Ligand Regulation of the Quaternary Organization of Cell Surface M₃ Muscarinic Acetylcholine Receptors Analyzed by Fluorescence Resonance Energy Transfer (FRET) Imaging and Homogeneous Time-resolved FRET^*

Introduction

Monomeric G protein-coupled receptors (GPCRs)² have the capacity to bind and activate G proteins (1–3). Despite this, GPCRs also have the capacity to exist as dimers and/or oligomers in living cells (4–7). Such quaternary organization has been suggested, among other functions, to play a key role in effective folding and cell surface trafficking of GPCRs (8–10). However, a series of key questions related to GPCR quaternary structure remain to be explored fully. These include the proportion of a GPCR that is dimeric/oligomeric at steady state, how this may be affected by receptor expression level, if this is regulated at different stages in the life cycle of a GPCR, and the ability of receptor ligands to alter GPCR-GPCR interactions.

Five individual muscarinic GPCRs respond to the neurotransmitter acetylcholine (11, 12). Despite overlapping tissue distribution and very limited selective ligand pharmacology, studies on mouse lines that have selective knock-out of individual muscarinic receptor genes mean that a good deal is known about the roles of the individual subtypes (12). For example, the M₃ receptor mediates a wide variety of functions, including vasodilation, bronchoconstriction, stimulation of pancreatic insulin and glucagon release, modulation of salivary gland function, and smooth muscle contraction. An alternative means to study the function of individual muscarinic receptor subtypes is based on the generation of receptor mutants that are often designated Receptors activated solely by synthetic ligand (RASSLs) (13, 14). The introduced mutation(s) result in loss of affinity for the endogenous ligand but enhanced affinity for one or more small synthetic ligands that have little affinity/potency at the wild type receptor (15, 16). In the case of muscarinic receptors, introduction of mutations at conserved amino acids in transmembrane domains III and V greatly alters the agonist potency ratio between acetylcholine and the synthetic ligand clozapine N-oxide (17, 18). Expression of such a modified receptor, in either cell lines or in animals via transgenesis, can allow selective activation and analysis of the function and regulation of the RASSL variant.

The quaternary structure of muscarinic receptor family members has been explored extensively (19–24). However, whether ligand binding regulates such interactions is an area of considerable controversy. Using bioluminescence resonance energy transfer (BRET)-based approaches, Goin and Nathanson (21) did not observe short term agonist regulation of either homo- or hetero-interactions involving M₁, M₂, or M₃ receptors. By contrast, Ilien et al. (25) have reported that the selective M₁ receptor antagonist pirenzepine enhances M₁ receptor dimerization, from a situation where M₁ receptor monomers predominate in the absence of ligand but dimerize upon pirenzepine binding. This is of particular interest because Hern et al. (26) have recently employed modified forms of the antagonist ligand telenzepine in single molecule tracking studies in Chinese hamster ovary cells expressing the M₁ receptor. These studies concluded that at any given time, ∼30% of the receptor molecules exist as dimers and that this is a dynamic process with the M₁ receptor undergoing inter-conversion between monomers and dimers within seconds. However, if antagonist ligand binding inherently alters such receptor-receptor interactions, then data interpretation may be difficult in studies that rely on imaging of a bound ligand. GPCR quaternary structure has been explored by a number of approaches, but in recent times, these have been dominated by combinations of intact cell BRET and fluorescence-resonance energy transfer (FRET). These techniques are based upon energy transfer between either two autofluorescent proteins (in FRET studies) or an autofluorescent protein and an enzyme able to generate bioluminescence (in BRET studies). These probes are usually attached to the intracellular C-terminal tail of the GPCRs of interest (27–30). Very recently, a novel, homogeneous time-resolved (htr)FRET approach has been developed. This takes advantage of the capacity of small proteins based on mammalian O⁶-alkylguanine-DNA-alkyltransferase to be covalently modified with fluorescent and other small molecule labels (31). Such “SNAP” tagging can allow detection of cell surface, as well as intracellular protein-protein interactions. As a proof of concept, this has been used to explore aspects of GPCR quaternary structure, with particular focus on members of the class C, metabotropic glutamate receptor family (32). Herein, we use combinations of dual color FRET imaging and SNAP tag-based htrFRET of both wild type and RASSL forms of the human M₃ muscarinic receptor to explore the quaternary structure of this receptor and its potential regulation by ligands.

EXPERIMENTAL PROCEDURES

RESULTS

The human M₃ muscarinic acetylcholine receptor was modified at the N terminus to incorporate the FLAG epitope tag and at the C terminus via in-frame fusion of the modified yellow fluorescent protein citrine. This generated the FLAG-hM₃WT-Citrine construct. Incorporation of Y149C (position 3.33) and A239G (position 5.46) mutations has been reported to generate a form of the M₃ muscarinic receptor with substantially reduced potency for the endogenous agonist acetylcholine but that is able to bind and respond to the synthetic ligand clozapine N-oxide (25). This form of the M₃ muscarinic receptor was modified at the N terminus to incorporate the c-Myc epitope tag and at the C terminus via in-frame fusion of the modified cyan fluorescent protein cerulean. This generated the myc-hM₃RASSL-Cerulean construct. Each of these constructs was inserted into the Flp-In locus of Flp-In^TM T-REx^TM 293 cells and pools of positive cells selected. No visible expression of either construct was observed in the absence of the antibiotic doxycycline (Fig. 1, A and B). However, sustained addition of doxycycline resulted in expression of each form as monitored by fluorescence corresponding to the citrine and cerulean fluorescent proteins, respectively (Fig. 1, A and B). Imaging of such cells suggested that the bulk of each construct was located at the plasma membrane (Fig. 1, A and B). In functional studies measuring ligand-induced elevation of [Ca²⁺]_i cells harboring FLAG-hM₃WT-Citrine at the Flp-In locus, but in which expression of the receptor construct had not been induced, clozapine N-oxide was without effect at concentrations up to 1 × 10⁻⁴ m. This was unaltered by treatment with doxycycline (up to 1 μg·ml⁻¹, 24 h) to induce receptor expression (Fig. 1C). By contrast, a weak response to carbachol could be recorded in untreated cells, with estimated EC₅₀ >1 × 10⁻⁴ m. This may reflect either low levels of endogenous expression of the M₃ muscarinic receptor, as has been reported previously in HEK293 cells (38), or low level expression of the construct in the absence of doxycycline. Following induction of FLAG-hM₃WT-Citrine, a robust response to carbachol was observed with EC₅₀ 4.0 ± 0.8 × 10⁻⁷ m (mean ± S.E., n = 4) (Fig. 1C). As anticipated, both in the absence and presence of doxycycline, Flp-In^TM T-Rex^TM cells harboring myc-hM₃RASSL-Cerulean displayed no response to carbachol at concentrations up to 1 × 10⁻⁴ m (Fig. 1D). A very weak response to clozapine N-oxide was observed in these cells in the absence of doxycycline (Fig. 1D). However, a robust response to clozapine N-oxide with EC₅₀ = 1.5 ± 0.3 × 10⁻⁸ m (mean ± S.E., n = 3) developed following induction of myc-hM₃RASSL-Cerulean by treatment with doxycycline (Fig. 1D). Saturation [³H]QNB binding studies demonstrated that FLAG-hM₃WT-Citrine bound this ligand with K_d = 68 ± 33 pm (mean ± S.E., n = 4), similar to values reported for the unmodified receptor. myc-hM₃RASSL-Cerulean had almost 40-fold lower affinity (K_d = 2.64 ± 0.24 nm, mean ± S.E., n = 4) for this ligand. To assess if this reduction in affinity for [³H]QNB was also noted for other muscarinic antagonists, the affinity of atropine as a competitive antagonist at both FLAG-hM₃WT-Citrine and myc-hM₃RASSL-Cerulean was assessed by Schild analysis. Herein, the ability of a range of concentrations of atropine to shift concentration-response curves to carbachol or clozapine N-oxide, respectively, was measured. Analysis of such studies produced estimates of affinity for atropine of 3.3 × 10⁻⁹ m for the wild type receptor and 9.5 × 10⁻⁹ m for the RASSL, confirming the somewhat lower affinity of atropine for the RASSL mutant that had been described previously (17).

Cells harboring myc-hM₃RASSL-Cerulean at the Flp-In locus were subsequently further transfected with FLAG-hM₃WT-Citrine. Clones expressing this construct constitutively and stably were then isolated. FLAG-hM₃WT-Citrine was predominantly present at the plasma membrane (Fig. 2A), whereas treatment with doxycycline was required for turn-on of expression of myc-hM₃RASSL-Cerulean in these cells (Fig. 2A). Induction of expression of myc-hM₃RASSL-Cerulean was time-dependent, reaching maximal levels within 24 h (Fig. 2B). This, however, had little effect on the expression level of FLAG-hM₃WT-Citrine (Fig. 2B). As anticipated, based on the pharmacological characteristics of cells able to express each variant individually, in these cells carbachol was an effective and potent agonist in both the absence (EC₅₀ = 2.4 ± 1.3 × 10⁻⁸ m, mean ± S.E., n = 3) and presence (EC₅₀ = 3.7 ± 0.14 × 10⁻⁸ m, mean ± S.E., n = 3) of doxycycline (Fig. 2C). By contrast, clozapine N-oxide was more than 650-fold more potent (EC₅₀ = 5.5 ± 0.8 × 10⁻⁸ m, mean ± S.E., n = 3) following treatment with doxycycline (Fig. 2C). Merging of images of the location of FLAG-hM₃WT-Citrine and myc-hM₃RASSL-Cerulean in cells induced to express the RASSL construct indicated almost perfect overlap of localization of the two forms at the cell surface (Fig. 2D).

Such “co-localization” studies can only define proximity within a distance of some 300 nm because of the current limits of light microscopy. To explore potential direct interactions between these two forms of the hM₃ receptor, FRET imaging studies (Fig. 3) were performed and both corrected and ratiometric FRET values calculated (Fig. 3, A and B). Both of these calculations indicated that the visual overlap of distribution of the two hM₃ receptor variants was consistent with the formation of FLAG-hM₃WT-Citrine-myc-hM₃RASSL-Cerulean dimers/oligomers. The ability of differing concentrations of doxycycline to induce varying amounts of the FRET energy donor myc-hM₃RASSL-Cerulean was assessed by each of cell imaging (Fig. 3A), direct measures of fluorescence intensity corresponding to the cerulean fluorescent protein (Fig. 3C), and anti-c-Myc immunoblotting studies (Fig. 3D). Maintained levels of FLAG-hM₃WT-Citrine in these cells were similarly confirmed by each of cell imaging (Fig. 3A), assessing fluorescence intensity corresponding to the citrine fluorescent protein (Fig. 3C) and anti-FLAG immunoblotting (Fig. 3D). Both corrected FRET and ratiometric FRET were then calculated at varying acceptor to donor ratios in these cells. These initially increased, from a lack of measurable signal in the absence of donor to reach a peak after exposure to 100 ng·ml⁻¹ doxycycline, after which FRET signals were reduced with turn-on of higher levels of myc-hM₃RASSL-Cerulean (Fig. 3B). Interestingly, addition of carbachol (1 × 10⁻³ m), but neither clozapine N-oxide (1 × 10⁻⁴ m) nor atropine (1 × 10⁻⁵ m), reduced substantially the calculated ratiometric FRET signals (Fig. 3E). This is consistent with carbachol altering the organizational structure of the oligomeric complex.

FRET between pairs of fluorescent proteins linked to polypeptides of interest is a well characterized means to observe protein-protein interactions in living cells (28, 29). However, a novel form of htrFRET, based on SNAP tagging, and marketed as TagLite^TM (32), has recently been introduced. This is suitable to monitor cell surface protein-protein interactions without the need for antibodies that, because of their potential to induce clustering, have been suggested to limit interpretation of more traditional time-resolved FRET techniques (28). We generated a generic plasmid to allow expression of constructs containing an N-terminal leader sequence, derived from the metabotropic glutamate 5 receptor, linked in-frame to the VSV-G epitope tag sequence, the 20-kDa SNAP tag (31), and then the receptor of interest. Both the wild type and the RASSL forms of the hM₃ receptor were cloned into this plasmid, and these were used to generate lines able to express, in an inducible fashion, VSV-G-SNAP-hM₃WT or VSV-G-SNAP-hM₃RASSL (Fig. 4A). Addition of an anti-SNAP-Alexa-594-labeled antibody to nonpermeabilized cells identified, in both cases, cell surface receptors following induction (Fig. 4A). Furthermore, addition of the cell-permeant SNAP-505 substrate, which links covalently to the SNAP tag, allowed detection of a small pool of intracellular receptors as well as confirming the extensive population of cell surface receptors (Fig. 4A). Merging of images derived from cells labeled with either the anti-SNAP antibody or SNAP-505 confirmed both cell surface and intracellular pools of the hM₃ receptor variants (Fig. 4A). Importantly, the presence of the N-terminal leader and SNAP tag did not alter basic pharmacological characteristics of the receptors. Saturation [³H]QNB binding studies indicated a K_d of 54 ± 5 pm (mean ± S.E., n = 6) for VSV-G-SNAP-hM₃WT and 2.44 ± 0.16 nm (mean ± S.E., n = 4) for VSV-G-SNAP-hM₃RASSL. Addition of carbachol (1 × 10⁻³ m) resulted in internalization of 17.9 ± 1.2% of cell surface VSV-G-SNAP-hM₃WT over a 40-min period, whereas clozapine N-oxide (1 × 10⁻⁴ m) produced loss of 17.6 ± 3.5% of VSV-G-SNAP-hM₃RASSL over the same time period (Fig. 4B). By contrast, treatment of either cell line with the agonist selective for the other receptor variant did not produce significant internalization (Fig. 4B).

To establish optimal conditions for the Tag-Lite^TM htrFRET studies, cells induced to express either VSV-G-SNAP-hM₃WT or VSV-G-SNAP-hM₃RASSL were incubated with the terbium-cryptate energy donor SNAP-Lumi4-Tb. The extent of labeling with this reagent was dependent on the level of receptor expression because treatment of cells with concentrations of doxycycline between 0 and 10 ng·ml⁻¹ increased the binding of a range of concentrations of this label (5–30 × 10⁻⁹ m) in an essentially linear fashion (Fig. 5A and data not shown). Optimization of htrFRET signals corresponding to cell surface hM₃ dimers/oligomers was achieved by varying the added concentration of the corresponding energy acceptor, SNAP-Red, in the presence of a fixed concentration of SNAP Lumi4-Tb (Fig. 5, A and B). For both the VSV-G-SNAP-hM₃WT and VSV-G-SNAP-hM₃RASSL cell lines (Fig. 5B), this was ∼8 × 10⁻⁸ m (Fig. 5, A and B) and essentially unaffected by the level of receptor expression (Fig. 5A). htrFRET signals were then monitored in cells induced to express VSV-G-SNAP-hM₃WT after addition of a combination of SNAP Lumi4-Tb and SNAP-Red. Basal signal, consistent with the presence of constitutive cell surface hM₃ receptor dimers/oligomers, remained stable over a period of at least 40 min (Fig. 6A) and was unaffected by the presence of atropine (Fig. 6A). Interestingly, however, the htrFRET signal was increased substantially by the addition of carbachol (1 × 10⁻³ m). This effect was time-dependent (Fig. 6A), reaching a maximal level by 20 min before subsequently declining. The extent of the effect of carbachol was dependent on the level of receptor expression, with lower levels of doxycycline associated with a greater effect of carbachol (Fig. 6B). This reflected that at higher receptor expression levels the basal htrFRET signal was higher than at lower expression levels (Fig. 6B), and in this situation carbachol produced a more limited effect on the htrFRET signal (Fig. 6B). The effect of carbachol on the htrFRET signal corresponding to cell surface VSV-G-SNAP-hM₃WT dimers/oligomers was selective. Clozapine N-oxide was without effect on the htrFRET signal (Fig. 6A), and although atropine (1 × 10⁻⁵ m) produced a small reduction in the basal htrFRET signal in some experiments (Fig. 6A), this was not observed consistently. The effect of carbachol was concentration-dependent with EC₅₀ = 3.3 ± 1.1 × 10⁻⁴ m (mean ± S.E., n = 3) (Fig. 6C). Studies in which carbachol was allowed to compete with [³H]QNB to bind to VSV-G-SNAP-hM₃WT in membranes of cells induced to express this construct showed that this potency corresponded to the lower of two affinity states for carbachol (Fig. 6D). Acetylcholine, the endogenous agonist of the M₃ muscarinic receptor, was also able to increase the htrFRET signal as extensively as carbachol in cells expressing VSV-G-SNAP-hM₃WT and was some 60-fold more potent than carbachol in so doing (EC₅₀ = 5.8 × 10⁻⁶ m) (Fig. 6E). Interestingly, in cells induced to express VSV-G-SNAP-hM₃RASSL, clozapine N-oxide increased the basal htrFRET signal in a concentration-dependent fashion (Fig. 7, left), whereas neither carbachol nor atropine had any effect (Fig. 7, right).

DISCUSSION

It is now well established that many, and perhaps all, rhodopsin-like family A GPCRs can form dimers and/or higher oligomers (4–7). However, the significance of this remains uncertain because it is not inherently required to allow interaction with a G protein (1–3). Indeed, there is evidence that monomeric family A GPCRs may actually mediate G protein-dependent signals more effectively than dimers (2). Signals from GPCRs may also be generated in G protein-independent fashion (39), but it remains to be established if such signals can also be transduced by monomeric GPCRs.

A further topic that has attracted considerable attention is the question of whether ligands modulate the quaternary organization of GPCRs. Published data on this topic are highly variable (5–8). Given that interactions between individual protomers of class A receptor dimers/oligomers are not generally based on covalent interactions (4, 5), then there is clear potential for monomer-multimer equilibria to be altered by the binding of receptor ligands. This reflects that agonist binding must alter receptor conformation (40) to induce states of the receptor able to interact more effectively with G proteins and other GPCR-interacting proteins. Although many studies have suggested that ligand binding to receptors does not produce substantial effects on GPCR quaternary structure (4–7), a considerable number of observations of ligand modulation of GPCR dimers/oligomers exist in the literature. These include effects of an antagonist/inverse agonist drug to promote the production of a high affinity dimeric state of the M₁ muscarinic receptor from receptor monomers (25) and the concept that the β₂-adrenoreceptor exists predominantly in a basal tetrameric state that is unaffected by either agonist or antagonist ligands, whereas inverse agonists enhance tighter packing of the protomers and/or the formation of more complex oligomers by reducing conformational fluctuations in individual protomers (41). Other studies are consistent with agonist ligands promoting conformational alterations in pre-existing GPCR dimers/oligomers (40) and both enhancing and reducing dimerization (4–7, 25, 42–44).

It is clearly possible that such variation reflects intrinsic differences in the organization and affinity between protomers of different GPCRs. For example, fluorescence recovery after photobleaching microscopy has been applied to conclude that the β₂-adrenoreceptor forms stable complexes, although the β₁-adrenoreceptor displays only transient interactions (45). However, given the range of approaches used to detect interactions between GPCRs and potential regulation by ligands, it is also possible that different techniques might be more or less well suited. Resonance energy transfer based approaches have dominated the field in recent years (27–29, 46), and studies have generally added energy acceptor and donor moieties to the intracellular C-terminal tail of the GPCR(s) being studied. Strengths and weaknesses of this have been reviewed (28) and debated (47, 48). One of the recent additions to the armorium of approaches is an htrFRET method that takes advantage of the capacity of small proteins based on mammalian O⁶-alkylguanine-DNA-alkyltransferase to be covalently modified with FRET-competent energy donor and acceptors (31). Via addition of such a SNAP tag to the extracellular N terminus of class C GPCRs, Maurel et al. (32) were able to confirm the presence of cell surface dimers of metabotropic glutamate receptor subtypes. They also used this approach to provide some preliminary data on dimerization of class A receptors (32). Herein, we have extended this to explore the presence and regulation of dimeric/oligomeric complexes at the surface of cells stably expressing forms of the M₃ muscarinic acetylcholine receptor and compared the results with those obtained via more conventional FRET imaging-based studies. A key feature of the FRET imaging studies was to employ the inducible expression of DNA located at the Flp-In^TM locus of Flp-In^TM T-REx^TM 293 cells (33–35). This allowed us to regulate expression of one form of the M₃ muscarinic receptor in the presence of an unaltered amount of a second form in the same cells, rather than attempting to control relative expression levels in a substantial series of transient co-transfection studies. Transient transfection into heterologous cell lines of GPCRs in general and, in particular, of the type of highly modified forms used for resonance energy transfer studies often results in incomplete folding and their retention in the endoplasmic reticulum and Golgi. This is a major issue in efforts to explore dimerization in so-called “saturation” resonance energy transfer studies (28). These require a series of measurements in cells expressing differing ratios of energy-donor and energy-acceptor species. Therefore, the linkage of the energy donor and acceptor species to the C-terminal tail of the GPCRs in question results in signal being recorded from intracellular locations as well as the cell surface. A second key feature of this study was the combined use of wild type and RASSL forms of the M₃ muscarinic receptor to take advantage of selective agonist pharmacology. One minor limitation of the M₃ muscarinic receptor RASSL is that it binds conventional antagonist ligands less well than the wild type receptor. This increased the concentration of [³H]QNB needed in the ligand binding studies that were used to define expression levels and receptor pharmacology. However, with good practice the pharmacological characteristics of this variant can be fully defined. This is important because although the affinity of [³H]QNB was 40-fold lower for the RASSL forms of the receptor compared with the wild type, variation in affinity for the widely used muscarinic receptor antagonist atropine was only 3-fold. Variation in ligand affinity at the modified receptor is therefore defined by the identity of the specific ligand and must be determined directly for each compound being studied.

Both wild type and RASSL forms of the M₃ muscarinic receptor suitable for FRET imaging were expressed predominantly at the cell surface of Flp-In^TM T-REx^TM 293 cells when they were induced. Furthermore, strong FRET signals between these forms at the cell surface membrane were obtained in cells engineered to allow their co-expression. However, it should be noted that increasing FRET donor amounts in the presence of a fixed level of FRET acceptor resulted in a bell-shaped curve of ratiometric FRET signal. This is not surprising. Higher levels of donor would be expected to result in a greater proportion of non-FRET-productive donor-donor interactions that are anticipated to eventually limit or out-compete productive donor-acceptor interactions. In these studies, carbachol caused a substantial reduction in ratiometric FRET signal. This is consistent with either alteration in the organizational structure of preformed WT-RASSL M₃ receptor complexes or the dissociation of such complexes. By contrast, the antagonist atropine did not modulate such signals substantially. Intramolecular GPCR FRET sensors, constructed in a single GPCR protomer, are often used to detect agonist-induced alterations in FRET signal corresponding to relative movements of the intracellular ends of transmembrane helices that are believed to promote interaction with a G protein (49–51). Furthermore, relative re-orientation of the transmembrane helices that are believed to be contact interfaces within GPCR dimers/oligomers have been detected upon addition of agonist by a number of means (41, 52, 53). However, in many studies employing FRET or BRET, limited overall effects of GPCR ligands have been noted and interpreted to indicate that GPCR dimers/oligomers are present constitutively and not regulated acutely (5, 21, 54). Surprisingly, unlike carbachol, clozapine N-oxide did not alter FRET signal in cells induced to co-express the hM₃ receptor variants. Given that FRET signals in these cells must reflect complexes containing at least one wild type and one RASSL mutant M₃ receptor, it might have been anticipated that agonist occupancy of either type of protomer would result in equivalent effects on aspects of complex re-organization. This reflects that there should be reciprocity in regulation between components of a protein complex (55). We do not have a clear explanation for why this was not observed. A number of possibilities exist. For example, although clozapine N-oxide acted as an apparent full agonist at the RASSL variant in the Ca²⁺ mobilization studies, it may, due to issues of receptor reserve, not truly have the same efficacy as carbachol at the wild type receptor. However, it was as effective at causing internalization of the RASSL variant from the surface of the cells as was carbachol in promoting internalization of the wild type receptor. Equally, it may be that the cell surface complex detected by these studies is not a simple 1:1 wild type/RASSL dimer. Indeed, there is growing evidence that at least certain class A GPCRs, including muscarinic receptors, can exist as higher order complexes containing at least four protomers (41, 52, 53, 56).

In contrast to the FRET imaging studies, those based on the Tag-Lite^TM system produced evidence consistent with either significant agonist-induced re-organization within constitutive M₃ receptor quaternary structure or, perhaps more interestingly, agonist-induced enhancement of dimerization/oligomerization. First, these effects were produced only by pharmacologically appropriate agonists, i.e. carbachol and acetylcholine at the wild type receptor and clozapine N-oxide at the RASSL variant. Moreover, agonist potencies in these studies were consistent with receptor occupancy. However, as with the FRET imaging studies, clozapine N-oxide was less effective at the RASSL variant than either carbachol or acetylcholine at the wild type receptor complex. Second, effects of carbachol were most pronounced at lower levels of receptor expression. This is at least consistent with the concept that the M₃ receptor displays a greater propensity to form oligomeric structure at higher density. In such a situation, agonist might not be able to promote this further, although at lower receptor density there is less quaternary organization, but this is promoted by agonist binding. One further curiosity of these observations was the time dependence of the effects. These were much slower than the anticipated association constant for the ligand. Interestingly, FRET studies on purified β₂-adrenoceptors have also shown slow time-dependent effects of ligands on intramolecular movement between transmembrane helices (57), but this is assumed to reflect the artificial environment needed to perform the studies. By contrast, intact cell intramolecular (50) and intermolecular (58) FRET studies on the movement of receptor helices are much more consistent with time courses of physiological function.

These results have major implications for our understanding of ligand regulation of GPCR quaternary structure, and it will be of great interest to see if similar observations are seen when studying other closely and less closely related GPCRs, both in terms of homo- and heteromeric pairings. They also indicate that conclusions as to the direction of effect of ligands on such GPCR complexes may depend on the approach used.