Introduction

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₂-Adrenoceptors (₂-ARs) belong to the rhodopsin-like class of G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) -helices with an extracellular N-terminus and a cytoplasmic C-terminus (Kobilka et al., 1987; Bikker et al., 1998). The TM helices in ₂-ARs form a water-accessible binding site for ligands in a pocket or crevice between the helices in the interior of the receptor. Residues within this cavity directly participate in ligand binding, which stabilizes the conformation of the receptor. Agonist ligands, whose pharmacological activity is manifested as an activation of downstream signaling, shift the equilibrium between the inactive and active receptor conformations in favor of the active form (Gether and Kobilka, 1998). Through their interactions with naturally occurring and synthetic ligands, ₂-ARs mediate a variety of physiological and pharmacological effects, and are thus key targets for pharmaceutical development. ₂-ARs have therapeutic applications in a variety of diseases, for example, in the treatment of hypertension, pain, and depression (Ruffolo et al., 1993; MacDonald et al., 1997).

For _2A-ARs, as well as all other types of - and -adrenoceptors, a conserved aspartate in the third transmembrane helix (TM3) has been established as a residue critical for phenethylamine ligand binding. In the human _2A-AR, this residue corresponds to Asp113 [D_3.32 according to the indexing system of Ballesteros and Weinstein (1995)]. The negatively charged aspartate in TM3 provides an anchoring point for ligands containing positively charged amine groups (Ruffolo, 1991; Kobilka, 1995). Other residues suggested to be involved in the binding of phenethylamine ligands in _2A-AR include Cys201 (C_5.43) in TM5 (Marjamäki et al., 1999; Marjamäki et al., 1998), Ser200 (S_5.42) and Ser204 (S_5.46) also in TM5 (Marjamäki et al., 1998, 1999; Rudling et al., 1999; Salminen et al., 1999), and aromatic residues in TM6 (Kobilka, 1995). Binding of the -OH group of the phenethylamines to adrenergic receptors has been suggested to involve a serine (S_2.61) in TM2 (Li et al., 1995; Hieble et al., 1998), a serine (S_4.57) in TM4 (Strader et al., 1989; Trumpp-Kallmeyer et al., 1992), a serine in TM7 (S_7.46; Hieble et al., 1998), and an asparagine (N_6.55) in TM6 of the ₂-AR (Wieland et al., 1996). The latter hydrogen bonding interaction could also be possible in _2A-AR, where residue 6.55 is a tyrosine. However, Hieble and colleagues (1998) have shown that this residue has no effect on the stereospecific binding of the -OH in phenethylamines. None of these proposals is compatible with our docking results, and we will suggest an alternative determinant for the stereospecific binding of -OH-phenethylamines to _2A-AR.

The first successful structural explanation for phenethylamine agonist binding to adrenergic receptors was the three-point attachment hypothesis outlined by Easson and Stedman in the 1930s (see Ruffolo, 1991). The original hypothesis was formulated without any empirical information on the structure of the binding site. Although no X-ray structure of _2A-AR has so far been reported, the functional, structural, and experimental data that exist in the literature for multiple classes of GPCRs can be combined to make an atomic resolution model of a particular member of the receptor family (Bikker et al., 1998). Receptor binding and activation assays, combined with a three-dimensional model of the receptor, allow us to study the receptor in great detail, which improves understanding of the conformational changes that take place upon receptor activation. The common location of the ligand binding site for many rhodopsin-like GPCRs between TM3, TM5, and TM6, and the accumulated functional and structural evidence suggest that the activation of GPCRs is connected to the movement of these transmembrane helices with respect to each other (Kobilka, 1995; Beck-Sickinger, 1996; Gether et al., 1997; Unger et al., 1997; Gether and Kobilka, 1998). We have previously introduced cysteine substitutions along TM5 and demonstrated that two alkylating agents with different chemical structures, chloroethylclonidine and 2-aminoethyl methanethiosulfonate, most likely recognize two different conformations of the human _2A-AR (Marjamäki et al., 1999). Molecular modeling (Salminen et al., 1999) of the different receptor variants with these alkylating agents supported this assertion and suggested to us that TM5 might rotate when _2A-AR is activated.

We now have a structural model for _2A-AR (Salminen et al., 1999) whose functionality has been verified through experimental studies that include site-directed mutagenesis and ligand binding assays (Marjamäki et al., 1999; Salminen et al., 1999) and that offers a better structural explanation for small molecule ligand binding in comparison to models based on the bacteriorhodopsin X-ray structure (Marjamäki et al., 1999; Salminen et al., 1999). The model is based on an -carbon template for the backbone of the receptor, and derives from the low-resolution electron microscopy structure of frog rhodopsin and sequence alignments of hundreds of GPCRs (Baldwin et al., 1997). We also have a new model based on the X-ray structure of bovine rhodopsin (Palczewski et al., 2000). The model based on this new structure positions most of the same residues within the binding cavity as seen in our current model. However, bovine rhodopsin is not an adrenergic receptor, and TM5, demonstrated by many groups to play an essential role in catecholamine binding, is apparently less important in photoactivation of bovine rhodopsin.

In the current study, we use two sets of ligand binding experiments, a functional receptor activation assay, and molecular modeling to predict the binding modes of 12 phenethylamine ligands, and present a model of how they could promote the activation of _2A-AR. We present an atomic resolution model for the binding mode of epinephrine, norepinephrine, and their close structural analogs based on the results of these experiments, and provide a structural explanation for the binding affinity differences of the R- and S-enantiomers of these molecules. In the model, the most critical interactions for the binding of the agonists exist between the ligands and residues in TM3, TM5, TM6, and TM7 of _2A-AR, which is consistent with many earlier reports. The structural basis for the roles of the charged amine group, the N-methyl group of epinephrine, the -OH group, the aromatic ring, and the catecholic para- and meta-hydroxyls for ligand binding and receptor activation in _2A-AR are also revealed by this study.

	Materials and Methods

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Experimental Materials. [³H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline] was obtained from Amersham (Buckinghamshire, UK; specific activity 52 Ci/mmol). [³H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine] (62.5 Ci/mmol) and [³⁵S]GTPS (1,225 Ci/mmol) were obtained from NEN (Boston, MA). p-Aminoclonidine, dopamine, (R)-norepinephrine (bitartrate), and unlabeled UK-14,304 were supplied by RBI/Sigma (Natick, MA). (R)-2-Amino-1-phenyl-ethanol and (S)-norepinephrine (hydrogen L-tartrate) were purchased from Fluka Sigma-Aldrich (Buchs, Switzerland). (R)-Epinephrine was from Sigma Chemical (St. Louis, MO). (R,S)-Norphenephrine and (R,S)-octopamine were obtained from Aldrich Chemical (Milwaukee, WI). The enantiomers of norphenephrine and octopamine were prepared using Pseudomonas cepacia lipase-catalyzed resolution of the racemates (Fmoc-protected in the case of octopamine) through enantioselective acylation in toluene/tetrahydrofurane (3:1). NH₃ treatment provided the free (R)- and (S)-norphenephrine counterparts (enantiomeric excess > 98%). Candida antarctica lipase B-catalyzed ethanolysis and treatment with piperidine (5% (v/v)) in tetrahydrofurane provided the free (R)- and (S)-octopamine enantiomers (enantiomeric excess > 98% and 91%, respectively). Cell culture reagents were supplied by Life Technologies (Gaithersburg, MD). Other chemicals were of analytical or reagent grade, and were purchased from commercial suppliers.

Transfection and Cell Culture. Adherent Chinese hamster ovary cells (American Type Culture Collection, Manassas, VA) were cultured as reported previously (Pohjanoksa et al., 1997). Cells were transfected with a pMAMneo-based expression construct encoding the human _2A-AR and standard methods (Pohjanoksa et al., 1997). Neomycin (G418; Sigma)-resistant (750 µg/ml) cell cultures were examined for their ability to bind the _2A-AR antagonist [³H]RX821002. The transfected clonal cell lines were cultured in medium containing 250 µg/ml G418. The cell clone chosen for the experiments expressed _2A-AR at a density of 1.3 pmol/mg total cellular protein as determined with saturation binding experiments with [³H]RX821002 (Pohjanoksa et al., 1997).

Competition Ligand Binding Assays. Competition binding assays with [³H]RX821002 were performed as reported previously (Halme et al., 1995; Marjamäki et al., 1999), using a radioligand concentration close to its affinity constant (K_d) for _2A-AR and 13 to 15 concentrations of the competitive ligands. The assay buffer was 50 mM K⁺-phosphate buffer supplemented with 10 mM MgCl₂. For agonist competition assays with [³H]UK-14,304 as radioligand, cell membranes (about 10 µg protein/sample) and 0.6 nM [³H]UK-14,304 were incubated in 50 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl₂, and 30 µM ascorbic acid, pH 7.4, with 12 concentrations of the test compounds covering 5.5 log units. Nonspecific binding was defined using 100 µM oxymetazoline. After 45 min at room temperature, incubations were terminated by rapid vacuum filtration through glass fiber filters. Filters were washed 3 times with 5 ml of ice-cold buffer (20 mM Tris-HCl, 1 mM EDTA, 5 mM MgCl₂, pH 7.4), dried, and counted for radioactivity in a scintillation counter. Analysis of the experiments was conducted by nonlinear least-square curve fitting with Prism software (GraphPad Software, San Diego, CA) with simultaneous analysis of three separate experiments. IC₅₀ values were converted to K_i values by use of the Cheng-Prusoff equation (Cheng and Prusoff, 1973).

Functional [³⁵S]GTPS Binding Assay. Agonist-induced stimulation of [³⁵S]GTPS binding to isolated membranes from Chinese hamster ovary cells expressing recombinant _2A-AR was measured essentially as described previously (McKenzie, 1992; Tian et al., 1994; Peltonen et al., 1998). The [³⁵S]GTPS binding assay was conducted using a Beckman Biomek 2000 Laboratory Automation Workstation (Beckman Instruments, Inc., Palo Alto, CA) and 96-well plates. Harvested cell membranes were thawed and resuspended in the reaction buffer (25 mM Tris-HCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 20 mM NaCl, and 1 µM GDP; pH 7.4 at 25°C). The reaction was started by adding an aliquot of membrane suspension (5 µg of membrane protein per well) to microwells containing reaction buffer and 0.08 to 0.15 nM [³⁵S]GTPS with agonist in a total volume of 250 µl. The microwell plates were incubated for 25 min at room temperature. The incubation was terminated by rapid filtration through glass fiber filters using a Tomtec Harvester 96 Mach II (Tomtec, Inc., Hamden, CT). The filters were washed with 3 × 4 ml of cold wash buffer (20 mM Tris-HCl, 5 mM MgCl₂, and 1 mM EDTA; pH 7.4 at 4°C). The bound radioactivity was determined in a 1205 Betaplate liquid scintillation counter (Wallac Oy, Turku, Finland).

_2A-AR Model and Ligand Models. The previously reported model structure of _2A-AR was used in this study (Salminen et al., 1999). The model is based on an -carbon template derived from the low-resolution electron microscopy structure of frog rhodopsin and the alignment of a large number of GPCRs (Baldwin et al., 1997). The ligand structures (Table 1) were built with the program Hyperchem version 5.01 (Hypercube, Inc., Gainesville, FL) and optimized using a short 200 ps simulated annealing procedure described elsewhere (Salminen et al., 1999). After simulated annealing, the ligands were energy minimized in vacuo using the MM+ (extended MM2) force field.

View this table: [in this window] [in a new window]   TABLE 1 Structures and names of the ligands used in the theoretical and experimental studies involving 2A-AR (S)-Octopamine, (S)-norphenephrine, and (S)-2-amino-1-phenyl-ethanol were not included in the docking study since their behavior can be predicted from the docking of the corresponding R-isomers and (S)-norepinephrine and (S)-epinephrine. The percentage given in parentheses is the size of the population having the conformation used in the docking studies (see Materials and Methods for details).

Automated Docking. Norepinephrine and epinephrine and their analogs, a total of 12 different ligands, were automatically docked to the ligand binding site of the _2A-AR model. The atomic partial charges, required for the docking simulations, were assigned to atoms of both the _2A-AR model and the ligand set according to the Gasteiger method (Gasteiger and Marsili, 1980) implemented in Quanta 97 (Molecular Simulations, Inc., San Diego, CA). The computer program Autodock version 2.4 (Morris et al., 1996) was used to dock the flexible small molecule ligands in the rigid _2A-AR receptor model. Conformational searches were limited to a 25 Å³ volume containing the _2A-AR ligand binding site and nearby residues. To find low-energy conformations of ligands in the receptor binding site, Autodock uses Monte Carlo simulated annealing combined with a rapid, atomic resolution, grid-based method of energy evaluation using the AMBER forcefield and a distance-dependent dielectric constant to account for the solvent effects.

Docking Parameters. The following scheme was used to seek low-energy ligand conformations: 1) 500 to 800 separate docking simulations were performed for each ligand; 2) for each simulation, there were 100 constant temperature cycles with 8000 steps accepted or rejected; 3) the initial simulation temperature (RT = 300 cal/mol, where R = gas constant and T = absolute temperature) was reduced by a factor of 0.97 in each cycle; and 4) flexibility of both the ligand and the orientation of the ligand in the binding site was introduced by allowing torsional rotation and molecule translation steps for the ligands of 15° and 0.2 Å, respectively, reduced by a factor of 0.97 in each cycle. In this way, over 10⁷ docked ligand-receptor conformations were evaluated for each ligand. Next, the docked structures were clustered into similar groups that differ by less than 1 Å root-mean-square deviation from each other at the binding site.

GRID Calculations. The computer program GRID version 16 (Goodford, 1985) was used to map essential interactions in the binding site of the _2A-AR model. GRID calculates energies of interaction between a chemical probe and the receptor. The probes used in this study mimic charged and neutral amino groups, (phenolic) hydroxyl groups, methyl groups, aromatic carbons, and hydrophobic groups. Probes were placed at positions throughout a 30 Å × 30 Å × 30 Å cube (3 points/Å, 27 points/ų) centered at the _2A-AR ligand binding site, and the interaction energies were calculated at each point. The flexibility of amino acid side chains of the _2A-AR model was considered in the evaluation of the interaction energy. The GRID maps were visualized using the program CERIUS 2 (Molecular Simulations, Inc.) and gOpenMol (Bergman et al., 1997).

Strategy for Building Receptor-Ligand Complexes. Initially, Autodock was used to dock epinephrine, norepinephrine, and their close analogs (Table 1) to our model structure (Salminen et al., 1999) of _2A-AR. In general, the program will lead to the identification of highly populated clusters corresponding to similar low-energy ligand-receptor complexes. Because Autodock only considers the enthalpic binding energy in its search for favorable orientations of torsionally flexible ligands, the intermolecular interaction energy function is not necessarily accurate in predicting the true binding energy. Thus, the cluster with the absolute minimum energy does not necessarily represent the best or global energy minimum of the ligand in the receptor binding-site, especially if the cluster is sparsely populated. Nonetheless, the method has been proven accurate enough to identify the possible conformations of ligand molecules at receptor binding sites (Minke et al., 1999; Rao and Olson, 1999; Salminen et al., 1999). Indeed, a highly populated cluster of similar conformations whose members are the result of many independent docking simulations suggests that the cluster is located at an energy minimum that is easily accessible. Moreover, a cluster with a higher population is more likely to represent the natural conformations of the ligand, even if a less populated cluster having a slightly lower energy is present.

	Results

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A total of 12 phenethylamines (Table 1), (R)-norepinephrine and five analogs (Fig. 1, A and B) and (R)-epinephrine and five analogs (Fig. 1, C and D), were docked to the structural model (Salminen et al., 1999) of _2A-AR (as indicated in Table 1, some S-isomers were not docked to the receptor model). K_i values for competition with the antagonist radioligand [³H]RX821002 to probe the lower affinity binding state and with the agonist radioligand [³H]UK-14,304 to probe the higher affinity binding state and receptor activation results (E_max and EC₅₀ values for [³⁵S]GTPS binding in isolated membranes) were determined experimentally for dopamine, (R)-norepinephrine, (S)-norepinephrine, (R)-epinephrine, (R)-norphenephrine, (S)-norphenephrine, (R)-octopamine, (S)-octopamine, and (R)-2-amino-1-phenyl-ethanol. Two additional agonist ligands, UK-14,304 and p-aminoclonidine, which are not phenethylamines but aminoimidazolines, were included for purposes of comparison. The results of these experiments are summarized in Table 2. The competition binding results obtained with the antagonist radioligand [³H]RX821002 were calculated using both one-site and two-site models. Two-site models were statistically significantly superior (p < 0.05) to one-site models for (R)- and (S)-norepinephrine, (R)-epinephrine, UK-14,304, and p-aminoclonidine. These two-site competition binding results are shown in Table 3.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Norepinephrine, epinephrine, and their analogs docked to the ligand binding site of the 2A-AR model structure. A and B, norepinephrine and analogs. C and D, epinephrine and analogs. In A and C, the binding site is viewed (in stereo) from the extracellular face of the receptor. In B and D, the view is perpendicular to the extracellular face and in the plane of the lipid bilayer. The -carbon trace of the TM helices is colored blue, and selected side chains are depicted and labeled with the single letter amino acid code (D, aspartate; F, phenylalanine; Y, tyrosine; C, cysteine; V, valine; and L, leucine). Color codes for ligands: dark blue, (R)-norepinephrine (A and B), (R)-epinephrine (C and D), N-methyl-dopamine (C and D); orange, (R)-octopamine (A and B), (R)-N-methyl-octopamine (C and D); violet, (R)-norphenephrine, (R)-N-methyl-norphenephrine; green, (R)-2-amino-1-phenyl-ethanol (A and B), (R)-2-methylamino-1-phenyl-ethanol (C and D).

View this table: [in this window] [in a new window]   TABLE 2 Competition binding and functional assay results The binding (Ki) and activation (EC50) values are presented for molecules that were commercially available, either as pure enantiomers or as racemic mixtures that were subsequently resolved into their separate pure enantiomers. In the case of the epinephrines, only (R)-epinephrine was available from a commercial source. Two separate binding assays were used with competition against [3H]RX821002 and [3H]UK-14,304: the apparent Ki is the inhibition constant for a one-site model. In the assays with [3H]RX821002, two-site fits were statistically significantly superior to one-site fits only for (R)- and (S)-norepinephrine, (R)-epinephrine, UK-14,304 and p-aminoclonidine (Table 3). The functional assay is based on measurement of agonist-induced binding of [35S]GTPS to 2A-AR-coupled G-proteins in isolated membranes. EC50 values of agonists are reported. Ki and EC50 ratios are the values reported for a ligand divided by that seen for (R)-norepinephrine. Values in parentheses are Emax relative to (R)-norepinephrine. All experiments are the means ± S.E. from 3-4 separate experiments.

The phenethylamine analogs differ from each other by either missing one or more hydroxyl groups (-OH, and catecholic meta- and para-OH groups) and/or in their chirality about the -carbon atom. The docked epinephrine analogs have a methyl group attached to the amine group, which is absent in norepinephrine and its analogs. The ligand set can be divided into six chemical domains whose interactions with _2A-AR can be classified: the positively charged amine group, the hydrophobic N-methyl group in epinephrine, and the epinephrine analogs, the -OH, the aromatic ring, and the para- and meta-catecholic OH groups. By dividing the ligands into six individual domains, we sought to understand the effect that each of these six chemical groups has on binding affinity and activation of _2A-AR.

Over 10⁷ ligand conformations were studied to produce each ligand-receptor complex model. To obtain the representative receptor-ligand complexes, we selected optimal docked poses found after cluster analysis in Autodock and visualization of the binding modes on a graphics workstation together with the GRID ligand affinity maps of the receptor binding site. The predicted binding modes of this set of ligands based on docking simulations, when taken in combination with experimental results on binding and receptor activation, makes it possible to assign functional roles to the chemical groups of the ligands, as well as to the receptor itself. Figure 2 shows the overall location of the ligand binding site on the model structure.

View larger version (73K): [in this window] [in a new window]   Fig. 2.   Stereoview of the general model for (R)-epinephrine binding to the 2A-AR model. A, (R)-epinephrine docked to 2A-AR showing the relative disposition of the seven transmembrane helices (cylinders). Interactions with the ligand are mainly provided by amino acids from TM3, TM5, and TM6 (red). B, electrostatic surface of the 2A-AR model without the extracellular loops viewed from the extracellular surface; regions of negative charge are shown in red, positive charge in blue, and neutral regions are white. Note that the positively charged N-methyl amine group is oriented toward the negatively charged portion of the distinctive binding cavity where Asp113 (D3.32) is located. C, close-up view of the electrostatic surface of the binding cavity. Epinephrine is shown docked in the cavity formed by TM3, TM5, and TM6. Rotation of TM5 coupled with movement toward the ligand would substantially increase the number of interactions with the ligand and could be the mechanism by which the receptor conformation is switched to the G-protein activating state. The figure was prepared using GRASP (Nicholls et al., 1991), Molscript (Kraulis, 1991), and Raster 3D (Merrit and Bacon, 1997).

We have thus used a series of very similar ligand molecules that are sequentially missing different functional groups. Because we have an accurate structural model for the mode of binding between each ligand and the receptor, we can then correlate the observed structural differences of the ligands and ligand-receptor interactions with the similarities and differences in the experimental data. This then permits us to discriminate between those interactions that are important for binding affinity and those interactions that help to stabilize the activated form of the receptor.

The Positively Charged Amine Group. With norepinephrine and its analogs, the most important interaction is formed between the positively charged amine group (Fig. 3) of the ligands and the negatively charged side chain carboxyl group of Asp113 (D_3.32) in TM3 of the receptor (Wang et al., 1991; Kobilka, 1995). In general, the interactions between Asp113 (D_3.32) and the amine group in the ligands should be strong and anchor the amine group close to that residue, as observed in the docking simulations (Fig. 1). The GRID calculations also indicated a large volume near Asp113 (D_3.32) where charged and neutral amine groups could be placed with favorable interaction energy. The electrostatic surface potential shown in Fig. 2, B and C, clearly shows the region of negative charge about Asp113 (D_3.32) in the receptor's ligand binding site.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   A, key interactions of (R)-epinephrine with the amino acid side chains lining the 2A-AR binding site in the inactive and active forms of the receptor. B, the important amino acids that contribute to the binding of the phenethylamine ligands and possibly to the activation of the 2A-AR are mainly from TM3, TM5, and TM6. For epinephrine, the N-methyl group, not present in norepinephrine, has hydrophobic contacts with residues from TM7. C, the two possible orientations of the catechol ring found in this study (light color: alternative docked conformation; dark color: conformation in consensus with current literature). Experimental evidence shows (Rudling et al., 1999) that the meta-OH probably points "upward", toward the extracellular surface (rotamer with dark color) and thus interacts with Ser200 (S5.42) in the active receptor conformation.

The N-Methyl Group in Epinephrine and Analogs. The N-methyl group (Fig. 3) in epinephrine and its analogs packs against Phe411 and Phe412 (F_7.38 and F_7.39) in TM7 in the docking simulations (Fig. 1, C and D). GRID maps also show that those parts of TM7 exposed to the binding cavity are favorable for hydrophobic, aromatic and methyl contacts. The presence of the N-methyl group in epinephrine and analogs would be predicted to improve the binding affinity by making an additional hydrophobic contact with Phe411 (F_7.38) and Phe412 (F_7.39) in TM7. Indeed, the affinity of (R)-epinephrine is 4 times higher than the affinity of (R)-norepinephrine in [³H]RX821002 binding studies and 3 times higher in the [³H]UK-14,304 assay (Table 2). The rank order of affinity of (R)-epinephrine and (R)-norepinephrine is, however, the opposite for the high-affinity binding site population as revealed in a two-site analysis of the [³H]RX821002 competition results (Table 3). Furthermore, the EC₅₀ values measured for stimulation of [³⁵S]GTPS binding are quite similar for (R)-epinephrine and (R)-norepinephrine (Table 2). Thus, the N-methyl group affects the binding affinity to the low-affinity receptor conformation, but is not directly coupled to the activation process.

The -OH Group. With both (R)-epinephrine and (R)-norepinephrine, the -OH group (Fig. 3) is positioned in a way that it can form a hydrogen bond with one side chain oxygen of Asp113 (D_3.32) (Fig. 1). Consistent with this view, the GRID maps calculated for _2A-AR indicate a favorable interaction for a hydroxyl group within a volume above (toward the extracellular surface) and at the level of the plane of the side chain oxygens of Asp113 (D_3.32). The interactions between _2A-AR and the -OH are unlikely to be any further than 4 to 5 Å away from Asp113 (D_3.32) because of the restrictions imposed by the ligand geometry. If the amine group in (R)-norepinephrine interacts with Asp113 (D_3.32), and the catecholic end of the ligand is oriented toward residues in TM5, it is also not possible for the -OH to interact with Ser90 (S_2.61) in TM2. The -OH group would thus not point toward TM2, as suggested by previous mutagenesis studies (Li et al., 1995), but would be located on the opposite side of the ligand, pointing toward Asp113 (D_3.32) in TM3.

The Aromatic Ring. Optimal placement of the aromatic ring, present in all of the investigated ligands, is with the ring plane packed against TM6, with additional interactions with TM3 and TM5 (Figs. 1 and 3). GRID calculations predict favorable aromatic group interactions between TM5 and TM6, induced by the aromatic side chains of Phe391 (F_6.52) and Tyr394 (Y_6.55) in TM6, both of which are conserved in all ₂-AR subtypes, and partially by the side chains of Phe205 (F_5.47) and Cys201 (C_5.43) in TM5 (Fig. 1). On the opposite face of the docked conformation of the aromatic ring lies another conserved residue, Val114 (V_3.33) in TM3. Aromatic rings, for example the adenine ring, are often seen sandwiched between valine or another hydrophobic residue on one face of the ring and aromatic residues on the opposite face of the ring (Denessiouk and Johnson, 2000). The catecholic OH groups (see below) are very likely to be in contact with Thr118 (T_3.37) in TM3 and Cys201 (C_5.43) in TM5, and are oriented toward TM5 where Ser200 (S_5.42) and Ser204 (S_5.46) are located; interactions with these groups would affect the orientation of the aromatic ring when docked.

The Para- and Meta-Catecholic OH Groups. In the docking simulations, the two catecholic OH groups (Fig. 3) contact residue side chains within TM3 and TM5, and thus influence the orientation of the aromatic ring. In the docking simulations, coordination can take place between catecholic hydroxyls and the side chains of Thr118 (T_3.37) in TM3; and Ser200 (S_5.42), Ser204 (S_5.46), and Cys201 (C_5.43) in TM5. Of the latter three residues, we believe that only Cys201 is exposed to the binding cavity in the low-affinity or inactive form of the receptor (Fig. 1). In the activated or higher affinity form of the receptor, we suggest that rotation of TM5 occurs and exposes both Ser200 (S_5.42) and Ser204 (S_5.46) to the ligand-binding cavity (Marjamäki et al., 1999; Salminen et al., 1999). Because of this rotation, good contacts are formed between the meta-OH and para-OH and Ser200 (S_5.42) and Ser204 (S_5.46) (Fig. 3). Due to the inaccuracies in the docking forcefield, the distinction between the two possible orientations of the catecholic ring in _2A-AR is difficult. The calculated energy difference between two possible ligand conformations, which differ in that the catechol ring is flipped by 180°, is too small to distinguish between the two conformations. Because mutagenesis studies (Wang et al., 1991; Rudling et al., 1999) have indicated that the meta-OH is likely to interact with Ser200 (S_5.42) and the para-hydroxyl with Ser204 (S_5.46) in _2A-AR, and because this orientation also is in agreement with results obtained with other adrenergic receptors (Strader et al., 1989, Hwa and Perez, 1996), the modeled orientation of the catechol ring was adjusted to correspond to these results (Fig. 3).

	Discussion

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RX821002 and UK-14,304 Probe Different Activation States of the Receptor. We have used two different ligand-binding assays in this study. [³H]RX821002 is an antagonist ligand, which mainly probes the predominant inactive state of the receptor, whereas [³H]UK-14,304 is an _2A-AR agonist, which probes the activated receptor. Both ligands are superficially similar, especially with regard to the imidazole end, which is likely to bind to Asp113 (D_3.32). At its opposite end, RX821002 has an unsubstituted phenyl ring, which can only participate in hydrophobic and aromatic interactions. These interactions are presumably not capable of stabilizing the activated form of the receptor. In contrast, UK-14,304 has two aromatic ring nitrogens that can participate in interactions that can help to promote or to maintain an activated conformation. This contrasts with p-aminoclonidine, a less potent and efficacious agonist than UK-14,304: p-aminoclonidine has a single amine group extending from the phenyl ring that can make one stabilizing set of polar interactions with the receptor, instead of the two possible with UK-14,304. p-Aminoclonidine is also shorter than UK-14,304. It is important to consider these aspects in the analysis of the data presented here.

Ligand Binding Affinity. The predicted location of the ligand binding site of _2A-AR and a set of representative phenethylamine binding modes are presented in Figs. 2 and 3. According to the Easson-Stedman hypothesis, an adrenoceptor should have three functional points in its binding site to accommodate the charged aliphatic nitrogen, the -OH group, and the aromatic ring of phenethylamine ligands [see Ruffolo (1991) for a detailed discussion]. In our model (Fig. 2), these interactions are primarily contributed by residues in TM3, TM5, and TM6 (Fig. 3). In addition, we suggest that residues in TM7 should have a role in ligand binding in the case of epinephrine and analogs (Fig. 3).

Receptor Activation. Gether and Kobilka (1998) have suggested a general model for the activation of rhodopsin-like GPCRs. We propose a similar but more detailed scheme for the activation of _2A-AR by (R)-phenethylamines (Figs. 3 and 4). We suggest that binding of a phenethylamine to _2A-AR is initiated by the formation of a hydrogen bond between the negatively charged carboxylate in Asp113 (D_3.32) in TM3 and the positively charged aliphatic amine group in the ligand. The N-methyl group of epinephrine increases the binding affinity through nonpolar interactions with hydrophobic residues in TM7. The interaction between the ligand and _2A-AR is enforced by the formation of another hydrogen bond between the -OH of the ligand and the other oxygen in the Asp113 (D_3.32) side chain. The agonist is oriented within the binding site so that the aromatic ring is sandwiched between the aromatic interactions with Tyr394 (Y_6.55) and Phe391 (F_6.52) in TM6 and possibly Phe205 (F_5.47) in TM5, and hydrophobic interactions with Val114 (V_3.33) in TM3. The orientation of the aromatic ring is locked into place by these nonpolar interactions and very probably through interactions between the catecholic OH groups and Thr118 (T_3.37) in TM3; and Ser200 (S_5.42), Cys201 (C_5.43), and Ser204 (S_5.46) in TM5.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Proposed mechanism for agonist-independent activation and agonist-induced 2A-AR activation by norepinephrine and epinephrine. A, agonist-independent activation of receptor (R) proceeds through a conformational change involving TM3, TM5, and TM6. This conformational change would be transmitted to the intracellular loops that can then bind and activate the G-protein complex (). The energy barrier for this transition would probably be high such that the population of activated receptor (R*) capable of binding G-proteins (G) in the absence of ligand (L) would be low. This low concentration of activated receptor would, however, be able to bind tightly to agonists (stars) presented to them (B, right-hand side). In the case of agonist ligands, the equilibrium would favor the formation of the activated ligand-bound complex (L*R*), whereas antagonists (circles) with inverse agonist activity would shift the equilibrium in favor of the inactive receptor form (LR).