QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Voigtländer, U. Articles by Mohr, K. Search for Related Content PubMed PubMed Citation Articles by Voigtländer, U. Articles by Mohr, K.

Allosteric Site on Muscarinic Acetylcholine Receptors: Identification of Two Amino Acids in the Muscarinic M₂ Receptor That Account Entirely for the M₂/M₅ Subtype Selectivities of Some Structurally Diverse Allosteric Ligands in N-Methylscopolamine-Occupied Receptors

Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany (U.V., M.M., A.R., C.T., S.B., K.M.); Institute of Pharmaceutical Chemistry, University of Düsseldorf, Düsseldorf, Germany (K.J., H.-D. H.); and Departments of Psychiatry and Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania, USA (S.B., J.E.).

Received October 7, 2002; accepted March 21, 2003

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Two epitopes have been identified recently to be responsible for the high-affinity binding of alkane-bisammonium and caracurine V type allosteric ligands to N-methylscopolamine (NMS)-occupied M₂ muscarinic acetylcholine receptors, relative to M₅ receptors: the amino acid M₂-Thr⁴²³ at the top of transmembrane region (TM) 7 and an epitope comprising the second extracellular loop (o2) of the M₂ receptor including the flanking regions of TM4 and TM5. We aimed to find out whether a single amino acid could account for the contribution of this epitope to binding affinity. Allosteric interactions were investigated in wild-type and mutant receptors in which the orthosteric binding site was occupied by [³H]NMS (5 mM Na,K,P_i buffer, pH 7.4, 23°C). Using M₂/M₅ chimeric and point-mutated receptors, the relevant epitope was narrowed down to M₂-Tyr¹⁷⁷. A double point-mutated M₂ receptor in which both M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ were replaced by the corresponding amino acids of M₅ revealed that these two amino acids account entirely for the (approximately 100-fold) M₂/M₅ selectivity of the alkane-bisammonium and the caracurine V type allosteric ligands. At NMS-free M₂ receptors, the caracurine V derivative also displayed approximately 100-fold M₂/M₅ selectivity, but the double point mutation reduced the M₂ affinity by only 10-fold; thus, additional epitopes may influence selectivity for the free receptors. A three-dimensional model of the M₂ receptor was used to simulate allosteric agent docking to NMS-occupied receptors. M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ seem to be located near the junction of the allosteric and the orthosteric areas of the M₂ receptor ligand binding cavity.

It is striking that muscarinic allosteric ligands exhibit generally the highest affinity to the M₂ receptor subtype (Lee and El-Fakahany, 1991; Ellis et al., 1991; Ellis and Seidenberg, 2000). A number of studies aimed to identify receptor epitopes that are critical for the binding of allosteric agents to the M₂ receptor (Leppik et al., 1994; Gnagey et al., 1999; Buller et al., 2002). The use of M₂/M₅ chimeric receptors has turned out to be an effective approach to gain more insight into receptor areas that confer the high affinity of allosteric ligands to the M₂ receptor subtype (Ellis et al., 1993; Gnagey et al., 1999; Ellis and Seidenberg, 2000). In these experiments, we have focused on the formation of ternary complexes that are the characteristic feature of allosteric interactions. For this purpose, the interaction of the allosteric agents was measured with receptors in which the orthosteric site was occupied by [³H]N-methylscopolamine ([³H]NMS). Receptor binding of the allosteric agents is reflected by an inhibition of [³H]NMS dissociation. Using this procedure, we were able to identify two epitopes (Buller et al., 2002) that account fully for the high affinity of alkane-bisammonium–type and caracurine V-type allosteric ligands (Fig. 1) to the M₂ receptor, relative to the M₅ receptor. One of these epitopes consists of a single amino acid at the beginning of the seventh transmembrane region of the M₂ receptor, M₂-Thr⁴²³. The other epitope has not been so precisely located but lies either within the second outer loop of the M₂ receptor or in adjacent regions of TM4 and TM5.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structures of the applied allosteric agents. The molecules are shown in an energetically favorable conformation.

In the present study, chimeric and point mutated receptors were used to examine whether the second epitope likewise consists of a single amino acid that is pivotal for the binding of these allosteric agents. We identified M₂-Tyr¹⁷⁷, in the middle region of the second outer loop of the M₂ receptor, as the critical amino acid. A double point-mutated receptor revealed that these two amino acids, M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³, are sufficient to completely account for the M₂/M₅ selectivity of the applied allosteric agents in the NMS-occupied receptors. To check whether these amino acids play a role for the interaction with NMS-free receptors, we carried out equilibrium binding experiments with [³H]NMS that were analyzed according to the ternary complex model of allosteric interactions (Stockton et al., 1983; Ehlert, 1988). The findings suggest that the two amino acids are involved in the binding affinity of the allosteric modulators for the free M₂ receptor, but additional epitopes seem to contribute to the M₂/M₅-selectivity under this condition. A three-dimensional model of the M₂ receptor in the NMS-occupied state was built on the basis of the crystal structure of bovine rhodopsin. The model seems to be suitable to gain insight into the putative topography and the molecular mechanisms of allosteric-orthosteric interactions.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Receptor Mutagenesis and Expression
Site-directed mutagenesis was performed with the QuikChange kit (Stratagene, La Jolla, CA). In short, oligonucleotides containing the desired base changes were synthesized and allowed to anneal with pCD plasmids containing the appropriate muscarinic receptor DNA sequence. A high-fidelity polymerase then extended the synthetic oligonucleotides in a thermocycled reaction. The parental DNA was digested by a methylation-specific endonuclease, leaving only the mutated product, which was confirmed by sequencing. Subsequently, the mutated products were transformed in Escherichia coli cells and amplified overnight. Plasmids containing the human M₂ or M₅ wild-type or mutated receptor genes were purified from bacterial cultures and transiently transfected into COS-7 cells by calcium phosphate precipitation. Cells were harvested 72 h after transfection by scraping into 5 mM Na,K,P_i buffer (PB), pH 7.4. After homogenization and centrifugation at 50,000g for 20 min, membranes were resuspended in 5 mM PB and stored as aliquots at -80°C. Schematic diagrams of the chimeric receptors are shown in Fig. 2. The exact sequences of the chimeras are as follows:

View larger version (26K): [in this window] [in a new window]   Fig. 2. Concentration-effect curves for the allosteric delay of [3H]NMS dissociation induced by W84 at the indicated wild-type and mutant receptors. A, chimeric substitutions in o2 of the M2 receptor. B, converse chimeric substitutions in o2 of the M5 receptor. The receptors were prelabeled with [3H]NMS, dissociation was then observed in the absence or presence of the allosteric modulator. Ordinate, apparent rate constant of [3H]NMS dissociation in the presence of test compound (kobs) divided by the parameter value found with [3H]NMS alone (k0), expressed in percent. Abscissa, log concentration of allosteric modulator. Experiments were carried out with membranes from transiently transfected COS-7 cells in 5 mM PB. Indicated are means ± S.E. of two to four separate experiments. Curve fitting based on a four parameter logistic function.

• M₂("A"M₅): hM₂ 1–160, hM₅ 168–175, hM₂ 169–466
  
• M₂("B"M₅): hM₂ 1–171, hM₅ 179–184, hM₂ 178–466
  
• M₂("C"M₅): hM₂ 1–180, hM₅ 188–193, hM₂ 187–466
  
• M₅("A"M₂): hM₅ 1–167, hM₂ 161–168, hM₅ 176–532
  
• M₅("B"M₂): hM₅ 1–178, hM₂ 172–177, hM₅ 185–532
  
• M₅("C"M₂): hM₅ 1–187, hM₂ 181–186, hM₅ 194–532
  

Binding Assays
Dissociation Binding Assays. Dissociation binding assays were conducted in 5 mM PB, pH 7.4, at 23°C. Membranes were preincubated with 1 nM [³H]NMS for 30 min. Net dissociation of [³H]NMS was initiated by the addition of 3 µM atropine, with or without the indicated concentrations of allosteric modulator. After the appropriate time interval, the dissociation was terminated by filtration through Whatman GF/B glass fiber filters that had been soaked in 0.1% polyethylenimine, using a Brandel cell harvester; the filtration was immediately followed by two rinses with 40 mM PB (0°C). The filter-bound radioactivity was quantitated by liquid scintillation counting. Nonspecific binding was defined as the radioactivity bound in the presence of 3 µM atropine.

Equilibrium Binding Assays. [³H]NMS equilibrium binding was measured in 5 mM PB, pH 7.4, at 23°C. The [³H]NMS concentration was 0.2 nM. The incubation time necessary for attaining equilibrium binding of [³H]NMS in the presence of allosteric modulator was calculated according to Lazareno and Birdsall (1995; eq. 31 therein). Five observed half-life times of association were taken to be sufficient to attain equilibrium binding. Incubation times amounted for M₂ and the indicated double point mutant to 3 to 5 h and for M₅ to 24 h. Filtration and further processing was carried out as described above.

Data Analysis
Dissociation data were analyzed using the monoexponential decay equation, and apparent rate constants were obtained. The rate constant of [³H]NMS dissociation in the presence of a given concentration of allosteric modulator (k_obs) was divided by the rate constant for the control condition [i.e., [³H]NMS dissociation in the absence of the allosteric modulator (k₀)]. Curve fitting was based on a four-parameter logistic function as described previously (Tränkle and Mohr, 1997). The concentration-effect curve for the delay of [³H]NMS dissociation reflects the binding curve of the allosteric agent at the NMS-occupied receptor (Lazareno and Birdsall, 1995; Ellis, 1997; Tränkle et al., 1998). The concentration for a half-maximum effect on [³H]NMS dissociation (EC_0.5,diss) can be taken as a measure of affinity of the allosteric modulator for the NMS-occupied receptor.

Equilibrium binding data were analyzed according to the ternary complex model of allosteric interactions (Ehlert, 1988) using the equation

(1)

where L and A denote the concentration of the orthosteric ligand and the allosteric agent, respectively; K_L and K_A indicate the equilibrium dissociation constants for the free receptors of the ligand L and of the allosteric modulator A, respectively; and is the factor of cooperativity representing the change in affinity of the orthosteric ligand for binding to the receptor occupied by an allosteric agent compared with the free receptor and vice versa. If an allosteric agent alters [³H]NMS equilibrium binding only slightly, curve fitting with and K_A as variable parameters is difficult. In that instance, we replaced K_A in eq. 1 by EC_0.5,diss/ assuming that x K_A/EC_0.5,diss = 1 (Raasch et al., 2002). EC_0.5,diss was known from the dissociation experiments and the curve fit yielded . K_A was then calculated as EC_0.5,diss/. Nonlinear regression analysis was performed using the Prism program (ver. 3.02; GraphPad Software, San Diego, CA).

Chemicals
Atropine sulfate and polyethylenimine were obtained from Sigma Chemical (München, Germany). Diallylcaracurine V (Zlotos et al., 2000) and dimethyl-W84 (Tränkle et al., 1998) were generously provided by Prof. Dr. Ulrike Holzgrabe and Dr. Darius P. Zlotos (Institute of Pharmacy, University of Würzburg, Germany). W84 is commercially available at Tocris Cookson Inc. (Ellisville, MO). [³H]NMS (83.5 Ci/mmol) was purchased from PerkinElmer Life Sciences Inc. (Boston, MA).

Three-Dimensional Modeling and Docking Simulations
Homology Modeling. The model is based on the X-ray structure of bovine rhodopsin (Protein Data Base accession no. 1F88 [PDB] /1HZX; Palczewski et al., 2000); the sequence of the human M₂ receptor was extracted from SwissProt (code no. P08172 [GenBank] ; Bonner et al., 1987). Transmembrane regions of the M₂ receptor were detected using so-called pinpoints, identified by Baldwin et al. (1997). The extracellular and intracellular loops were created employing a combination of different methods [i.e., secondary structure prediction and the application of a loop search routine based on homology aspects, implemented in the Homology module of Insight II (Accelrys, San Diego, CA)]. 3D-coordinates for N and C termini were built in analogy to the X-ray structure of bovine rhodopsin. A detailed description of the modeling procedure has been given elsewhere (Jöhren and Höltje, 2003).

Receptor-Ligand Complexes. The geometries of interaction for the allosteric modulators diallylcaracurine V and W84 were found in a stepwise manner. In a first step, the free volume located between the extracellular loops and the upper part of the transmembrane helices was explored using the program SURFNET (Laskowski, 1995). In the next step, the large cavity, which was found, was investigated using different GRID-probes, imitating the functional groups present in the ligands. Subsequently, the allosteric modulators were manually docked into this cavity according to the favorable positions detected by GRID (Stoll et al., 2002). The two resulting wild-type complexes (diallylcaracurine V + NMS, W84 + NMS) were virtually mutated (M₂-Tyr¹⁷⁷Gln, M₂-Thr⁴²³His, M₂-Tyr¹⁷⁷Gln + Thr⁴²³His), and all newly generated complexes were minimized to obtain energetically acceptable geometries.

Molecular Dynamics Simulation. To check whether the protocol for the molecular dynamics simulation was suitable, identical simulations for the X-ray structure of bovine rhodopsin as well as for the M₂ receptor model were performed. The result was confirmed that a backbone tether of 200 kcal/Ų, a distance dependent dielectric constant of 2 x r and the use of the physiological pH of 7.4 give acceptable results when using the consistent valence force field (Accelrys). A simulation time of 300 ps is adequate for equilibration and simulation of receptor-ligand-complexes.

Model Quality Check. All molecular dynamics simulation runs were checked for equilibration of the system and protein stability. The potential energy and the root-mean-square deviation were taken as measures for complex equilibration. During the molecular dynamics simulation, the protein geometry, which was monitored with PROCHECK (Laskowski et al., 1993), was stable. The program NMRCLUST (Kelley et al., 1996) was applied to achieve representative structures of the protein-ligand complex after equilibration. The interaction energies between the allosteric ligands and the M₂ receptor during molecular dynamics simulation were determined using the DeCipher module in Insight II (Accelrys).

	Results

Top Abstract Materials and Methods Results Discussion References

 
In a first step, we aimed to narrow down the epitope in o2 by dividing the amino acid strand into three parts: A, B, and C. Thus, M₂/M₅ chimeric receptors were created, in which part A, B, or C of the M₂ receptor was substituted with the corresponding sequences of the M₅ receptor and vice versa. Schematic representations of the chimeric receptors are shown in Fig. 2, and the exact sequences are given under Materials and Methods.

The chimeric receptors were investigated for their sensitivities to the allosteric actions of the test compounds. The experiments were performed with receptors in which the orthosteric site was occupied by [³H]NMS. The allosteric delay of [³H]NMS-dissociation served to assess the binding affinity of the allosteric test compounds for the NMS-occupied receptors. The half-lives of [³H]NMS-dissociation measured in the absence of test compounds are compiled in Table 1. In agreement with previous studies (Ellis et al., 1991), [³H]NMS dissociated much more rapidly from the M₂ subtype than from the M₅ subtype. The dissociation rates found for the M₂ receptors with small chimeric substitutions or point mutations were near the rate of the parent M₂ receptor. The dissociation rates for the M₅ wild-type and M₅ mutant receptors were much slower.

The dependence of the allosteric action of W84 on parts A to C of o2 is shown in Fig. 2. Only the replacement of part B of the M₂ receptor with the corresponding sequence of the M₅ receptor resulted in a significant loss of affinity of W84 (Fig. 2A). The converse mutation (Fig. 2B) (i.e., replacement of the corresponding part B of the M₅ receptor with part B of the M₂ receptor) led to a reciprocal effect; the affinity toward W84 is increased, relative to the M₅ wild-type receptor. In contrast, substitutions of part A or C had no effect on the affinity toward W84. The same pattern of effects was observed with the other allosteric modulators, dimethyl-W84 and diallylcaracurine V (Table 2). These results suggest that the crucial amino acid is located in part B. The sequences of part B in the M₂ and in the M₅ receptor are given in Fig. 3.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Amino acid sequences of the corresponding "parts B" of the human M2 and M5 receptor. Sequences are from Bonner et al. (1987).

Subsequently, site-directed mutagenesis was applied to gain more insight into the contribution of the amino acids for the binding affinity of the allosteric agents. We focused on three amino acids of part B of the M₂ receptor: Glu¹⁷², Asp¹⁷³ and Tyr¹⁷⁷. The neutral amino acid Gly¹⁷⁴ was not expected to contribute to the binding affinity of the allosteric ligands, and Glu¹⁷⁵ and Cys¹⁷⁶ are conserved at the corresponding positions in the M₅ receptor.

In the cases of Asp¹⁷³ and Tyr¹⁷⁷ of M₂, the corresponding amino acids of the M₅ receptor were substituted. However, the corresponding amino acid of Glu¹⁷² is proline, which is known to disrupt the conformation of peptide strands (MacArthur and Thornton, 1991). Therefore, Glu¹⁷² was replaced by alanine. Thus, three point-mutated receptors were generated: M₂-Glu¹⁷² Ala, M₂-Asp¹⁷³ Leu, and M₂-Tyr¹⁷⁷ Gln. Neither the replacement of Glu¹⁷² with Ala nor the substitution of Asp¹⁷³ with Leu resulted in any significant change of affinity of W84 (Fig. 4), relative to the M₂ wild-type receptor. In contrast, replacement of M₂-Tyr¹⁷⁷ by glutamine of M₅ markedly reduced the affinity of W84 (Fig. 4) relative to the M₂ wild-type receptor. The loss of affinity to this mutant receptor is equivalent to the loss of affinity observed when substituting the whole part B of the M₂ receptor by the corresponding part of M₅. Similar findings were obtained for dimethyl-W84 and diallylcaracurine V (Table 2). The converse mutation was also constructed, M₅-Gln¹⁸⁴ Tyr, and the result was an increase of affinity toward all of the allosteric modulators that were studied, relative to the M₅ wild-type receptor (Table 2). The loss of binding affinity that was caused by removing Tyr¹⁷⁷ from the M₂ receptor and the gain of binding affinity caused by introducing tyrosine into the corresponding position of M₅ suggested that M₂-Tyr¹⁷⁷ is the essential amino acid within the o2 epitope of the M₂ receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Allosteric effects of W84 at the indicated wild-type and point mutated receptors. Experiments were conducted and analyzed as described in the legend to Fig. 2. Values are means ± S.E. of two to four separate experiments.

In a preceding study, we found that M₂-Thr⁴²³ is a critical epitope for the M₂/M₅ selectivity of all the allosteric test compounds examined here. Therefore, we further investigated whether M₂-Thr⁴²³ and M₂-Tyr¹⁷⁷ might fully account for the M₂/M₅ selectivity of these allosteric compounds. A double point-mutated receptor was generated, replacing the tyrosine and threonine of the M₂ receptor with the corresponding amino acids of the M₅ receptor, glutamine and histidine (M₂-Tyr¹⁷⁷ Gln + Thr⁴²³ His). As shown in Fig. 4, the affinity of W84 toward this mutant receptor matched the affinity of W84 for the wild-type M₅ receptor. The double mutation in M₂ likewise reduced the affinities toward dimethyl-W84 and diallylcaracurine V to M₅ levels (Fig. 5 and Table 2). The reverse mutation, insertion of tyrosine and threonine in the M₅ receptor, resulted in a significant increase of affinity toward the allosteric agents, although the affinity level of M₂ was not obtained with any of the ligands (Table 2). This outcome is not altogether surprising. When an essential residue is removed from a receptor, its contribution is likely to be completely lost if the substituted residue is incapable of mimicking the lost interaction. On the other hand, the effectiveness of the insertion of such a residue may depend upon other aspects of the recipient receptor's structure (Ellis et al., 1993). In the present case, the proline at M₅¹⁷⁹ might contribute to a less-than-optimal orientation of the inserted tyrosine at position 184, for example. Or there could be important structural features even further removed, as suggested by previous studies in which the entire second outer loop of M₂, along with TM4 and TM5 residues, was inserted into M₅; when the threonine was additionally inserted into TM7 of this chimera, affinities were observed for several allosteric ligands that were essentially like those for M2 (Buller et al., 2002).

View larger version (28K): [in this window] [in a new window]   Fig. 5. Allosteric effects of dimethyl-W84 (A) and diallylcaracurine V (B) at the indicated wild-type and point mutated receptors. Experiments were carried out and analyzed as described in the legend to Fig. 2. Indicated are means ± S.E. of two to four separate experiments.

The aforementioned experiments have all been conducted with NMS-occupied receptors. We aimed to check the role of Tyr¹⁷⁷ and Thr⁴²³ for allosteric agent binding in unoccupied M₂ receptors. Therefore, equilibrium binding experiments were carried out with [³H]NMS. In the absence of test compound, the applied wild-type and mutant receptors revealed the following pK_D-values for NMS binding (single values derived from separate [³H]NMS/NMS competition experiments): M₂, 10.40 and 10.32; M₅, 9.89 and 9.88; M₂-Tyr¹⁷⁷ Gln + Thr⁴²³ His, 10.80 and 10.14. Diallylcaracurine V was chosen to study the M₂/M₅ selectivity in the unoccupied receptors because the compound is stable (Wedig et al., 2002) in contrast to the alkane-bisammonium compounds, which are prone to undergo spontaneous degradation over the long incubation periods (24 h in this study) that are required to reach binding equilibrium in the presence of allosteric agents in M₅ receptors (t_1/2 of W84 degradation in PB is 10–14 h; Schulz, 1998). As shown in Fig. 6, the caracurine derivative slightly enhanced [³H]NMS binding in M₂, whereas in M₅, the [³H]NMS binding was reduced, and concentrations of the modulator 100-fold higher were needed for this effect. To verify that the upper plateau of [³H]NMS binding is stable in M₂, we carried out two additional experiments and included concentrations of diallylcaracurine V at which equilibrium binding was reached within an incubation time of 24 h (instead of 3 h). As expected according to the ternary complex model of allosteric interactions, the level of [³H]NMS equilibrium binding was maintained at the higher concentrations of up to 3 x 10^-7 M (data not shown). Figure 6 indicates that both the binding affinity of diallylcaracurine V and its cooperativity with [³H]NMS are subtype-selective. The pK_A values of diallylcaracurine V binding to the free receptors and the factors of cooperativity with NMS are compiled in Table 3. In the double mutant receptor, the cooperativity with NMS was at least as negative as in M₅, whereas the affinity of diallylcaracurine V binding remained halfway between M₂ and M₅. Additional epitopes seem to be involved in the M₂/M₅ selectivity of the binding of this allosteric agent to unoccupied receptors. In cases of W84 and dimethyl-W84, the double point mutation of the M₂ receptor led to losses in binding affinity of >10-fold relative to the wild-type M₂ receptor; the cooperativity with NMS was negative. Taken together, the findings indicate that the pair of amino acids plays a major role for the binding of the alkane-bisammonium–type and the caracurine V-type allosteric agents in free M₂ receptors. Future studies will have to elucidate the relative contribution of the single amino acids to the loss of binding affinity seen with all agents and to the shift in cooperativity seen with the caracurine V type agent. Furthermore, it will be interesting to find out in how far the reverse mutation M₅-Gln¹⁸⁴ Tyr + His⁴⁷⁸ Thr induces a gain of binding affinity for the free receptor and restores cooperativity.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of diallylcaracurine V on the equilibrium binding of [3H]NMS to the indicated wild-type and point mutated receptors. Ordinate, specific [3H]NMS binding in percentage of the control value in the absence of the allosteric agent. Abscissa, concentration of the allosteric agent. Indicated are means ± S.E. of three to four separate experiments with duplicated values. Curve fitting based on the ternary complex model of allosteric interactions.

View this table: [in this window] [in a new window]   TABLE 3 Affinities (pKA) of the allosteric agents to bind to the indicated wild-type and mutant receptors and factors of cooperativity (p) for the interaction with [3H]NMS equilibrium binding pKA is the -log equilibrium dissociation constant for allosteric agent binding to NMS-free receptors. p is the -log value of the cooperativity factor; a positive or negative p reflects an elevation or reduction of [3H]NMS equilibrium binding, respectively. Data are presented as means ± S.E. of three to four separate experiments with duplicated values.

To gain more insight into the topography of allosteric agent binding to the M₂ receptor, a 3-dimensional model of the receptor protein was built (Jöhren and Höltje, 2003). The model was derived from the crystal structure of bovine rhodopsin in the inactive state. Thus, the transmembrane helices should be in the same position as if stabilized through an orthosteric antagonist. For the modeling, NMS as the orthosteric agent offers favorable features: 1) it is relatively large and rigid and 2) the amino acids, which are important for its binding, are well known but different from the amino acids important for agonist binding (Heitz et al.,1999; Lu et al., 2002). The model of the ligand binding cavity of the M₂ receptor is shown in Fig. 7. The extracellular loops form a cleft-like vestibule that is connected through a narrow corridor with a chamber in which the orthosteric binding site is located, lined by the transmembrane helices.

View larger version (111K): [in this window] [in a new window]   Fig. 7. Positions of the orthosteric binding site with bound N-methylscopolamine (NMS) and of the common allosteric binding site in the M2 receptor model; view from the membrane. Helices are dark gray, extracellular loops are light gray, accessible volumes are in a gray grid, and NMS is a ball-and-stick model.

To model the ternary complex characteristic for allosteric interactions, NMS was placed in the orthosteric site as described previously (Vogel et al.,1997; Heitz et al., 1999). Then diallylcaracurine V in the conformation illustrated in Fig. 1, which is based on the 3D structure of caracurine V as determined by Zlotos (2000), could easily be docked into the entrance of the ligand binding cavity (Fig. 8). The molecular dynamics simulation performed subsequently indicated that the aromatic ring of M₂-Tyr¹⁷⁷ is prone to make an upward movement toward the allosteric agent. Thereby, the aromatic ring of M₂-Tyr¹⁷⁷ approaches the adjacent aromatic ring of the allosteric agent, leading to the formation of a - interaction (Fig. 9). The movement of M₂-Tyr¹⁷⁷ takes place at the expense of free volume in the region connecting the two binding sites; consequently, the corridor between the orthosteric site and the allosteric site is closed and NMS is trapped (Fig. 8). In the NMS-liganded mutant M₂-Tyr¹⁷⁷ Gln, the conformation of the allosteric site is not changed compared with the wild-type M₂. In this mutant, docking of diallylcaracurine V is followed by a movement of Gln away from the allosteric agent [i.e., into the opposite direction compared with the movement of Tyr in the wild-type receptor (Fig. 10)]. This is accompanied by a considerable loss of interaction energy (Fig. 11, top).

View larger version (90K): [in this window] [in a new window]   Fig. 8. Positions of NMS and diallylcaracurine V in the wild-type M2 receptor; view from the membrane. Protein: helices, red; o1 and N terminus, gray; o2, cyan; EDGE (residues 172–175 in M2) green; o3, yellow; disulfide bridge, magenta; orthosteric binding site, blue; free volumes, gray grid. Tyr177/Thr423: carbon, green; nitrogen, dark blue; oxygen, red; hydrogen, white. NMS/diallylcaracurine V: carbon, white; nitrogen, dark blue; oxygen, red; hydrogen, cyan.

View larger version (36K): [in this window] [in a new window]   Fig. 9. - interaction between M2-Tyr177 and diallylcaracurine V; view from the bottom of the ligand binding cavity. Protein: helices, red; o2, cyan; EDGE, green; o3, yellow; disulfide bridge, magenta. Tyr177/Thr423: carbon, green; nitrogen, dark blue; oxygen, red; hydrogen, white. Diallylcaracurine V: carbon, white; nitrogen, dark blue; oxygen, red; hydrogen, not shown.

View larger version (77K): [in this window] [in a new window]   Fig. 10. Difference between the orientation of M2 wild-type Tyr177 and mutant Gln177 in the ternary complex with NMS and diallylcaracurine V. Wild-type: helices, red; o1 and N terminus, gray; o2, cyan; EDGE, green; o3, yellow; disulfide bridge, magenta; orthosteric binding site, blue; free volumes, gray grid. Tyr177: carbon, green; nitrogen, dark blue; oxygen, red; hydrogen, white. Tyr177 Gln mutant: protein backbone, orange. Gln177: carbon, orange; nitrogen, dark blue; oxygen, red; hydrogen, cyan.

View larger version (70K): [in this window] [in a new window]   Fig. 11. Interaction energies during molecular dynamics simulations. Top, interaction between M2 residue Tyr177 and diallylcaracurine V in the NMS-occupied M2 receptor: Wild-type Tyr, black; Gln mutant, gray. Bottom, interaction between M2 residue Thr423 (M2) and diallylcaracurine V in the NMS-occupied M2 receptor: wild-type Thr, black; His mutant, gray.

We have also studied the M₂-Thr⁴²³ His mutant receptor. M₂-Thr⁴²³ is located near one of the ether oxygens of diallylcaracurine V. In the mutant, the interaction energy converts from an attractive to a repulsive term (Fig. 11, bottom). Taken together, these results are in line with the experimental findings; i.e., the loss of binding affinity of diallylcaracurine V is greater in M₂-Tyr¹⁷⁷ Gln than in M₂-Thr⁴²³ His. Investigation of the double mutant (M₂-Tyr¹⁷⁷ Gln + Thr⁴²³ His) yielded both effects described above (data not shown), in analogy to the increased loss of affinity seen in the binding experiments.

The quaternary nitrogens and the allyl substituents of diallylcaracurine V are placed in two lipophilic pockets. One is formed by M₂-Ala¹³, Val²³, Glu²², Ile²⁶, and Val⁸⁵, the other one by Met¹, Asn⁶, Thr¹⁷⁰, Val¹⁷¹, and Ile⁴¹⁷. The charge of the quaternary nitrogen, which is dissipated on the surrounding aliphatic carbon atoms, interacts favorably with the negative charge of Glu²² or with the polar Asn⁶. Except for the three nearest the N-terminal (Met¹, Asn⁶, and Ala¹³), the residues comprising these pockets lie in or near regions of the receptor that are significantly conserved across the muscarinic family. Furthermore, these residues are themselves highly conserved between M₂ and M₅ and throughout the family.

To assign a realistic low energy conformation for the highly flexible W84, a conformational analysis was performed. For this purpose molecular dynamics simulations in an aqueous environment were employed (Höltje and Folkers, 1996). This procedure leads to a conformation of W84 that differs from the S-shape low energy conformer detected by Holzgrabe and Hopfinger (1996) in the course of a systematic analysis in vacuo. The conformation resulting from the dynamical treatment of W84 was docked to the NMS-occupied receptor. W84 fills the ligand binding cavity and one phthalimido-group approaches M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ (Fig. 12); the quaternary nitrogens are found in similar positions as described above for diallylcaracurine V. The M₂-Tyr¹⁷⁷ Gln mutant leads to a comparable loss of interaction energy and a geometry comparable to that described for diallylcaracurine V. Mutating M₂-Thr⁴²³ to His causes nearly no difference in the interaction energy in this position but reduces the interaction energy between W84 and the conserved tryptophans by influencing their side-chain conformation (data not shown). Taken together, the results of the docking simulations carried out with the three-dimensional model correspond with the experimental findings from the binding assays in the wild-type M₂ and the mutant M₂ receptors.

View larger version (88K): [in this window] [in a new window]   Fig. 12. Position of NMS and W84 in the wild-type M2 receptor. Protein: helices, red; o1 and N terminus, gray; o2, cyan; EDGE, green; o3, yellow; disulfide bridge, magenta; orthosteric binding site, blue; free volumes, gray grid. Tyr177/Thr423: carbon, green; nitrogen, dark blue; oxygen, red; hydrogen, white. NMS/W84: carbon white; nitrogen, dark blue; oxygen, red; hydrogen, cyan.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In this study, we have identified M₂-Tyr¹⁷⁷ in o2 as a second essential epitope for the high-affinity binding of some structurally diverse muscarinic allosteric agents in M₂ receptors whose orthosteric site is occupied by the muscarinic antagonist N-methylscopolamine. This amino acid, together with the previously identified M₂-Thr⁴²³ at the beginning of TM7 (Buller et al., 2002), account entirely for the M₂/M₅ selectivity. For each of the three ligands investigated in the present study, the substitution of M₂-Tyr¹⁷⁷ resulted in a more pronounced loss of affinity than substitution of M₂-Thr⁴²³ (M₂-Y¹⁷⁷ Q, M₂-T⁴²³ H, Table 2). Thus, M₂-Tyr¹⁷⁷ seems to be more important for the binding affinity of these allosteric test compounds than the previously identified M₂-Thr⁴²³. Yet, this is not a general phenomenon, because the allosteric affinity of the methyl analog of diallylcaracurine V has been shown to be affected much more by mutation at the M₂-Thr⁴²³ than by substitution of the entire o2 loop (Buller et al., 2002).

Multiple studies have indicated that residues in the second outer loop or near the junction of the third outer loop and the top of TM7 are important in the binding of muscarinic allosteric ligands. The EDGE sequence (residues 172–175 in M₂) markedly affects the binding of gallamine (Leppik et al., 1994; Gnagey et al., 1999). The asparagine residue at M₂⁴¹⁹ has been implicated in the subtype selectivities of gallamine and alcuronium (Gnagey et al., 1999; Kreji and Tuek, 2001). Elimination of a tryptophan residue that is conserved among all five muscarinic receptor subtypes (located at position 422 in M₂) reduces the affinity of gallamine and other allosteric ligands (Matsui et al., 1995). More general studies using chimeric receptors have also suggested that these two regions of the receptor (i.e., o2 and o3) are critical to the binding or selectivity of many muscarinic allosteric ligands, although the relative importance of the regions varies from ligand to ligand (Ellis and Seidenberg, 2000). Nevertheless, many of these compounds seem to interact competitively at a common allosteric site (Ellis and Seidenberg, 1992; 2000; Tränkle and Mohr, 1997), perhaps reflecting the close proximity of the residues involved, either near the middle of o2 or near the o3/TM7 junction. In keeping with the above studies, the agents investigated in the present study were sensitive to residues in these two regions: the recently described threonine near o3/TM7 and the tyrosine in o2 that is the main finding of the present study. It remains to be seen whether another group of compounds, which does not seem to interact competitively with gallamine and the others (Tränkle and Mohr, 1997; Lazareno et al., 2000), may bind to regions other than o2 and o3.

The present findings made with diallylcaracurine V reveal that the pair of amino acids, M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³, is also involved in the M₂/M₅ selectivity of the binding affinity for NMS-free receptors. The affinity of diallylcaracurine V for the NMS-free M₂ double mutant receptor does not reach the level of M₅. Thus, the pair of amino acids does not completely account for the M₂/M₅ selectivity in free receptors, and one or more additional epitopes seem to be involved.

Nevertheless, for NMS-occupied receptors, the present study is the first to identify epitopes that fully explain the M₂/M₅ selectivity of structurally different allosteric agents. The finding is a good starting point to develop a three-dimensional model of the M₂ receptor and to check its validity. Docking NMS into the orthosteric site has the advantage that NMS stabilizes an inactive conformation of the receptor protein; in addition, the possibility is excluded that the allosteric agent may use elements of the orthosteric site. The two amino acids identified in the present study can serve as probes to check the model in that these amino acids should be involved in a meaningful fashion in the docking of the allosteric agents. In fact, the model provides a hypothesis of how the alkane-bisammonium–type agent W84 and the caracurine derivative, although structurally diverse, could interact with the same two epitopes.

In principle, the amino acids identified here as essential epitopes for the high binding affinity of the allosteric agents in M₂ relative to M₅ receptors could either be important for the overall conformation of the allosteric site or they could be docking points. In the 3D model, the exchange of M₂-Tyr¹⁷⁷ by the corresponding Gln of M₅ did not affect the conformation of the allosteric site. In contrast, a mutation from the acidic M₂ sequence EDGE, located near M₂-Tyr¹⁷⁷, into the corresponding sequence PLDE of M₅ would change the proposed -I'-turn and hereby the loop conformation and the allosteric binding site (data not shown). Upon docking of diallylcaracurine V to the wild-type M₂ receptor, M₂-Tyr¹⁷⁷ makes a movement in the sense of an induced fit, whereas in the mutant receptor M₂-Tyr¹⁷⁷ Gln, the glutamine moves in the opposite direction (i.e., away from the allosteric agent). M₂-Thr⁴²³ is situated just between two tryptophans that are conserved in muscarinic receptors. The effect of the M₂-Thr⁴²³ His mutation seems to be a direct interaction in the case of diallylcaracurine V, whereas in the case of W84, it is more likely to be a steric influence on the relative orientation of the two tryptophans. Taken together, the model recognizes M₂-Tyr¹⁷⁷ and M₂-Thr⁴²³ as essential sites of attachment. At the same time, the allosteric ligands also possess significant affinity for the M₅ receptor subtype. We have speculated previously that a core binding region common to all of the receptor subtypes is likely to exist and that allosteric ligands bind to the different subtypes with similar orientations (Ellis, 1997; Buller et al., 2002). It remains to be seen whether the conserved residues that make up the lipophilic pockets, which we have identified in the model of the M₂ receptor, contribute to a core binding region of the allosteric site for the receptor family.

In summary, we have identified a second amino acid, M₂-Tyr¹⁷⁷ in o2, which, together with M₂-Thr⁴²³ at the top of TM7, accounts entirely for the M₂/M₅ selectivity of alkane-bisammonium–type and caracurine V-type allosteric modulators at muscarinic acetylcholine receptors that are occupied by the antagonist N-methylscopolamine. In free receptors, the pair of epitopes clearly contributes to the binding affinity of the modulators for M₂ receptors, but it does not seem to fully account for the M₂/M₅ selectivity. A three-dimensional model of the NMS-occupied M₂ muscarinic acetylcholine receptor has been developed that accommodates the experimentally observed phenomena and may provide insight into the topography and the molecular mechanisms of allosteric-orthosteric-interactions.

	Acknowledgements

 
We thank Prof. Dr. Ulrike Holzgrabe and Dr. Darius Paul Zlotos for synthesizing and providing dimethyl-W84 and diallylcaracurine V.

	Footnotes

 
This article is dedicated to the late Dr. Stanislav Tuek, Academy of Sciences of the Czech Republic, Prague.

This work was supported by the Deutsche Forschungsgemeinschaft (GRK 677/1 and Mo821/1-2), by a grant of the University of Bonn (to S.B.), and by U.S. Public Health Service grant R01-AG05214 (to J.E.).

3D complex coordinates are available on request from Kirstin Jöhren, Institute of Pharmaceutical Chemistry, University of Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf (E-mail: joehren{at}pharm.uni-duesseldorf.de).

ABBREVIATIONS: NMS, N-methylscopolamine; TM, transmembrane region of the receptor; o1, o2, o3, first, second, third outer (extracellular) loop of the receptor; PB, sodium-potassium phosphate buffer; 3D, three-dimensional.

Address correspondence to: Dr. Klaus Mohr, Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, 53121 Bonn, Germany. E-mail: k.mohr{at}uni-bonn.de

	References

Top Abstract Materials and Methods Results Discussion References

 
Baldwin JM, Schertler GFX, and Unger VM (1997) An alpha carbon template for the transmembrane helices of the rhodopsin family of G-protein-coupled receptors. J Mol Biol 272: 144-164.[CrossRef][Medline]

Bonner TI, Buckley NJ, Young AC, and Brann MR (1987) Identification of a family of muscarinic acetylcholine receptor genes. Science (Wash DC) 237: 527-532.[Abstract/Free Full Text]

Buller S, Zlotos DP, Mohr K, and Ellis J (2002) Allosteric site on muscarinic acetylcholine receptors: a single amino acid in transmembrane region 7 is critical to the subtype selectivities of caracurine V derivatives and alkane-bisammonium ligands. Mol Pharmacol 61: 160-168.[Abstract/Free Full Text]

Christopoulos A and Kenakin T (2002) G protein-coupled receptor allosterism and complexing. Pharmacol Rev 54: 323-374.[Abstract/Free Full Text]

Ehlert FJ (1988) Estimation of the affinities of allosteric ligands using radioligand binding and pharmacological null methods. Mol Pharmacol 35: 187-194.

Ellis J (1997) Allosteric binding sites on muscarinic receptors. Drug Dev Res 40: 193-204.[CrossRef]

Ellis J, Huyler JH, and Brann MR (1991) Allosteric regulation of cloned m1–m5 muscarinic receptor subtypes. Biochem Pharmacol 42: 1927-1932.[CrossRef][Medline]

Ellis J and Seidenberg M (1992) Two allosteric modulators interact at a common site on cardiac muscarinic receptors. Mol Pharmacol 42: 638-641.[Abstract]

Ellis J, Seidenberg M, and Brann MR (1993) Use of chimeric muscarinic receptors to investigate epitopes involved in allosteric interactions. Mol Pharmacol 44: 583-588.[Abstract]

Ellis J and Seidenberg M (2000) Interactions of alcuronium, TMB-8 and other allosteric ligands with muscarinic acetylcholine receptors: studies with chimeric receptors. Mol Pharmacol 58: 1451-1460.

Gnagey AL, Seidenberg M, and Ellis J (1999) Site-directed mutagenesis reveals two epitopes involved in the subtype selectivity of the allosteric interactions of gallamine at muscarinic acetylcholine receptors. Mol Pharmacol 56: 1245-1253.[Abstract/Free Full Text]

Heitz F, Holzwarth JA, Gies JP, Pruss RM, Trumpp-Kallmeyer S, Hibert MF, and Guenet C (1999) Site-directed mutagenesis of the putative human muscarinic M₂ receptor binding site. Eur J Pharmacol 380: 183-195.[CrossRef][Medline]

Höltje H-D and Folkers G (1996) Conformational analysis using molecular dynamics, in Molecular Modeling: Basic Principles and Applications, pp 29-36, VCH, Weinheim, Germany.

Holzgrabe U and Hopfinger AJ (1996) Conformational analysis, molecular shape comparison and pharmacophore identification of different allosteric modulators of muscarinic receptors. J Chem Inf Comp Sci 36: 1018-1024.[CrossRef][Medline]

Hulme EC, Birdsall NJ, and Buckley NJ (1990) Muscarinic receptor subtypes. Annu Rev Pharmacol Toxicol 30: 633-673.[CrossRef][Medline]

Jöhren K and Höltje H-D (2003) A model of the human M₂ muscarinic acetylcholine receptor. Proceedings of 17th International Conference on Medicinal Chemistry; 2002 Sep 1–5; Barcelona, Spain. J Comput Aided Mol Des, in press.

Kelley LA, Gardner SP, and Sutcliffe MJ (1996) An automated approach for clustering an ensemble of NMR-derived protein structures into conformationally-related subfamilies. Protein Eng 9: 1063-1065.[Free Full Text]

Kreji A and Tuek S (2001) Changes of cooperativity between N-methylscopolamine and allosteric modulators alcuronium and gallamine induced by mutations of external loops of muscarinic M₃ receptors. Mol Pharmacol 60: 761-767.[Abstract/Free Full Text]

Laskowski RA, MacArthur MW, Moss DS, and Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst 26: 283-291.

Laskowski RA (1995) SURFNET: A program for visualizing molecular surfaces, cavities and intermolecular interactions. J Mol Graph 13: 323-330.[CrossRef][Medline]

Lazareno S and Birdsall NJ (1995) Detection, quantitation and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors. Mol Pharmacol 48: 362-378.[Abstract]

Lazareno S, Popham A, and Birdsall NJM (2000) Allosteric interactions of staurosporine and other indolocarbazoles with N-[methyl-³H]scopolamine and acetylcholine at muscarinic receptor subtypes: identification of a second allosteric site. Mol Pharmacol 58: 194-207.[Abstract/Free Full Text]

Lee NH and El-Fakahany EE (1991) Allosteric interactions at the m1, m2 and m3 muscarinic receptor subtypes. J Pharmacol Exp Ther 256: 468-479.[Abstract/Free Full Text]

Leppik RA, Miller RC, Eck M, and Paquet JL (1994) Role of acidic amino acids in the allosteric modulation by gallamine of antagonist binding at the m2 muscarinic acetylcholine receptor. Mol Pharmacol 45: 983-990.[Abstract]

Lu ZL, Saldanha JW, and Hulme EC (2002) Seven-transmembrane receptors: crystals clarify. Trends Pharmacol Sci 23: 140-146.[CrossRef][Medline]

MacArthur MW and Thornton JM (1991) Influence of proline residues on protein conformation. J Mol Biol 218: 397-412.[CrossRef][Medline]

Matsui H, Lazareno S, and Birdsall NJ (1995) Probing of the location of the allosteric site on m1 muscarinic receptors by site-directed mutagenesis. Mol Pharmacol 47: 88-98.[Abstract]

Palczewski K, Kumaska T, Hori T, Behnke CS, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, et al. (2000) Crystal structure of rhodopsin: a G-protein-coupled receptor. Science (Wash DC) 289: 739-745.[Abstract/Free Full Text]

Raasch A, Scharfenstein O, Tränkle C, Holzgrabe U, and Mohr K (2002) Elevation of ligand binding to muscarinic M₂ acetylcholine receptors by bis(ammonio)alkane-type allosteric modulators. J Med Chem 45: 3809-3812.[CrossRef][Medline]

Schulz U (1998) Antagonismus-Untersuchungen zur Topographie der Bindung allosterischer Modulatoren an muskarinischen Acetylcholinrezeptoren des Subtyps M₂. Thesis. Faculty of Mathematics and Natural Sciences, University of Bonn, Bonn, Germany.

Stockton JM, Birdsall NJM, Burgen ASV, and Hulme EC (1983) Modification of the binding properties of muscarinic receptors by gallamine. Mol Pharmacol 23: 551-557.[Abstract]

Stoll F, Liesener S, Hohlfeld T, Schrör K, Fuchs PL, and Höltje H-D (2002) Pharmacophore definition and three-dimensional quantitative structure-activity relationship study on structurally diverse prostacyclin receptor agonists. Mol Pharmacol 62: 1103-1111.[Abstract/Free Full Text]

Tränkle C and Mohr K (1997) Divergent modes of action among cationic allosteric modulators of muscarinic M2 receptors. Mol Pharmacol 51: 674-682.[Abstract/Free Full Text]

Tränkle C, Mies-Klomfass E, Cid MH, Holzgrabe U, and Mohr K (1998) Identification of a [³H]ligand for the common allosteric site of muscarinic acetylcholine M2 receptors. Mol Pharmacol 54: 139-145.[Abstract/Free Full Text]

Tuek S and Proka J (1995) Allosteric modulation of muscarinic acetylcholine receptors. Trends Pharmacol Sci 16: 205-212.[CrossRef][Medline]

Vogel WK, Sheehan DM, and Schimerlik MI (1997) Site-directed mutagenesis on the M₂ muscarinic acetylcholine receptor: the significance of Tyr403 in the binding of agonists and functional coupling. Mol Pharmacol 52: 1087-1094.[Abstract/Free Full Text]

Wedig M, Novatchev N, Worch T, Laug S, and Holzgrabe U (2002) Evaluation of the impurity profile of alcuronium by means of capillary electrophoresis. J Pharm Biomed Anal 28: 983-990.[CrossRef][Medline]

Wess J (1993) Molecular basis of muscarinic acetylcholine receptor function. Trends Pharmacol Sci 14: 308-313.[CrossRef][Medline]

Zlotos DP (2000) Stereochemistry of caracurine V. J Nat Prod 63: 864-865.[CrossRef][Medline]

Zlotos DP, Buller S, Tränkle C, and Mohr K (2000) Bisquaternary caracurine V derivatives as allosteric modulators of ligand binding to M2 acetylcholine receptors. Bioorg Med Chem Lett 10: 2529-2532.[CrossRef][Medline]

This article has been cited by other articles:

				F. J. Ehlert and M. T. Griffin Two-State Models and the Analysis of the Allosteric Effect of Gallamine at the M2 Muscarinic Receptor J. Pharmacol. Exp. Ther., June 1, 2008; 325(3): 1039 - 1060. [Abstract] [Full Text] [PDF]

				D. Jager, C. Schmalenbach, S. Prilla, J. Schrobang, A. Kebig, M. Sennwitz, E. Heller, C. Trankle, U. Holzgrabe, H.-D. Holtje, et al. Allosteric Small Molecules Unveil a Role of an Extracellular E2/Transmembrane Helix 7 Junction for G Protein-coupled Receptor Activation J. Biol. Chem., November 30, 2007; 282(48): 34968 - 34976. [Abstract] [Full Text] [PDF]

				V. A. Avlani, K. J. Gregory, C. J. Morton, M. W. Parker, P. M. Sexton, and A. Christopoulos Critical Role for the Second Extracellular Loop in the Binding of Both Orthosteric and Allosteric G Protein-coupled Receptor Ligands J. Biol. Chem., August 31, 2007; 282(35): 25677 - 25686. [Abstract] [Full Text] [PDF]

T. Steinfeld, M. Mammen, J. A. M. Smith, R. D. Wilson, and J. R. Jasper A Novel Multivalent Ligand That Bridges the Allosteric and Orthosteric Binding Sites of the M2 Muscarinic Receptor Mol. Pharmacol., August 1, 2007; 72(2): 291