Constitutive Activation of the δ Opioid Receptor by Mutations in Transmembrane Domains III and VII*

We have investigated whether transmembrane amino acid residues Asp128 (domain III), Tyr129(domain II), and Tyr308 (domain VII) in the mouse δ opioid receptor play a role in receptor activation. To do so, we have used a [35S]GTPγS (where GTPγS is guanosine 5′-3-O-(thio)triphosphate) binding assay to quantify the activation of recombinant receptors transiently expressed in COS cells and compared functional responses of D128N, D128A, Y129F, Y129A, and Y308F point-mutated receptors to that of the wild-type receptor. In the absence of ligand, [35S]GTPγS binding was increased for every mutant receptor under study (1.6–2.6-fold), suggesting that all mutations are able to enhance constitutive activity at the receptor. In support of this finding, the inverse agonistN,N-diallyl-Tyr-Aib-Aib-Phe-Leu (where Aib represents α-aminobutyric acid) efficiently reduced basal [35S]GTPγS binding in the mutated receptor preparations. The potent agonist BW373U86 stimulated [35S]GTPγS binding above basal levels with similar (D128N, Y129F, and Y129A) or markedly increased (Y308F) efficacy compared with wild-type receptor. BW373U86 potency was maintained or increased. In conclusion, our results demonstrate that the mutations under study increase functional activity of the receptor. Three-dimensional modeling suggests that Asp128 (III) and Tyr308 (VII) interact with each other and that Tyr129 (III) undergoes H bonding with His278(VI). Thus, Asp128, Tyr129, and Tyr308 may be involved in a network of interhelical bonds, which contributes to maintain the δ receptor under an inactive conformation. We suggest that the mutations weaken helix-helix interactions and generate a receptor state that favors the active conformation and/or interacts with heterotrimeric G proteins more effectively.

Opiates elicit their potent biological actions through three classes of opioid receptors, , ␦, and . Three homologous opioid receptor genes have been identified that encode each of the previously characterized receptor sites (for a review see Ref. 1). The cloned receptors belong to the G protein-coupled receptor superfamily, endowed with a seven transmembrane (Tm) 1 to-pology. All three opioid receptors are coupled to heterotrimeric G proteins of the G i /G o type, and receptor stimulation by an opioid agonist modulates a number of intracellular pathways (2)(3)(4) leading to inhibition of neuronal activity. Little is known about the molecular mechanisms of transmembrane signaling at the receptor level.
Activation mechanisms of G protein-coupled receptors (GPRs) are being extensively investigated both by computer modeling and mutagenesis or spectroscopy (for reviews see Refs. [5][6][7][8]. Rhodopsin, muscarinic, and adrenergic receptors have been most studied, and a number of hypotheses have been proposed. In particular it has been suggested that transmembrane motions are involved in processes that would make intracellular loops of the receptor accessible to G proteins. Tm-Tm interactions seem to be critical in maintaining the receptor in an inactive conformation, and modifications of H bonding by proton transfer have been suggested as part of the activation phenomenon. The question of how binding interactions do propagate through Tm domains down to intracellular loops and trigger G protein activation has not been solved. It is nevertheless predictable that some residues that belong to the binding pocket may also participate in signaling. According to low structure resolution of bovine rhodopsin (9,10), Tm III and VII are the most buried regions within the helical bundle, with limited access to the lipidic membrane environment, and they most probably interact with each other. We have previously mutated single amino acid residues of Tm III (Asp 128 and Tyr 129 ) and VII (Tyr 308 ) of the mouse ␦ opioid receptor (mDOR) (Fig. 1) and proposed that these residues belong to the opioid binding site and participate in ligand recognition (11,12). To investigate whether these residues also participate in the transduction process itself, we have examined the effect of mutations on receptor signaling.
The determination of ligand potency and efficacy at mutant recombinant G i /G o -coupled receptors usually requires the establishment of stable cell lines for each receptor under study. We have recently developed a method to measure opioid receptor activation using transient expression in COS cells and [ 35 S]GTP␥S binding (13). We now have used this approach to evaluate the effect of D128N and D128A (Tm III), Y129F and Y129A (Tm III), and Y308F (Tm VII) mutations on functional activation of the ␦ receptor. An important result from this study is the finding that structural modifications in Tm III and Tm VII enhance ligand-independent [ 35 S]GTP␥S binding. This is the first report of constitutively active mutant (CAM) opioid receptors.

EXPERIMENTAL PROCEDURES
Materials-DADLE, D-Ala 2 deltorphin II, naloxone, GDP, and GTP␥S were purchased from Sigma. Bremazocine was from Research Biochemicals International (Natick, MA). SNC80 and ICI174864 were from Tocris Cookson (Bristol, United Kingdom). BW373U86 was kindly provided by Dr. K. J. Chang (Burroughs Wellcome Co., Research Triangle Park, NC). [ 3 H]NTI (specific activity, 44.5 Ci/mmol) was provided by A. Borsodi (Hungarian Academy of Sciences, Szeged, Hungary), and [ 35 S]GTP␥S (specific activity, 1250 Ci/mmol) was from NEN Life Science Products. mDOR cDNA (14) was subcloned into pcDNAI/Amp (Invitrogen) for site-directed mutagenesis and transient expression in COS cells. The carrier plasmid used in the electroporation procedure (pBluescript) was from Stratagene. K d , K i , and B max values were calculated using the EBDA/Ligand program (G. A. McPherson, Biosoft, UK), and EC 50 values were determined using the Prism software (GraphPad, San Diego, CA).
Mutagenesis of mDOR and Expression of Wild-type and Mutant Receptors in COS Cells-mDOR cDNA was modified by site-directed mutagenesis, as described in previous studies (11,12), to obtain point mutations in Tm III (D128N, D128A, Y129F, and Y129A) and in Tm VII (Y308F). COS cells were transfected using an electroporation procedure as described previously (13). Briefly, 2 ϫ 10 7 COS cells were seeded the night before transfection at a density of 10 7 cells/140-mm dish. Cells were washed two times with phosphate-buffered saline, detached by applying 5 ml of trypsin/EDTA (Eurobio) for 5 min at 37°C, and diluted with 5 ml of Dulbecco's modified Eagle's medium, and an aliquot was used to determine the total cell number. Cells were collected by centrifugation for 10 min at 1000 rpm and resuspended at a 10 8 cell/ml density in EP 1ϫ buffer (50 mM K 2 HPO 4 , 20 mM CH 3 CO 2 K, 20 mM KOH, pH 7.4). Plasmidic DNA, prepared using Nucleobond columns (Macherey Nagel) and consisting of 4 g (standard condition) or a variable amount of receptor-encoding plasmid and a carrier plasmid (pBluescript) up to a final 20-g DNA quantity was diluted into EP 1ϫ buffer to a total volume of 300 l. The DNA mix was then supplemented with 13 l of MgSO 4 1 M and incubated with 200-l cell suspension for 20 min at room temperature. The cell/DNA mixture was then transferred to a 0.4-cm cuvette (Bio-Rad) and electroporated using a Gene Pulser apparatus (Bio-Rad) at a capacitance setting of 2000 microfarads and a voltage setting of 240 V. Cells were then immediately transferred into 50 ml of Dulbecco's modified Eagle's medium with 10% fetal calf serum and seeded into two 140-mm dishes. After 72 h growth the cells were harvested, and membranes were then prepared as described previously (13).
Membrane Preparation-The same procedure was used for either [ 3 H]naltrindole or [ 35 S]GTP␥S binding. Transfected cells (four 140-mm dishes at a 50 -100% confluency) were washed two times with phosphate-buffered saline, scrapped off the plates in phosphate-buffered saline, pelleted by spinning at 1500 rpm for 10 min at 4°C, frozen at Ϫ80°C for at least 30 min, and thawed in 30 ml of cold 50 mM Tris-HCl, pH 7, 2.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride added extemporaneously. The cell lysate was put on ice for 15 min, processed in a Dounce homogenizer, and spun at 2500 rpm for 10 min at 4°C. The pellet was resuspended in 15 ml buffer, processed in a Dounce homogenizer, and spun again at 2500 rpm for 10 min at 4°C. Both supernatants were pooled and centrifuged at 40 000 rpm for 30 min at 4°C. The supernatant was removed, and the pellet was resuspended in 25 ml of 50 mM Tris-HCl, pH 7, processed in a Dounce homogenizer, and spun again at 40000 rpm for 30 min at 4°C. The pellet was then resuspended in 4 ml of 50 mM Tris-HCl, pH 7, sucrose 0.32 M, and the protein concentration was measured using the Bradford assay. Membranes were aliquoted and stored at Ϫ80°C.
Saturation and Competition Analysis-Opioid binding experiments were done as described previously (12). For saturation experiments, 0.8 -5 g of membrane proteins were diluted in 50 mM Tris-HCl, pH 7.4, in a final volume of 0.5 ml and incubated with variable concentrations of [ 3 H]naltrindole (0.03-3 nM) for 1 h at 25°C. Nonspecific binding was determined in the presence of 2 M naloxone. For competition studies membrane preparations were incubated for 1 h at 25°C with [ 3 H]naltrindole (0.5 nM) in the presence of various concentrations (10 Ϫ5 to 10 Ϫ13 M) of the opioid competing ligand (SNC80). In both cases, incubation mixtures were rapidly washed using a Brandell cell harvester with cold 50 mM Tris buffer on 0.1% polyethylenimine presoaked GF/B filters (Whatman).
[ 35 S]GTP␥S Binding Assays-Ligand-stimulated [ 35 S]GTP␥S binding on COS cell membrane preparation expressing mDOR and mDOR mutants was examined as described previously (13). 5 g of membrane proteins were incubated for 2 h at 4°C both with and without the agonist (10 Ϫ5 to 10 Ϫ13 M) or inverse agonist ICI174864 (10 Ϫ5 M) in assay buffer containing 50 mM Hepes, pH 7.6, 5 mM MgCl 2 , 100 mM NaCl, 10 mM EDTA, 1 mM dithiothreitol, 0.1% bovine serum albumin, 10 M GDP, and 0.2 nM [ 35 S]GTP␥S. Nonspecific binding was determined in the presence of 10 M GTP␥S, and basal binding was assessed in the absence of agonist. As for saturation experiments, the incubation was terminated by rapid filtration through H 2 O presoaked GF/B filters, followed by three washes with ice-cold 50 mM Tris-HCl, pH 7, 5 mM MgCl 2 , 50 mM NaCl. Bound radioactivity was determined by scintillation counting. Mock transfected control cells showed no ligand-induced [ 35 S]GTP␥S binding.  Table I) showed high affinity sites for the ligand (K d ϭ 0.33 Ϯ 0.08 nM) and a B max value of 51.2 Ϯ 4.2 pmol/mg protein, indicating high receptor expression level under our standard transfection conditions. These membrane preparations were then subjected to stimulation by agonists, and specific [ 35 S]GTP␥S binding was measured using a 10 Ϫ13 to 10 Ϫ5 M concentration range of the following agonists: BW373U86 and SNC80 (two ␦ selective alkaloids), deltorphin II and DADLE (two ␦ selective peptides), and bremazocine (a nonselective alkaloid). Results are shown in Fig. 2 and Table II [ 35 S]GTP␥S binding increased 1.7-3-fold above basal level upon agonist stimulation. BW373U86, SNC80, and deltorphin II were most efficacious with 275%, 273%, and 264% activation at saturating agonist concentrations. Bremazocine was the least effective compound (167% of basal level) and behaved as a partial agonist. The order of potencies was BW373U86 Ͼ deltorphin II Ͼ DADLE Ͼ bremazocine Ͼ SNC80, and EC 50 values correlate with previously determined affinity values (11). BW373U86, which displays highest affinity for the receptor, is also most potent at stimulating [ 35 S]GTP␥S binding (EC 50 0.62 Ϯ 0.18 nM), in accordance with previous data (15). Together these results show that our assay conditions are able to discriminate ligands with distinct activation properties. Recently, [ 35 S]GTP␥S binding has been successfully used to evaluate agonists potencies and efficacies at the cloned (16) and (17) receptors using Chinese hamster ovary cell lines stably expressing the receptors. Here we demonstrate that this assay     (Fig. 4), but this action was only significant for membrane preparations WT II and WT IV. This confirms the weak ligand-independent activity of mDOR transiently expressed in COS cells suggested by our measurements of [ 35 S]GTP␥S binding at different receptor densities (see above). Our data contrast with the high efficacy of ICI174864 for inhibiting constitutive activity in other cell lines expressing the endogenous receptor (50% inhibition of GTPase activity in NG108-15 (20)) or stably expressing the WT cloned receptor (40% inhibition of GTPase or basal [ 35 S]GTP␥S binding activity in Rat-1-DOR cells (21); 4-fold increase of forskolin-stimulated cAMP accumulation in HEK 293-DOR cells (22)). The difference could arise from different experimental procedures or from distinct G protein content and receptor/G protein stoichiometry in host cells. The type of G␣ subunits present in COS cells, as well as the high receptor/G protein ratio under transient expression conditions, may be less appropriate for efficient coupling to G proteins than in the other systems that have been studied so far. Alternatively, the presence of other proteins or the membrane composition of COS cells may be less favorable to conformational modifications within the WT receptor.

[ 35 S]GTP␥S Binding in COS Cells
In mutant receptors ICI174864 decreased [ 35 S]GTP␥S binding below basal levels but to variable extends depending on the mutant (Fig. 4). ICI174864 markedly reduced [ 35 S]GTP␥S binding in mutant receptors with high constitutive activity, namely D128N (36% inhibition) and D128A (51% inhibition). This result therefore provides additional evidence for constitutive activity at Asp 128 mutant receptors.
For mutant receptors exhibiting lower ligand-independent activity (Y129F, Y129A, and Y308F), the effect of ICI174864 was less evident. Basal levels were moderately diminished for mutations at Tyr 129 , with a significant 19% inhibition at the Y129A mutant. No ICI174864 activity was detectable at Y308F mutant receptor. Hence the analysis of ICI174864 activity at Tyr 129 and Tyr 308 receptors is less conclusive, presumably because of the low constitutive activity of the latter mutants compared with Asp 128 mutant receptors. ICI174864 activity may also be less well detected in these mutant receptors for another reason: we observed that the ICI174864 compound was not able to decrease [ 35 S]GTP␥S binding in mutant receptor preparations to levels as low as in their corresponding WT preparations. Thus, in the presence of ICI174864, [ 35 S]GTP␥S binding levels remain 1.3-2.1-fold higher in mutant preparations than in WT preparations (Fig. 4). One explanation could be that although ICI174864 binding to mutant receptors is maintained (not shown), the point mutations introduced into the receptor protein have altered some interactions between ICI174864 and the receptor that are critical for the inverse agonist activity. Alternatively, the active states of the mutant receptors may be structurally different from the active state of the WT receptor, and mutations may have produced receptor conformations that cannot be fully inactivated by an inverse agonist.

Stimulation of [ 35 S]GTP␥S Binding at WT and Mutant
Receptors by the Agonist BW373U86 -To measure agonist-induced stimulation of mutant receptors we used the BW373U86 compound because its binding affinity was not dramatically impaired by the mutations under study (Table III) and because it is one of the most potent agonist in our assay system (Fig. 2). We first compared BW373U86-evoked stimulation of [ 35 S]GTP␥S binding to mutant and WT receptor membrane preparations at a saturating agonist concentration (Fig. 4). A strong increase of [ 35 S]GTP␥S binding levels was observed for all high expressing receptor preparations (WT II, WT III, WT IV, D128N, Y129F, Y129A, and Y308F), clearly indicating that BW373U86 remains a potent agonist at D128N, Y129F, Y129A, and Y308F mutant receptors. Extremely weak agonist-induced activation could be measured for the D128A preparation, as well as its corresponding WT preparation (WT V). Because of the low receptor densities in those membrane preparations, it is difficult to conclude whether  (Table I). BW373U86-induced activation is altered in the D128A mutant receptor. Transfecting higher amount of cDNA (up to 20 g) failed to increase expression levels of the D128A receptor (data not shown). Therefore agonist activity at this mutant receptor could not be investigated further.
Dose-response curves for D128N, Y129F, Y129A, and Y308F mutants are shown in Fig. 5. These data from a representative experiment show both the constitutive activity and the agonistinduced activation at mutant receptors. Indeed, their enhanced agonist-independent activity combined with the efficient BW373U86-induced activation leads to maximal [ 35 S]GTP␥S binding levels that largely exceed those of WT receptors. The efficacy of BW373U86 was evaluated as the difference of [ 35 S]GTP␥S binding in the presence and absence of agonist and is shown for each mutant receptor and its WT counterpart (Table III). Results show that agonist efficacy was not impaired in any mutant receptor. On the contrary, a tendency to increased efficacy (1.1-1.5-fold) could be detected for Tm III mutants, and a marked augmentation was measured for the Tm VII mutant (2.7-fold).
The potency of BW373U86 to activate [ 35 S]GTP␥S binding was determined from the dose response-curves (Fig. 5), and EC 50 values are shown in Table III together with the previously determined affinity values of the agonist for each mutant receptor. For the Y129A receptor, the EC 50 value was decreased to the same extent than affinity (5-fold). At the D128N receptor BW373U86 potency was reduced to a lesser extent (7.6-fold) than affinity (27-fold). Finally no significant change in potency was observed for mutants Y129F and Y308F, although BW373U86 affinity was reduced approximately 8-fold at both mutants. These results suggest that, similar to efficacy, the potency of BW373U86 was generally maintained or even enhanced at the mutant receptors. DISCUSSION Here we have investigated both agonist-dependent and agonist-independent activity of point-mutated ␦ opioid receptors.
Our results show that all the structural changes that we have introduced enhance receptor activity. The most noticeable feature is the observation that each mutation significantly augments ligand-independent receptor-mediated [ 35 S]GTP␥S binding activity.
Point mutations at adrenergic receptors originally demonstrated that structural modifications of a GPR can trigger agonist-independent activation (23)(24)(25). Mutations that produce constitutive activity have since been found for other  GPRs, both from site-directed mutagenesis studies and among spontaneous mutations associated with human diseases (for reviews see Refs. 6 and 26). For rhodopsin, adrenergic, muscarinic, thyroid-stimulating hormone, luteinizing hormone, and melanocortin receptors, constitutive activity mostly arises from mutations clustered in Tms II, III, VI, and VII, as well as in the cytoplasmic extensions of Tms III and VI. It has been suggested that these specific receptor domains form a key structure that is central for the activation process and would operate among most GPRs (for reviews see Refs. [5][6][7][8]. CAM were also identified in other receptor areas, including extracellular loops for thrombin (27) and thyroid-stimulating hormone (28) receptors or the C-terminal tail for two prostaglandin E receptor splice variants (29). This suggests the possible existence of receptorspecific activation mechanisms.
In this study we have generated constitutive activity at the ␦ receptor by modifying amino acid residues located in the upper portion of putative helices III (Asp 128 and Tyr 129 ) and VII (Tyr 308 ). CAM localized in these particular receptor regions have been described in other GPRs. Some are found at a position homologous to Asp 128 and were characterized in rhodopsin (Glu 113 ; see Refs. 30 and 31) and in the ␣ 1B -adrenergic receptor (Asp 125 ; see Ref. 32). Mutations at vicinal positions have also been shown to enhance receptor basal activity both for the ␣ 1B -adrenergic receptor (C128F; see Refs. 32 and 33) and the AT1A angiotensin II receptor (N111A; see Ref. 34). Thus our data showing constitutive activity at D128N, D128A, Y129F, and Y129A mutant ␦ receptors further strengthen the notion that the exofacial portion of Tm III is critical in receptor signaling. CAMs located at a position homologous to Tyr 308 in Tm VII have also been reported. These include mutations at Lys 296 in rhodopsin (30,31,35,36) and Lys 331 in ␣ 1B -adrenergic receptor (32). Again our finding of an enhanced, although modest, basal activity at the Y308F mutant is consistent with findings at other GPRs. Altogether our data suggest that receptor domains that have been shown to be critical for the activation process in previously studied GPRs are also important in opioid receptors.
The initial finding that replacement of Ala 293 in the ␣ 1Badrenergic receptor by any other amino acid residue increases agonist-independent activity, has led to the notion that a number of critical intramolecular interactions would maintain the receptor under an inactive conformation (23). In this hypothesis, agonists would act by disrupting these conformational constraints and induce a "relaxed" receptor state that in turn would productively interact with G proteins. Compelling evi-dence has now accumulated, from a wide variety of methodological approaches, to show the importance of interhelical interactions in the allosteric transition between inactive and active receptor states (for a recent review see Ref. 8). On the basis of the current hypothesis, it is likely that the three amino acid residues of the ␦ receptor that we have studied may be part of the stabilizing interhelical network that keeps the receptor inactive. Our results show that conservative mutations in Tm III (D128N and Y129F) and Tm VII (Y308F) trigger constitutive activation of the receptor. Those structural modifications may alter (D128N) or suppress (D128A, Y129F, and Y308F) one hydrogen bond within the helical bundle, and this may be sufficient to enhance conformational flexibility, allowing the receptor to undergo transition from inactive to active state more easily. Further, it seems that the alanine substitution at Tyr 129 produces a slightly higher constitutive activation than does the conservative phenylalanine substitution (Fig. 4). We therefore cannot exclude the possibility that the benzene ring of Tyr 129 also participates in interhelical stabilization through aromatic-aromatic interaction, as previously suggested (12,37).
Possible interacting partners of Asp 128 , Tyr 129 , and Tyr 308 can be hypothesized based on computer three-dimensional modeling. Most models that have been proposed for the ␦ opioid receptor poorly describe the empty receptor and mainly identify amino acid residues that could potentially interact with opioid ligands (38 -46). From these models Asp 128 , Tyr 129 , or Tyr 308 could establish direct hydrogen bonding with a number of agonists, including BW373U86 (Asp 128 and Tyr 129 ; see Ref. 38; Asp 128 ; see Footnote 2). On the assumption that agonist binding induces receptor activation by disrupting pre-existing interhelical interactions, those amino acid residues are good candidates for participating in the structural stabilization of the unliganded receptor. We have further investigated possible Tm-Tm interactions in the absence of ligand using the model of Yue et al. (43). The model indicates that Asp 128 in Tm III and Tyr 308 in Tm VII could directly interact with each other and be critical in stabilizing the entire helical core (Fig. 6). A similar interaction may exist in other GPRs. For rhodopsin, it has been suggested that a salt bridge between the Tm III glutamic acid residue (Glu 113 ), and the Tm VII lysine bond visual chromophore 11-cis-retinal (Lys 296 ) would help maintain the constrained inactive conformation of the native protein (30,31 Table III. the ␣ 1B -adrenergic receptor, the Tm III residue homologous to Asp 128 has been proposed to serve as a counterion for the Tm VII residue homologous to Tyr 308 , and disruption of this bond was postulated to initiate receptor activation (32). There is therefore good indication that Tm III and Tm VII are located in close proximity and also that the existence of a Asp 128 -Tyr 308 interaction could play a role in the activation process. In the model by Yue et al. (Fig. 6) Tyr 129 , the other Tm III residue that we have studied, forms a hydrogen bond with His 278 in Tm VI. The same interhelical hydrogen bond has also been hypothesized in the three-dimensional model proposed by Poda and Maigret (46). This specific Tm III-Tm VI interaction may be opioid-receptor specific because no homologous interhelical interaction has been reported for other GPRs. In summary, we propose that Asp 128 and Tyr 129 interact with Tyr 308 and His 278 , respectively, within the unliganded receptor. The mutations would mimic the ligand action by altering those interhelical interactions, thereby facilitating receptor isomerization into an active conformation and/or its interaction with G proteins.
The originally proposed ternary agonist-receptor-G protein complex theory (47), as well as the now commonly accepted allosteric ternary complex model (24), predict that agonist binding increases binding affinity of the G protein for the receptor and vice versa. As a consequence, CAM receptors presumably imitate the active state of the receptor and are likely to bind agonist ligands with higher affinity, the so-called "high affinity state." In accordance with this hypothesis, most previously reported CAM GPRs exhibit enhanced affinity for agonists (23,24,32,48,49). Our data are not concordant with these findings. In a previous report, we have shown that agonist binding is diminished in D128N, Y129F, Y129A, and Y308F mutant ␦ opioid receptors, and we have suggested that this may be due to the loss of appropriate interactions in the binding pocket (11,12). Because we have modified amino acid residues that seem to be involved both in ligand recognition and in the activation process, one cannot exclude that a putative gain of affinity associated with constitutive activity may be masked by the loss of binding interactions between the ligand and the receptor.
Further, this observation is not necessarily in opposition with the data obtained at other GPRs. Receptors undergo several processes that include agonist binding, receptor isomerization between active and inactive conformations, and G protein association or dissociation. The relationship between these different steps is complex, the extent to which they are interconnected most certainly depends on the receptor under study and its cellular environment. Each step could be differently influenced by chemical modifications of the receptor (for a review see Ref. 5), and accumulating data now tend to demonstrate increasing diversity in the properties of CAM receptors. For example, the extent of agonist-induced stimulation appears highly variable, depending on the receptor and the mutation under study. As for the Y308F mutant receptor investigated here, agonist-induced stimulation was augmented in a CAM AT1A angiotensin II mutant receptor (34), but agonist efficacy was shown diminished for CAM ␣ 1B -adrenergic (23), luteinizing hormone 32 (50), and VIP (51) receptors or abolished in CAM thyrotropin (48) and prostaglandin EP3 (29) receptors. This suggests that the active conformations of agonist bond receptors may differ widely from one CAM to another, as well as from that of their wild-type counterparts. Both the localization and nature of the mutation certainly are critical to explain the pharmacological properties of a CAM receptor. Our mutations modify amino acid residues that are involved in at least two steps, namely ligand binding and receptor isomerization. The mutations may have suppressed a number of anchor points for the agonist, as well as Tm-Tm hydrogen bonds, impairing both the ligand recognition process and the stability of the inactive conformation. Full agonist-induced G protein activation, however, is preserved. Therefore, modifications of Asp 128 , Tyr 129 , and Tyr 308 side chains may have modified the ligand-receptor complex and the receptor itself as well as the receptor-G protein complex, but none of these mutations prevents productive interactions within the ligand-receptor-G protein ternary complex. The activity profile of the Y308F mutant receptor (Tm VII) differed to some extent from that of the Tm III mutant receptors. Most remarkably, Y308F responded significantly better to agonist stimulation. Currently the BW373U86-induced hyperactivation remains unexplained. The finding that Asp 128 mutants behave differently from the Y308F receptor suggests that breakage of the proposed Asp 128 -Tyr 308 hydrogen bond is not the only event induced by the site-directed mutagenesis. In addition to the hypothesized direct interaction, Asp 128 and Tyr 308 could also be involved in other interactions that, in turn, would implicate them differently in the activation process. The role of amino acid residues homologous to Tyr 308 has been investigated in other GPRs, by examining agonist-induced responses in mutant receptors. In the angiotensin AT1 receptor, substitution of the equivalent tyrosine (Tyr 292 ) by a phenylalanine did not modify agonist and antagonist peptide binding but impaired angiotensin II-stimulated inositol triphosphate hydrolysis (52). A different result was obtained for the m3 muscarinic receptor, where a tyrosine is also found at this position. Mutation into phenylalanine (Y533F) clearly reduced agonist affinity, but maximal stimulation of inositol triphosphate hydrolysis was comparable with that of the WT receptor (53,54). Similar results were obtained for the homologous residue of adenosine receptor (His 278 ), in which substitution with several amino acid residues abolished high affinity binding of agonist and antagonist, whereas the cAMP production was comparable with that of the WT receptor in the H278A mutant (55,56). Therefore our finding that the Y308F mutation reduces opioid binding affinity parallels what was observed for muscarinic and adenosine receptors. We also found constitutive activity at the Y308F receptor, as described previously for homologous mutations in rhodopsine and adrenergic receptors (see above), but the enhanced agonist-induced stimulation in Y308F receptor contrasts with findings at angiotensin AT1A, m3, and adenosine mutant receptors. We conclude that there is no obvious common role for this Tm VII amino acid residue. The distinct effect of homologous substitutions at different GPRs suggests the existence of receptor-specific mechanisms at the level of Tm VII, presumably because of distinct structural characteristics of this specific area of the helical bundle.
In conclusion CAMs have already proven to be useful tools to explore activation mechanisms of GPRs. We anticipate that the further exploration of constitutive activity in opioid receptors will help to clarify the general mechanisms of GPRs activation and also highlight opioid receptor-specific processes that accompany receptor signaling.