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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}T3–1 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. 1AGo). 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.



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Figure 1. Effects of Ligand Binding on Signal Transduction and Lateral Diffusion of GnRHR-GFP

CHO cells stably expressing GnRHR-GFP were treated with increasing doses of either GnRH ({bullet}), D-Ala6-GnRH (), or Antide (). Intracellular concentrations of cAMP were determined by ELISA after 1-h treatment (A). To assess the effects of ligand binding on rate of lateral diffusion (diffusion coefficient) (B) and percentage of laterally mobile receptors (% Recovery) (C), each ligand was added at the indicated concentration 5 min before data acquisition and remained at this concentration for the duration of data acquisition, which was approximately 45 min. Data were analyzed by one-way ANOVA, and means were separated using least significant differences (LSD) criteria. Values indicated with a * were different (P < 0.01) from untreated cells. The mean and SDs for diffusion coefficient and %R were calculated from at least 60 measurements on individual cells.

 
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. 1BGo). 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. 1CGo). 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. 1CGo). 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. 2Go, 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.



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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 ({bullet}) 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 {alpha}T3–1 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. 3Go). 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).



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Figure 3. Signal Transduction by GnRHR-YFP and GnRH-GFP/GnRHR-YFP Cell Lines

CHO cells stably expressing either GnRHR-YFP alone (A) or in combination with GnRHR-GFP (B) were treated with increasing concentrations of either GnRH ({bullet}), D-Ala6-GnRH (), or Antide (). Intracellular concentrations of cAMP were determined by ELISA after 1 h of treatment. Data (mean ± SD) were analyzed by one-way ANOVA, and means were separated using LSD criteria. *, Values were different (P < 0.01) from untreated cells.

 
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. 4Go 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. 4Go). 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.



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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 ({bullet}) 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. 4Go, we observed essentially no energy transfer between GnRHR-GFP and GnRHR-YFP before the introduction of ligand (Fig. 5Go). 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).



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Figure 5. Effects of Ligand Binding on Energy Transfer Efficiency between GnRHR-GFP and GnRHR-YFP

CHO cell lines stably expressing GnRHR-GFP and GnRHR-GFP and -YFP were treated with increasing concentrations of either GnRH ({bullet}), D-Ala6-GnRH (), or Antide (). Fluorescence decay (cps) was measured and % energy transfer efficiency calculated as described in Materials and Methods. Hormone treatments were applied 5 min before data acquisition and remained at the indicated concentrations 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 untreated cells.

 
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. 6Go, 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. 5Go, 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.



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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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 6–7 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. 6Go). 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 receptor’s 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 {delta}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Eagle’s 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 5.2–5.7 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.


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