diffusion coefficient was
evident at 0.1 nM GnRH and continued to decrease to a
minima of 5.5 x
10-10cm2sec-1
at 10 nM GnRH. A similar dose-dependent decrease in the
rate of receptor lateral diffusion was evident for
D-Ala6-GnRH. However, as with
signaling, the dose-response curve was shifted to the left relative to
GnRH. Treatment with Antide also reduced the rate of lateral diffusion
in a concentration-dependent manner and, like the dose-response curve
for D-Ala6-GnRH, was left-shifted
relative to that for GnRH. This observation is consistent with higher
affinity binding of both D-Ala6-GnRH
and Antide for the GnRHR as compared with GnRH (18, 19).
As previously reported, the fraction of mobile receptors for
the unoccupied GnRHR was high (%r = 73 ± 1%)
(Fig. 1C
). Thus, the majority of GnRHRs are laterally mobile and
display rapid lateral diffusion in the membrane. Increasing doses of
GnRH led to a dose-dependent reduction in the fraction of mobile
receptors (Fig. 1C
). Similarly,
D-Ala6-GnRH also decreased
the fraction of laterally mobile receptors in a dose-dependent manner;
however, as with signaling and rate of lateral diffusion a lower dose
of the superagonist caused a significant reduction in the mobile
fraction (0.01 nM for
D-Ala6-GnRH vs.
1.0 nM for GnRH). The fraction of laterally
mobile receptors was unaffected at any dose of Antide tested. Thus, a
reduced rate of lateral diffusion of the GnRHR-GFP fusion protein
appears to be independent of the "nature" of the ligand
(i.e. agonist, superagonist, or antagonist). In contrast,
only agonist or superagonist (GnRH or
D-Ala6-GnRH) is capable of
effectively reducing the percentage of laterally mobile receptors in
the plasma membrane and activating signal transduction. Furthermore, it
is interesting to note the relationship between signaling and the
reduction in the fraction of mobile receptors. In Fig. 2
, data for cAMP production or signaling
(S) and percentage of laterally mobile receptors (%R) were normalized
to the maximal values for each parameter. For GnRH and
D-Ala6-GnRH, the point of
convergence of the two lines (R/S50) is
coincident with a 50% reduction in %R and a 50% increase in cAMP
production. Consistent with the increased affinity of the superagonist,
the R/S50 for
D-Ala6-GnRH is
approximately 0.1 nM, as compared with 1.0
nM for GnRH.

View larger version (32K):
[in this window]
[in a new window]
|
Figure 2. Relationship Between Ligand-Induced cAMP Production
(S) and Decrease in Fraction of Laterally Mobile Receptors (%
Recovery)
Data for both cAMP production and %R were normalized to
maximal response or to maximal recovery, respectively. The normalized
decrease in recovery ( ) and normalized increase in intracellular
cAMP ( ) were plotted
against increasing concentrations of GnRH (A),
D-Ala6-GnRH (B), or Antide (C). Where the two
data traces converge indicates a 50% reduction in fluorescence
recovery and a 50% increase in cAMP production (R/S50).
|
|
Construction and Analysis of Cell Lines Coexpressing GnRHR-GFP and
GnRHR-YFP
The reduction in the fraction of laterally mobile receptors may
reflect receptor self-association and the formation of
receptor-receptor complexes. If this is correct, then treatment of
cells with GnRH or D-Ala6-GnRH, but
not with Antide, should result in receptor self-association, which can
be detected as fluorescence resonance energy transfer (FRET) between
intrinsically fluorescent proteins fused to GnRHRs. The use of FRET to
address GnRHR self-association required the fusion of a second
fluorophore to the GnRHR. Based on the separation of excitation and
emission wavelengths, we chose the red-shifted variant of GFP, YFP, to
serve as the acceptor fluorophore in FRET experiments. As with the
GnRHR-GFP fusion protein, we fused YFP to the carboxyl terminus of the
GnRHR and constructed CHO cell lines that express either GnRHR-GFP or
GnRHR-YFP alone or in combination. Based on Scatchard analyses, the
GnRHR-YFP fusion receptor bound
D-Ala6-GnRH with a
Kd of 0.8 nM. CHO cells expressing
both GFP- and YFP-tagged GnRHRs had a Kd of 1.1
nM (data not shown). These values are similar to that
determined for the GnRHR-GFP fusion protein and for wild-type GnRHR
expressed in
T31 cells (9). Thus, both GnRHR-GFP and GnRHR-YFP
bind D-Ala6-GnRH with affinities
similar to the wild-type GnRHR. No specific binding was detected for
nontransfected CHO cells.
To confirm that the fusion receptors were capable of signal
transduction, the intracellular concentration of cAMP in the CHO cells
expressing GnRHR-YFP or GnRHR-GFP/GnRHR-YFP was measured after 1 h
incubation with increasing concentrations of GnRH,
D-Ala6-GnRH, or Antide (Fig. 3
). In both cell lines, the addition of
GnRH and D-Ala6-GnRH to the cells
resulted in a dose-dependent increase in intracellular cAMP
concentration with an ED50 similar to the
Kd of the wild- type GnRHR and the GnRHR-GFP
fusion receptor (9). There was no effect of Antide on intracellular
concentrations of cAMP at any dose tested. There was no effect of GnRH
on cAMP levels in nontransfected CHO cells (data not shown).
Agonist, but Not Antagonist, Elicits GnRHR Self-Association
To assess receptor self-association, we used a FRET method based
on the reduced rate of irreversible photobleaching of donor
fluorophores when acceptor fluorophores are present (20). The rate of
donor fluorophore photobleaching was measured with cells expressing
only the GnRHR-GFP fusion protein (donor fluorophore) and compared with
the rate of donor photobleaching in cells expressing both donor and
acceptor (GnRHR-YFP) fluorophores. A slower rate of fluorescence decay
for cells expressing both the donor and acceptor molecules than for
cells expressing only the donor is indicative of energy transfer from
fluorescence donor to acceptor (20). Energy transfer occurs when the
fluorescence donor, GFP, and the fluorescence acceptor, YFP, which have
a Förster distance of approximately 50 Å (21), are separated by
a distance of less than approximately 100 Å. The magnitude of energy
transfer is quantitated as % energy transfer efficiency (%E) as
described in Materials and Methods. Presented in Fig. 4
are representative fluorescence data
and the fitted decay curves for CHO cells expressing fluorescence donor
GnRHR-GFP only or the fluorescence donor and acceptor, GnRHR-GFP and
GnRHR-YFP. In the absence of ligand, there was essentially no energy
transfer between donor and acceptor fluorophores. Treatment of CHO
cells with 10 nM GnRH resulted in energy transfer
efficiency of approximately 16 ± 1%, a value consistent with
aggregation of the donor and acceptor molecules in the plasma membrane
(Fig. 4
). In contrast, 10 nM Antide had no
significant effect on %E. Therefore, the binding of agonist, but not
antagonist, induced self-association of GnRHR-GFP and GnRHR-YFP.

View larger version (31K):
[in this window]
[in a new window]
|
Figure 4. Representative Data Traces from Measurements of
FRET
Fluorescence decay (normalized counts per second) were
determined for CHO cells stably expressing GnRHR-GFP alone ( ) and
CHO cells coexpressing GnRHR-GFP and -YFP fusion proteins
( ). Values for energy
transfer efficiency are based on the initial photobleaching rate
constant for cells expressing only GnRHR-GFP or coexpressing GnRHR-GFP
and GnRHR-YFP. Fluorescence decay was determined after treatment with
vehicle alone (top panel), 10 nM GnRH
(middle panel), or 10 nM Antide
(bottom panel). Calculated values for energy transfer
efficiency are shown in the upper right hand corner of
each panel.
|
|
Next, we sought to determine whether receptor self-association
was dependent on the concentration of available ligand. As in Fig. 4
, we observed essentially no energy transfer between GnRHR-GFP and
GnRHR-YFP before the introduction of ligand (Fig. 5
). The addition of increasing
concentrations of GnRH led to a dose-dependent increase in %E to a
maximum value of approximately 16 ± 1% at 10 nM
GnRH. Increasing concentrations of
D-Ala6-GnRH produced a similar
increase in %E. However, as with signaling, rate of lateral
diffusion, and fraction of laterally mobile receptors, the %E
dose-response curve for D-Ala6-GnRH
was shifted to the left relative to GnRH. The GnRH antagonist Antide
had no significant effect on energy transfer efficiency at any
concentration tested (1.0, 10, and 100 nM).
To determine whether agonist-occupied GnRHRs associate
only with other agonist-occupied receptors, we examined
fluorescence energy transfer between GnRHR on cells that were treated
with both D-Ala6-GnRH and
Antide (Sigma, St. Louis, MO) at various ratios. As
shown in Fig. 6
, GnRHR exhibited maximum
energy transfer efficiency when cells were treated with 9
nM
D-Ala-6-GnRH and 1
nM Antide suggesting that the presence of Antide
at low concentrations did not block energy transfer between receptors.
However, energy transfer efficiency decreased by nearly 50% when
the amount of D-Ala6-GnRH
was reduced to 7 nM and Antide was increased to 3
nM. As the concentration of Antide was increased
and D-Ala6-GnRH was
decreased, energy transfer efficiency decreased further. No significant
energy transfer was detected when concentrations of Antide were 9
nM even in the presence of 1
nM
D-Ala6-GnRH which, as shown
in Fig. 5
, was sufficient to cause maximum energy transfer between
GnRHR in the absence of Antide. Together, these results suggest
that within the population of GnRHRs only agonist-occupied receptors
will self-aggregate and that the presence of Antide, even at low
concentrations, significantly reduces receptor-receptor
interactions.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 6. Energy Transfer Efficiency between GnRHR When
Treated with Both Agonist and Antagonist
CHO cell lines stably expressing GnRHR-GFP and GnRHR-GFP
and -YFP were treated with both D-Ala6-GnRH and
Andide at various ratios. The rate constant of irreversible
photobleaching was measured, and % energy transfer efficiency was
calculated as described in Materials and Methods. The
ligand treatments were applied 5 min before data acquisition and
remained at total concentration of 10 nM for the duration
of data acquisition ( 20 min). Data (mean + SD) were
analyzed by one-way ANOVA, and means were separated using LSD criteria.
*, Values were different (P < 0.01) from Antide
alone-treated cells.
|
|
 |
DISCUSSION
|
---|
Of the different hormones that directly mediate gonadotrope
function, none are more fundamental than GnRH (22). Quite simply, in
the absence of GnRH input, gonadotrope function ceases. Thus, the GnRHR
is the site that ultimately receives and mediates the primary
stimulatory input to gonadotropes. In the past 67 yr, the isolation
of cDNAs that encode the GnRHR has led to the identification of
structural motifs in this receptor that mediate expression, ligand
binding, signaling, and internalization (23, 24, 25, 26, 27, 28, 29). Unfortunately, the
inability to detect the unbound form of the GnRHR has precluded any
direct analysis of the effects of ligand binding on the
organization of this receptor in the plasma membrane. Toward this end,
we have fused the coding sequence for GFP to the carboxyl terminus of
the murine GnRHR. This GnRHR-GFP fusion receptor was appropriately
trafficked to the plasma membrane of both gonadotrope and
non-gonadotrope-derived cell lines, bound hormone with an affinity
similar to the wild-type GnRHR, and was capable of signal transduction
(9). Here, we have used laser optical methods to more closely examine
the differential effects of agonist, superagonist, and antagonist on
the organization of GnRHR in the plasma membrane of living cells.
We find that the majority of unoccupied GnRHRs display lateral
movement in the plasma membrane. Additionally, the rate of lateral
diffusion of the unoccupied GnRHR is very rapid. Increasing
concentrations of either agonist or antagonist slows the rate of
lateral diffusion in a dose-dependent fashion. In contrast, only
agonist and superagonist lead to a dose-dependent decrease in the
fraction of laterally mobile receptors. Thus, a reduced rate of lateral
diffusion of the GnRHR is apparent after the binding of either agonist,
superagonist, or antagonist whereas a reduction in the percentage of
laterally mobile receptors is observed only after the binding of a
functional ligand that is capable of initiating intracellular
signaling.
The slowing of diffusion could be attributed, at least in
part, to incorporation of the GnRHR into protein complexes within the
membrane. Even though antagonist-occupied receptors slow their rate of
lateral diffusion, perhaps by incorporation into protein
complexes, the mobile fraction does not change significantly. These
results would be consistent with formation of small, nonfunctional
complexes containing GnRHR.
The reduction in the fraction of mobile receptors may,
alternatively, result from "trapping" of the receptor within
microdomains in the plasma membrane. Recently, there has been increased
speculation that the plasma membrane contains small domains with
specialized functions (30). A number of plasma membrane receptors,
including the IgA receptor (31), epidermal growth factor
receptor (32), and the tissue factor receptor (31), are preferentially
distributed into discrete domains. In some cases these microdomains are
detergent-insoluble membrane fragments that exhibit high buoyancy after
isopycnic centrifugation and can contain high concentrations of
sphingomyelin and cholesterol as well as membrane proteins involved in
cell signaling such as G proteins (31). It is possible that
binding of hormone to the GnRHRs not only drives receptors into
microdomains within which receptor motions are constrained. If a large
number of receptors were constrained within these domains, the fraction
of immobile receptors would appear to be large in measurements of
fluorescence photobleaching recovery.
Another critical difference between functional and
nonfunctional complexes was the appearance of large extents of receptor
self-association under conditions in which receptors were capable of
activating adenylate cyclase. Janovick and Conn (17) have hypothesized
that the GnRHR is found within microaggregates after binding of agonist
and that it is the presence of the receptor within these protein
complexes that allows for the initiation of signal transduction. This
appears to be the case in our studies. However, by stably coexpressing
GnRHRs fused to GFP or YFP in CHO cells, we were able to examine
whether GnRHRs were self-associated both before and after exposure to
ligand. As predicted, the unoccupied GnRHR was not self-associated, and
treatment of cells with agonist led to an dose-dependent increase in
energy transfer efficiency. There was no significant increase in energy
transfer between receptors when treated with Antide, even at the
highest concentration. Interestingly, when GnRHRs are simultaneously
treated with both agonist and antagonist, the presence of excess
antagonist blocks energy transfer between agonist-occupied receptors
(Fig. 6
). Collectively these data are direct evidence that the GnRHR
self-associates due to binding of agonist but not antagonist.
From these data, we can suggest a general mechanism for signal
transduction by GnRHRs. We hypothesize that GnRHR exists as isolated
membrane proteins that diffuse freely in the plasma membrane. Upon
binding of hormone agonists, GnRHRs undergo a conformational change
that exposes contact sites on one or more of the receptors
transmembrane domains. A dimerization motif has been identified for the
ß-adrenergic receptor on the sixth transmembrane domain (11).
Interactions between GnRHRs via similar sites may result in the
formation of receptor aggregates. During receptor self-association, or
perhaps as a result of receptor self-association, other proteins
involved in signaling interact with the receptor. Thus, a complex is
formed containing both receptors and nonreceptor proteins that is
sufficiently large to exhibit significantly slower rates of lateral
diffusion than does the isolated, monomeric receptor. When the receptor
is functional, a large fraction of these complexes become laterally
immobile.
The hypothetical situation for nonsignaling receptors would be
somewhat different. GnRHRs bound with antagonist have diffusion
coefficients similar to those of their functional counterparts and
significantly slower diffusion than unoccupied receptors. We suggest
that GnRHRs, when bound by antagonist, are present in slowly diffusing
complexes that do not contain the full complement of proteins required
for signaling or, alternatively, do not reach membrane microdomains
containing necessary proteins for initiation of productive signaling.
In either case, the ability of GnRHR to transduce signal appears to
require receptor self-association. Further, our biophysical assessment
of GnRHR self-association is in agreement with other biochemical
studies in which G protein-coupled receptors, such as the
ß-adrenergic receptor and the
-opioid receptor (11, 33), form
dimers after binding of ligand. That this process is important in
receptor function can be demonstrated by functional rescue of
M3 muscarinic receptors containing two reciprocal
nonfunctional mutations (12).
These studies of receptor self-association and membrane
domains, together with the development of new biophysical methods to
examine membrane proteins in living cells, should considerably advance
our understanding of GnRHR signaling mechanisms. In particular, methods
for monitoring the organization and distribution of single receptors
within the membrane will establish whether the plasma membrane
environment of the GnRHRs and the organization of receptors within that
environment differs significantly after the binding of GnRH agonists
vs. antagonists.
 |
MATERIALS AND METHODS
|
---|
Chemicals
Unless otherwise noted, all enzymes were from New England Biolabs, Inc. (Beverly, MA). All other reagents were the highest
grade available.
Construction of pGnRHR-YFP
Plasmid pEYFP (CLONTECH Laboratories, Inc. Palo
Alto CA) was digested with NotI and NcoI to
liberate the YFP coding sequence. The YFP cDNA was then ligated into
pEGFP-N2 (CLONTECH Laboratories, Inc.), which had been
digested to completion with NotI followed by partial
digestion with NcoI to remove the GFP coding sequence. This
ligation yielded pEYFP-N2 in which YFP is placed in the correct reading
frame for fusion to the carboxyl terminus of the murine GnRHR cDNA.
The mGnRHR cDNA was liberated from pGnRHR-GFP (9) by digestion with
EcoRI and BamHI and then ligated into pEYFP-N2
digested with the same enzymes yielding pGnRHR-YFP.
Construction of Stable Cell Lines
pGnRHR-GFP (0.4 µ g) and pGnRHR-YFP (1.2 µg) were
transfected into CHO cells using Lipofectamine Plus Reagent (Life Technologies, Inc., Gaithersburg, MD) in a 35-mm plate (ISC
Bioexpress, Kaysville, UT) (9). After overnight culture, the cells were
transferred into a 150-mm plate and cultured with 600 µg/ml G418
(Gemini Bioproducts, Woodland CA) for 7 days, washed with PBS, and
cloned by limiting dilution in 96-well plates (ISC Bioexpress). Wells
were examined with epifluorescence for fluorescent cells. Clonal
cell lines expressing fluorescence were expanded and used for further
studies. CHO cells expressing GnRHR-YFP were produced by the same
method omitting pGnRHR-GFP from the transfection.
Cell Culture
Cells were cultured in Eagles modified media with high glucose
(DMEM) containing 10% FBS, 100 U/ml penicillin, 100 µg/ml
streptomycin sulfate, 292 µg/ml glutamine (Gemini Bioproducts), 1x nonessential amino acids, in
high-glucose DMEM (Life Technologies, Inc.), in a 5%
CO2, humidified atmosphere at 37 C.
Scatchard Analysis
Approximately 100,000 cells per well of the CHO GnRHR-YFP and
CHO GnRHR-GFP/YFP cell lines were plated in 24-well plates (BD
Biosciences, Bedford, MA) and cultured overnight. Varying
concentrations of freshly prepared
[125I]-des-Gly10,
D-Ala6-GnRH N-ethyl amide
(specific activity of 1.5 µCi/pmol) in the range of 4.5
nM to 20 pM in 150 µl of
ice-cold complete media were then added to each well in the presence or
absence of 260 nM of unlabeled
D-Ala6-GnRH
N-ethyl amide (9). Cells were incubated on ice for 4 h
and then washed twice with 1 ml of 170 mM NaCl, 3
mM KCl, 10 mM
Na2HPO4, and 4
mM
K2HPO4, pH 7.4 (PBS)
containing 2 mg/ml BSA. The cells were lysed in 100 µl of 1% SDS
(sodium dodecyl lauryl sulfate) and counted on an Apex Automatic Gamma
Counter (Micromedic Systems, Inc., Horsham, PA). Specific counts were
determined as total counts per minute bound less the counts per minute
bound in the presence of 260 nM of unlabeled
D-Ala6-GnRH N-ethyl amide.
Data were analyzed by a nonlinear regression using Prism Software
(GraphPad Software, Inc., San Diego, CA) using a one-site
model. A minimum of two independent experiments were conducted.
cAMP Assays
CHO (1 x 106) cells expressing
GnRHR-GFP, GnRHR-YFP, or GnRHR-GFP/GnRHR-YFP were incubated with 0,
0.01, 0.1, 1, 10, or 100 nM GnRH,
D-ala6-GnRH or Antide in a total
volume of 0.5 ml in HBSS for 1 h at 37 C. Cells were lysed with
ice-cold 10% trichloroacetic acid and the cAMP was extracted with
ether and dried under N2. The cAMP was assayed
for by enzyme-linked immunosorbent assay (ELISA) (PE Applied Biosystems, Framingham, MA) (9), and data were analyzed
by ANOVA (SAS Institute, Inc., Cary, NC). To reduce
well-to-well variations in measured levels of cAMP, 96-well plates
coated with antirabbit IgG were obtained from Pierce Chemical Co. (Rockford, IL) and were substituted for those provided in
cAMP kits.
Spot Fluorescence Photobleaching Recovery Analysis of GnRHR-GFP
Lateral Diffusion
The equipment and methods used for performing spot fluorescence
photobleaching recovery (FPR) measurements have been described in
detail elsewhere (34). In these studies, all measurements were
performed at room temperature using a Carl Zeiss
Axiomat-based instrument and a 40x microscope objective (Carl Zeiss, Thornwood, NY). For spot FPR measurements on individual
cells, a Coherent Radiation Innova 100 Argon ion laser interrogated an
area of the cell with a 1/e2 radius of 0.41 µm.
The laser provided power of 53 mW in the bleaching beam and 0.2 mW in
the probe beam at 488 nm. Fluorophore bleaching time was 150 msec in
the spot FPR measurements. In an individual FPR experiment, data were
acquired for 20 sec before fluorophore bleaching and for 30 sec
postbleach at a rate of 50 msec/point. FPR data were processed as
described previously (34). To assess the effect of GnRH,
D-Ala6-GnRH, or Antide on the
GnRHR-GFP lateral dynamics, each ligand was added to a final
concentration of 0, 0.01, 1.0, 10, or 100 nM 5 min before
the acquisition of data and remained at this concentration for the
duration of the data acquisition, which was approximately 45 min.
Spot Fluorescent Energy Transfer between GnRHR-GFP and
GnRHR-YFP
Fluorescence energy transfer between GnRHR-GFP and GnRHR-YFP
was evaluated based on the reduced rate of irreversible photobleaching
of GFP fluorophores when YFP fluorophores were present (20). Slower
rates of fluorescence decay for cells expressing the GnRHR-GFP donor
and GnRHR-YFP acceptor (D+A) than for cells expressing
GnRHR-GFP alone (D) are indicative of energy transfer from
fluorescence donor to acceptor and occurs only when the donor and
acceptor are separated by a distance less than approximately 100 Å.
The Forster distance or Ro for the GFP-YFP pair
is 51 Å (21). To perform these experiments, we used a fluorescence
microscope photometer based on the inverted-configuration Axiomat
microscope (Carl Zeiss). Fluorescence excitation was
provided by a Coherent Radiation Innova 100 argon ion laser operating
under light control at 488 nm. The intensity of the laser radiation
focused on the cell was 30 mW, and this was held constant between
measurements on GnRHR-GFP cells or on GnRHR-GFP/YFP cells. The
1/e2 Gaussian spot diameter was 0.41 µm. Donor
fluorescence from GFP was isolated with a standard fluorescein filter
set together with a short pass fluorescein-selective filter to remove
yellow fluorescence from the YFP-tagged GnRHR. This combination was
effective in eliminating YFP fluorescence. In individual experiments
cells were identified and centered in the microscope field. At time
zero, an electronically controlled shutter was opened to allow laser
radiation to impinge on the cell. Simultaneously, a computer program
was activated to record the output of the photomultiplier measuring
membrane fluorescence. Data were collected at 0.01-sec intervals for 10
sec. Typically about 20 cells in each sample were photobleached in this
manner. In each experiment, four sets of identically handled cells were
examined including untransfected CHO cells, CHO cells expressing
GnRHR-GFP alone, CHO cells expressing GnRH-YFP alone, and cells
expressing both GnRHR-GFP and GnRHR-YFP. Cells expressing GnRHR-YFP
alone produced signals that did not differ significantly from those of
untransfected CHO cells using the fluorescein-selective filter set.
Signal from CHO cells expressing GnRHR-GFP or GnRHR-GFP/YFP was greater
than 9-fold higher than background levels from untransfected CHO cells
or cells expressing GnRHR-YFP. Thus, the rate constants for
photobleaching of GFP on cells expressing GnRHR-GFP alone
(kD) or GnRHR-GFP/YFP (kDA)
were analyzed from data traces as described in detail previously
(35). There was no significant difference (P < 0.05)
between the rate constants for photobleaching of GFP in cells
expressing GnRHR-GFP alone. These rate constants were consistently
5.25.7 sec-1 and were not affected by binding
of ligand. The energy transfer efficiency was expressed as a percent
(%E) and was calculated from these rate constants using %E =
(1 - kDA/kD) x
100 (36). To assess the effect of GnRH,
D-Ala6-GnRH, or Antide on
the self-association of GnRHRs, each ligand was added to a final
concentration of 0, 0.01, 1.0, 10, or 100 nM 5
min before the acquisition of data and remained at this concentration
for the duration of the data acquisition, which was approximately 20
min.
Statistical Analysis
In photobleaching recovery and fluorescence energy transfer
experiments, diffusion coefficients and energy transfer efficiencies
were obtained through curve fitting appropriate mathematical models to
experimental data sets. These data sets contained hundreds of points,
and fitting is accomplished using the Marquardt algorithm (20). Since
each of the many observations in a single measurement provides
independent information on the parameter of interest, the
SE of the parameter was calculated at the same time as the
fitted parameter itself. However, because any real data set has some
systematic deviation from a model representing the parent experiment,
these standard errors calculated during the curve fitting procedure
almost certainly overestimate the reliability of parameters.
We thus present the uncertainties of a fitted parameter x as <x>±
2 s where s is the SEM of a set of three to
four complete, independent determinations of x. Uncertainties in
quantities such as percent efficiency of energy transfer, which involve
parameters obtained in at least three separate experiments, were
calculated by standard propagation of errors methods. Decisions as to
whether parameters differ significantly between multiple treatment
groups were made using single classification ANOVA methods (SigmaStat,
Jandel Scientific, San Rafael, CA).
The average cAMP, lateral diffusion, and energy transfer data
were analyzed by one-way ANOVA using SigmaStat (Jandel Scientific). If
the F test was significant (P < 0.01), means were
separated using the least significant difference (LSD) criterion. Data
are presented as the mean ± SD.
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr.Terry Nett for supplying radioiodinated
D-Ala6-GnRH for the binding studies
and Dr. Todd Farmerie for his assistance in review and preparation of
this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Colin M. Clay, Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523. E-mail:
cclay{at}cvmbs.colostate.edu
This research was supported by NIH Grants R01-HD-32416
(C.M.C.) and R01-HD-23236 (D.A.R.) and the Colorado State University
Experiment Station.
Received for publication August 17, 2000.
Revision received January 19, 2001.
Accepted for publication February 6, 2001.
 |
REFERENCES
|
---|
-
Clayton RN, Catt KJ 1981 Gonadotropin-releasing hormone
receptors: characterization, physiological regulation and relationship
to reproductive function. Endocr Rev 2:186209[Medline]
-
Kaiser UB, Conn PM, Chin WW 1997 Studies of
gonadotropin-releasing hormone (GnRH) action using GnRH
receptor-expressing pituitary cell lines. Endocr Rev 18:4670[Abstract/Free Full Text]
-
Hamernik DL, Nett TM 1988 Gonadotropin-releasing
hormone increases the amount of messenger ribonucleic acid for
gonadotropins in ovariectomized ewes after hypothalamic-pituitary
disconnection. Endocrinology 122:959966[Abstract]
-
Locker GY 1998 Hormonal therapy of breast cancer.
Cancer Treat Rev 24:221240[Medline]
-
Leondires MP, Berga SL 1998 Role of GnRH drive in
the pathophysiology of polycystic ovary syndrome. J Endocrinol Invest 21:476485[Medline]
-
Haviv F, Bush EN, Knittle J, Greer J 1998 LHRH
antagonists. Pharm Biotechnol 11:131149[Medline]
-
Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts
JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and
functional expression of a mouse gonadotropin- releasing hormone
receptor. Mol Endocrinol 6:11631169[Abstract]
-
Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning
and expression of cDNA encoding the murine gonadotropin-releasing
hormone receptor. J Biol Chem 267:2128121284[Abstract/Free Full Text]
-
Nelson S, Horvat RD, Malvey J, Roess DA, Barisas BG,
Clay CM 1999 Characterization of an intrinsically fluorescent
gonadotropin-releasing hormone receptor and effects of ligand binding
on receptor lateral diffusion. Endocrinology 140:950957[Abstract/Free Full Text]
-
Hebert TE, Loisel TP, Adam L, Ethier N, Onge SS,
Bouvier M 1998 Functional rescue of a constitutively desensitized
ß2AR through receptor dimerization. Biochem J 330:287293[Medline]
-
Hebert TE, Moffett S, Morello JP, Loisel TP, Bichet
DG, Barret C, Bouvier M 1996 A peptide derived from a ß2-adrenergic
receptor transmembrane domain inhibits both receptor dimerization and
activation. J Biol Chem 271:1638416392[Abstract/Free Full Text]
-
Maggio R, Vogel Z, Wess J 1993 Coexpression
studies with mutant muscarinic/adrenergic receptors provide evidence
for intermolecular "cross-talk" between G-protein-linked receptors.
Proc Natl Acad Sci USA 90:31033107[Abstract]
-
Conn PM, Rogers DC, McNeil R 1982 Potency enhancement
of a GnRH agonist: GnRH-receptor microaggregation stimulates
gonadotropin release. Endocrinology 111:335337[Abstract]
-
Conn PM, Rogers DC, Stewart JM, Niedel J, Sheffield T 1982 Conversion of a gonadotropin-releasing hormone antagonist to an
agonist. Nature 296:653655[Medline]
-
Hazum E, Cuatrecasas P, Marian J, Conn PM 1980 Receptor-mediated internalization of fluorescent gonadotropin-releasing
hormone by pituitary gonadotropes. Proc Natl Acad Sci USA 77:66926695[Abstract]
-
Conn PM, Venter JC 1985 Radiation inactivation
(target size analysis) of the gonadotropin-releasing hormone receptor:
evidence for a high molecular weight complex. Endocrinology 116:13241326[Abstract]
-
Janovick JA, Conn PM 1996 Gonadotropin releasing
hormone agonist provokes homologous receptor microaggregation: an early
event in seven-transmembrane receptor mediated signaling. Endocrinology 137:36023605[Abstract]
-
Bagatell CJ, Conn PM, Bremmer WJ 1993 Single-dose
administration of the gonadotropin-releasing hormone antagonist,
Nal-Lys (antide) to healthy men. Fertil Steril 60:680685[Medline]
-
Wise ME, Nieman D, Stewart J, Nett TM 1984 Effect of
number of receptors for gonadotropin-releasing hormone on the release
of luteinizing hormone. Biol Reprod 31:10071013[Abstract]
-
Young RM, Arnette JK, Roess DA, Barisas BG 1994 Quantitation of fluorescence energy transfer between cell surface
proteins via fluorescence donor photobleaching kinetics. Biophys J 67:881888[Abstract]
-
Patterson GH, Piston DW, Barisas BG 2000 Förster distances between green fluorescent protein pairs. Anal
Biochem 284:438440[CrossRef][Medline]
-
Gharib SD, Wierman ME, Shupnik MA, Chin WW 1990 Molecular biology of the pituitary gonadotropins. Endocr Rev 11:177190[Medline]
-
Sealfon SC, Weinstein H, Millar RP 1997 Molecular
mechanisms of ligand interaction with the gonadotropin-releasing
hormone receptor. Endocr Rev 18:180205[Abstract/Free Full Text]
-
Sealfon SC, Millar RP 1995 The
gonadotrophin-releasing hormone receptor: structural determinants and
regulatory control. Hum Reprod Update 1:216230[CrossRef][Medline]
-
Hoffmann SH, ter Laak T, Kuhne R, Reilander H,
Beckers T 2000 Residues within transmembrane helices 2 and 5 of the
human gonadotropin-releasing hormone receptor contribute to agonist and
antagonist binding. Mol Endocrinol 14:10991115[Abstract/Free Full Text]
-
Heding A, Vrecl M, Hanyaloglu A, Sellar R, Taylor PL,
Eidne KA 2000 The rat gonadotropin-releasing hormone receptor
internalizes via a ß-arrestin-independent, but dynamin-dependent,
pathway: addition of a carboxyl-terminal tail confers ß-arrestin
dependency. Endocrinology 141:306
-
Willars GB, Heding A, Vrecl M, Sellar R, Blomenrohr
M, Nahorski SR, Eidne KA 2000 Lack of a C-terminal tail in the
mammalian gonadotropin-releasing hormone receptor confers resistance to
agonist-dependent phosphorylation and rapid desensitization. J
Biol Chem 274:3014630153[Abstract/Free Full Text]
-
Arora KK, Krsmanovic LZ, Mores N, OFarrell H, Catt
KJ 1998 Mediation of cyclic AMP signaling by the first intracellular
loop of the gonadotropin-releasing hormone receptor. J Biol Chem 273:2558125586[Abstract/Free Full Text]
-
Arora KK, Chung HO, Catt KJ 2000 Influence of a
species-specific extracellular amino acid on expression and function of
the human gonadotropin-releasing hormone receptor. Mol Endocrinol 13:890896[Abstract/Free Full Text]
-
Pralle A, Keller P, Florin EL, Simons K, Horber JK 2000 Sphingolipid-cholesterol rafts diffuse as small entities in the
plasma membrane of mammalian cells. J Cell Biol 148:9971008[Abstract/Free Full Text]
-
Rietveld A, Simons K 1998 The differential
miscibility of lipids as the basis for the formation of functional
membrane rafts. Biochim Biophys Acta 1376:467479[Medline]
-
Waugh MG, Lawson D, Hsuan JJ 1999 Epidermal
growth factor receptor activation is localized within low-buoyant
density, non-caveolar membrane domains. Biochem J 337:591597[CrossRef][Medline]
-
Cvejic S, Devi LA 1997 Dimerization of the delta
opioid receptor: implication for a role in receptor internalization.
J Biol Chem 272:2695926964[Abstract/Free Full Text]
-
Munnelly HM, Roess DA, Wade WF, Barisas BG 1998 Interferometric fringe fluorescence photobleaching recovery
interrogates entire cell surfaces. Biophys J 75:11311138[Abstract/Free Full Text]
-
Young RM, Arnette JK Roess DA,
Barisas BG 1994 Quantitation of fluorescence energy transfer between
cell surface proteins via fluoresence donor photobleaching kinetics.
Biophys J 67:881888[Abstract]
-
Lackowicz J 1983 Quenching of fluoresence.
In: Principles of Fluoresence Spectroscopy, ed 1. Plenum
Press, New York, pp 257301