Gonadotropin-releasing Hormone Receptor Microaggregation RATE MONITORED BY FLUORESCENCE RESONANCE ENERGY TRANSFER*

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release and is a therapeutic tar-get 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 occu- pancy 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 m M vinblas- tin, 2 m M cytochalasin D, or 3 m M EGTA, an observation that distinguishes it from macro aggregation (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. multiple

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)(2)(3)(4)(5)(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 sug-gested 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 (Ͻ1 min) (7) and is distinguished from macroaggregation (patching, capping, internalization), an event that occurs later (Ͼ20 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.

EXPERIMENTAL PROCEDURES
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. pD-sRed 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 CLON-TECH. 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-* 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
butyl-D-Ser 6 -Pro 9 des Gly 10 EA GnRH, Hoechst), and antagonist, "Nal-Arg" (Ac-D-Nal 1 -D-4-chlorophenylalanine 2 -D-3-pyridylalanine 3 , Arg 5 -D-Arg 6 -D-Ala 10 -GnRH, Contraceptive Development Branch, NIH) were obtained as indicated. All GnRH analog solutions were prepared 10-fold concentrated, and 50 l of this solution was added to 450 l 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/CTGTC-CACTGGG) encoding for the RFP1-N1 and rGnRHR-C tail without the stop codon. The sequence for RFP including the full coding region and 3Ј-untranslated region was amplified from pRFP1-N1 vector using a 30-mer chimeric forward primer (GACCAACCCAGTGGACAG/ATGGT-GCGCTCC) 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 End-oFree Plasmid Maxi Prep kit (Qiagen Inc., Valencia, CA). The purity and identity of the plasmid DNA were further verified by restriction enzyme analysis.

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 (q) expressed on the membrane elicits a strong inositol 1,4,5-trisphosphate response to buserelin, similar to wild-type receptor (E), as was shown previously for GnRH-C tail-GFP. IPs, inositol phosphates.

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:

Transient Transfection of GH 3 Cells
For confocal microscopy of live cells, glass coverslips (24 ϫ 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 GH 3 cells were maintained in growth medium (DMEM (Irvine Scientific, Santa Ana, CA) containing 10% fetal calf serum (FCS; Life Technologies, Inc.) and 20 g/ml gentamicin (Gemini Bioproducts, Calabasas, CA)) in a humidified atmosphere (37°C) containing 5% CO 2 . The cells were plated at a density of 9 ϫ 10 5 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 g of cDNA for chimeric rGnRHR-C tail-RFP plus 0.45 g of cDNA for chimeric rGnRHR-C tail-GFP using 10 l 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 M 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 l of buserelin solution (10 Ϫ6 M) was added to 450 l of medium in the imaging chamber, for a final concentration of 10 Ϫ7 M, unless otherwise specified. When vinblastin or cy-tochalasin D were used, cells were incubated for 60 min in 0.1 mM vinblastin or 30 min in 2 M 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.

Inositol Phosphate Bioassay
An inositol phosphate bioassay was used to check the biological activity of the chimeric constructs. Briefly, GH 3 cells were plated in a 24-well Costar (Cambridge, MA) plate at 10 5 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 g of plasmid chimeric receptor DNA/ well or wt rGnRHR cDNA using 2 l 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 g/ml gentamicin and then preloaded with 0.5 ml of DMEM (inositol free) containing 4 Ci of [ 3 H]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 ϫ 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.

RESULTS AND DISCUSSION
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 GH 3 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 Ϫ5 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.
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 10 Ϫ7 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 ϳ10% 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 FIG. 4. The red:green fluorescence ratio as an indicator of FRET increases after exposure to buserelin but not to medium alone or to the GnRH antagonist Nal-Arg. A, a cell was first imaged in culture medium that was replaced with medium containing Buserelin at time 0. (छ) shows representative values of the red:green fluorescence ratio for a small region on the membrane for a cell imaged in medium alone, and (q) shows values for the same cell after medium was replaced by a solution containing 10 Ϫ7 M buserelin in the same medium. In B, the initial medium (छ) was changed at time 0 with fresh medium (छ) and, after 12 min, with medium plus buserelin (q). In C, the initial medium (छ) was replaced with a solution containing the GnRH antagonist Nal-Arg (10 Ϫ9 M) (‚) in the same medium for 10 min and then by a solution containing 10 Ϫ7 M buserelin (q). In D, a cell was imaged first in medium for 17 min (छ), in fresh medium changed at time 0 for 18 min (छ), and then in 10 Ϫ7 M Nal-Arg (‚) for an additional 22 min. red to green fluorescence intensity measured over time, observed only in the presence of buserelin, is not caused by the faster bleaching of GFP.
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 Ͼ30% 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 (छ) and after exposure to buserelin (q) 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 10 Ϫ7 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 10 Ϫ7 M buserelin, the FRET signal, as measured by the red: green intensity ratio, increased constantly for the next 20 min (Fig. 4B).
When cells were exposed to GnRH antagonist, Nal-Arg (10 Ϫ7 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 ϳ20 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 Ϯ 0.019 min Ϫ1 for buserelin, 0.005 Ϯ 0.010 min Ϫ1 for medium, and 0.003 Ϯ 0.014 min Ϫ1 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).
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 10 Ϫ10 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 Ͻ120 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 (Ͻ100 Å) and the geometry permits a productive event. It would not be unreasonable to imagine that Ͻ20% of all actual receptor-receptor collisions are measured, because even under optimal conditions only 50% of the collisions would be GFP⅐RFP.
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).