Introduction

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5-Hydroxytryptamine_2A (5-HT_2A) receptors are the major site of action for at least three classes of hallucinogens, including the ergolines (e.g., lysergic acid diethylamine), phenylisopropylamines (e.g., 4-iodo-2,5-dimethylphenylisopropylamine, DOI), and substituted tryptamines (e.g., N,N-dimethyltryptamine, DMT). Our limited, but expanding, understanding of 5-HT_2A receptor function has led to the development of novel clinical therapies, including the atypical antipsychotics (e.g., clozapine, risperidone, olanzepine, and quietiapine), and the atypical antidepressants (e.g., nefazadone, mirtazepine, and mianserin; for review, see Roth et al., 1998). Efforts to improve clinical therapies by developing more receptor-specific, novel therapeutic agents have been hampered in part by a lack of knowledge concerning the precise three-dimensional structure of ligand-receptor interactions. In the absence of a high-resolution crystal structure for 5-HT_2A (or any other G-protein-coupled receptor; GPCR), site-directed mutagenesis accompanied by molecular modeling has been used to uncover the details of ligand binding.

Based on analogies with rhodopsin and bacteriorhodopsin, a number of models of the 5-HT_2A and 5-HT_2C receptors have been constructed and, occasionally, subjected to testing by fitting of site-directed mutagenesis data (Choudhary et al., 1993; Westkaemper and Glennon, 1993; Holtje and Jendretzki, 1995; Almaula et al. 1996a,b; Kristiansen and Dahl, 1996; for example, see Roth et al., 1997a). Most models of agonist-5-HT_2A receptor complexes share the following features: a key stabilizing ionic interaction between the positively charged amine moiety of the agonist with a negatively charged aspartic acid residue (D155[3.32 using the numbering system of Ballesteros and Weinstein (1995), see below] in helix 3) and the OH group of S159[3.36] (helix 3), one or more serine residues (S207[4.57], S239[5.43] in helix 5, S372[7.45], S373[7.46] in helix 7), which serve to stabilize NH or OH moieties via hydrogen bonds, and a central core of aromatic residues, which stabilize aromatic and hydrophobic moieties of the ergoline nucleus (previously hypothesized to include F240[5.44], F340[6.52], W336[6.48], and W367[7.40]). Of these key residues, a great deal of attention has been focused on the ligand interactions involving D155[3.32] (Wang et al., 1993; Kristiansen et al., 2000) and F340[6.52] (Choudhary et al., 1993, 1995; Roth et al., 1995, 1997b).

In the present study, we have combined site-directed mutagenesis and molecular modeling techniques to identify additional residues in the rat 5HT_2A receptor that are important for agonist binding, antagonist binding, and agonist-stimulated phosphoinositide (PI) hydrolysis. The effects of four single-point mutations in transmembrane helix 5 (S239A, F240A, F243A, and F244A) of the rat 5HT_2A receptor on ligand binding and agonist-stimulated PI hydrolysis were examined. Our results suggest a key role for F243[5.47] in binding and activation of the 5HT_2A receptor. Depending on the nature of the bound ligand, F243[5.47] and F244[5.48] likely participate in a cluster of stabilizing, aromatic amino acids lining the binding pocket, exerting their influence through - stabilization, favorable van der Waals (vdW) interactions and, indirectly, by influencing the position of neighboring side chains. Our findings imply that changes in agonist binding not only affect binding pocket interactions but may also induce helical perturbations, which variously affect the receptor's ability to interact with G-proteins. Agonist binding was particularly sensitive to disruption by S239A, most likely interacting via formation of an OH-N1 hydrogen bond with the ring nitrogen of the indolealkylamines. Finally, the receptor binding and activation data from this and previous studies were utilized in molecular dynamics simulations to construct detailed molecular models for complexes between the 5-HT_2A receptor and structurally diverse agonists.

	Materials and Methods

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Receptor-Numbering Schemes. Where appropriate, amino acid residues have been labeled both by standard amino terminus-based numbering and by a numbering scheme introduced by Ballesteros and Weinstein (1995) in which relative amino acid positions are highlighted. This scheme facilitates the efficient comparison of residues within different GPCRs. With this method, every amino acid identifier starts with the transmembrane helix number and is followed by the position relative to a reference residue (arbitrarily assigned the number 50) among the most conserved amino acids in that transmembrane helix. In the seven transmembrane (TM) GPCRs, this generalized numbering scheme utilizes N1.50, D2.50, R3.50, W4.50, P5.50, P6.50, and P7.50 as seven reference positions, corresponding in rat 5-HT_2A receptors to N92, D120, R173, W200, P246, P338, and P377, respectively. F340, for example, lies two positions positive relative to P338, yielding the identifier [6.52].

Site-Directed Mutagenesis and Plasmid Construction. The mammalian expression vector pSVK3-SR2 was used as previously detailed (Roth et al., 1992; Choudhary et al., 1993, 1995). Mutants were developed using a polymerase chain reaction-based technique (Quikchange kit; Stratagene, La Jolla, CA ) following the recommendations of the manufacturer, with mutagenesis and sequencing primers from Life Technologies/BRL (Gaithersburg, MD). For each mutant made, the full length of the receptor insert was sequenced using the dideoxy method (T7 Sequenase Quick Denature kit; Amersham, Piscataway, NJ).

Cell Culture. COS-7 cells were grown as previously detailed (Choudhary et al., 1993, 1995; Roth et al., 1997b) and transiently transfected with various receptor mutants using Fugene6 (Roche, Indianapolis, IN) in 100-mm dishes by scaling up the recommended procedure supplied by the manufacturer.

Binding Assays. Transiently transfected cells were switched from medium containing dialyzed serum to serum-free medium for 24 h before harvest to remove residual 5-HT and then harvested using a cell scraper as previously described (Roth et al., 1995, 1997b). Binding assays were performed using membrane preparations in a total volume of 1.0 ml using [³H]ketanserin or [³H]N-methylspiperone as the labeled ligand. For saturation-binding assays, six concentrations of labeled ligand spanning a range of 100-fold (typically 0.1-10 nM) were used. For competition-binding assays, six to 10 concentrations of unlabeled ligand spanning a range of 10,000-fold (typically 1-10,000 nM) were used. Agonist and antagonist competition-binding assays and saturation-binding assays were performed in a buffer of the following composition: 50 mM Tris-Cl, 10 mM MgCl₂, 0.5 mM EDTA, 0.1% ascorbic acid, pH 7.4. (Roth et al., 1995, 1997b) Typically, specific binding represented 90% of total binding with no more than 10% of the total counts bound. Data were analyzed using the LIGAND program (Munson and Rodbard, 1980) as previously detailed (Roth et al., 1997a,b) with differences in binding parameters analyzed using the F test.

Phosphoinositide Hydrolysis Assays. For measurements of [³H]PI release, cells were loaded for 18-24 h with 1 Ci/ml [³H]inositol in serum-free and inositol-free medium as previously described (Roth et al., 1997a,b). Measurements of PI hydrolysis were performed as previously detailed (Roth et al., 1984, 1997a,b). K_act and V_max values were determined using a nonlinear curve-fitting routine as previously described (Roth et al., 1997a,b).

Molecular Modeling of the Rat 5-HT_2A Receptor. A model of the transmembrane domain in the rat 5-HT_2A receptor was constructed using computer graphics, molecular mechanics, and molecular dynamics simulations. The MIDASPLUS and SYBYL (6.4 beta) programs (University of California, San Francisco, CA) were used for computer graphics. Molecular mechanics and molecular dynamics simulations were performed with the AMBER 4.1/5.0 programs (Oxford Molecular, Oxford Science Park, UK) using the all atomic force field (Cornell et al., 1995). All calculations were performed with a distance-dependent dielectric function ( = r_ij) and a 10-Å cutoff radius for nonbonded interactions. Energy minimizations of ligand-receptor complexes were performed by 500 steps of steepest descent minimization followed by conjugate gradient minimization until convergence with a 0.02-kcal mol¹ Ź root mean square energy gradient difference between successive minimization steps.

Molecular Modeling of Ligand-5HT_2A Receptor Interactions. Energy minimizations of ligands excluding electrostatic interactions were performed by 500 steps of steepest descent minimization followed by conjugate gradient minimization until convergence with a 0.0001-kcal mol¹ Ź root mean square energy gradient difference between successive minimization steps. Electrostatic potentials around 5-HT, -methyl (Me)-5-HT, 5-methyl-DMT, N--methyl-5-HT, and (R)DOM were calculated at the HF/6-31G* level by using the GAMESS (October 31, 1996 version; Gordon Research Group, Ames, IA) and MOLDEN programs (Molden 3.6; Schaftenaar and Noordik, 2000) or by using the GAUSSIAN programs (Gaussian94, revision D.4; Gaussian, Inc., Carnegie, PA). Atomic point charges were projected from these potentials by using the RESP program of the AMBER program package.

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	Results

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Characterization of Mutant Receptor Expression. Saturation-binding isotherms were established for [³H]ketanserin and [³H]N-methylspiperone (NMSP) binding to wild-type and mutant receptors. [³H]NMSP was used for the F243A mutant because ketanserin did not bind with high affinity to this mutant. A summary of K_d and B_max values for each mutant is shown in Table 1. Receptor expression among the transfectants was variable, ranging from a low of 574 ± 111 fmol/mg (F243A) to a high of 2367 ± 583 fmol/mg (F244A). Native receptors bound [³H]ketanserin and [³H]N-methylspiperone with affinities of 1.7 ± 0.29 and 0.78 ± 0.17 nM, respectively. S239A and F240A had slightly increased affinities for ketanserin (2.7- and 2.9-fold, respectively), whereas the F244A mutant showed a 3.1-fold decrease in binding affinity. The F243A mutant had a 7.7-fold lower affinity than the native receptor for [³H]N-methylspiperone.

View this table: [in this window] [in a new window]   TABLE 1 [3H]Ketanserin and [3H]-N-methylspiperone binding by native and mutant 5-HT2A receptors Data represent means ± S.E. for three separate experiments of mean Kd (nM) and Bmax (fmol/mg protein). For the F243A mutant, [3H]NMSP was used as the radioligand because of the low affinity of ketanserin for F243A. For all other mutants, [3H]ketanserin was used. In a typical experiment, 90% of total binding was specific, and 10% of the total radioligand was bound.

Binding Affinities for the Wild-Type and Mutant Receptors. We next measured the K_i values for eight structurally diverse agonists and antagonists at native and mutant receptors. Structures of the various compounds used in this study are presented in Fig. 1A. The relative position of each residue along a theoretical -helix is presented in the helical net diagram in Fig. 1B. Binding affinities for the drugs tested at the various helix V mutants as well as the native receptor are summarized in Table 2. The F240A mutation had no significant effect on binding except for ritanserin, which displayed a 2-fold increase in binding affinity. The S239A mutant showed 6- to 13-fold decreases in binding affinity for 5-HT, -Me-5HT, 5-methyl-DMT, and N--methyl-5HT and 2- and 5-fold increases in binding affinity to ritanserin and DOI, respectively. The F244A mutant exhibited similar changes in ligand binding, ranging between 2.9- and 10.1-fold decreases in binding affinities for 5-HT, -Me-5HT, 5-methyl-DMT, N--methyl-5HT, and ketanserin but showing a 3.0-fold increase in binding affinity for ritanserin. There was no measurable difference in spiperone binding for the F244A mutant. The most striking alterations in ligand-binding affinities were seen with the F243A mutation, ranging from no effect (5HT and N--methyl-5HT) to 48.7- and 216.6-fold reductions in binding affinities for -Me-5HT and ritanserin, respectively.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Ligand structures and helical net diagram of TM V. Shown are the structure of ligands used in this study (A), and a helical net diagram of the relative orientations of residues within transmembrane region V in a theoretical -helix (B). Residues evaluated in this study are highlighted in bold (S239, F240, F243, F244, corresponding to 5.43, 5.44, 5.47, 5.48, respectively). Homologous residues in the human D2 receptor shown to be accessible to MTSEA (Javitch et al., 1995) are cross-hatched.

Effects of S239A, F240A, F243A, and F244A Mutations on Agonist-Mediated PI Hydrolysis. We next examined the ability of the various 5-HT_2A mutants to activate PI hydrolysis. None of the mutants altered the basal levels of PI hydrolysis, indicating that they did not alter the constitutive activity of the 5-HT_2A receptor (not shown). The abilities of four different 5-HT_2A agonists to stimulate PI hydrolysis in cells with each receptor mutation are presented in Table 3, and a representative activation isotherm is shown in Fig. 2.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Agonist-mediated PI hydrolysis in cells transfected with wild-type 5-HT2A receptors. Data represent means ± S.E.M. (of triplicate determinations) for the percentages of stimulation of PI hydrolysis compared with the native rat 5-HT2A receptors. Six log-serial dilutions of 5-HT were incubated in 24-well tissue culture plate wells containing transiently transfected COS-7 cells, and the accumulation of [3H]inositol monophosphate was determined as described in Materials and Methods. Data are expressed as percentages of native receptor stimulation.

DOM Interacts with the Rat 5HT_2A Receptor in a Manner Different from Serotonin. Figures 3 and 4 show the binding pockets of agonist-5HT_2A receptor complexes obtained after energy minimizations and 100-ps molecular dynamics simulations. In all of these models, the aromatic ring of the agonist molecule was surrounded by V156[3.33], F243[5.47], F244[5.48], and F340[6.52]. However, subtle differences (described below) in the detailed receptor-ligand interactions for the structurally diverse agonists were also apparent.

View larger version (66K): [in this window] [in a new window]   Fig. 3.   Serotonin and DOM-binding pockets in the rat 5HT2A receptor. Views of the transmembrane helices (represented as tubes) from the extracellular side are shown after various molecular dynamics simulations. These views show the agonists 5-HT or DOM and all residues within a 3.5-Å sphere radius of the agonist molecule in energy minimized complexes after 100-ps molecular dynamics simulations. The left panels represent simulations obtained with 5-HT, and the right panels represent simulations obtained with DOM. The top section represents simulation 1, and the bottom panel represents simulation 2. Color coding of ligand and receptor atoms: red, O; green, N; cyan, H; white, C in the receptor; green, C in the ligand.

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Molecular modeling and site-directed mutagenesis reveal differential binding of methyl 5HT to the 5-HT2A receptor. Views (from the extracellular side of the receptor) of the transmembrane helices (represented as tubes), showing various agonists (green) and all residues within a 3.5-Å sphere radius of the agonist molecule in energy-minimized complexes after 100-ps molecular dynamics simulations. The top two panels show the simulated binding of 5-methyl-DMT and N--methyl-5HT. The bottom two panels show two views of the simulated binding of -Me-5HT to the 5-HT2A receptor after two different molecular dynamics simulations (see Results and Discussion for details). Color coding of ligand and receptor atoms: red, O; green, N; cyan, H; white, C in the receptor; green: C in the ligand.

N1-Unsubstituted Tryptamines and -Methyl-5HT Differentially Interact with the Rat 5-HT_2A Receptor. Figure 4 shows the binding pocket of agonist-receptor complexes with N1-unsubstituted tryptamine analogs and -Me-5HT obtained after energy minimizations and 100-ps of molecular dynamics simulations. The NH group of the unsubstituted tryptamines interacted as a hydrogen bond donor with the OH group of S239[5.43]. As seen with the 5HT-receptor model, the secondary amine side chain of N--methyl-5HT interacted with both the carboxylate group of D155[3.32] and the side chain CO group of N343[6.55]. In contrast, the tertiary amine side chain of 5-methyl-DMT interacted with the carboxylate group of D155[3.32] only. The 5-OH substituent of N--methyl-5HT was hydrogen bonded between the hydroxyl groups of S159[3.36] and T160[3.37].

	Discussion

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Very little is known about the complex ligand-receptor interactions that regulate 5-HT receptor actions, primarily because no direct structural information is currently available regarding any details of the receptor or the ligand-receptor complexes. The best characterized receptor belonging to family A of G-protein-coupled receptors is rhodopsin for which electron density maps and models to approximately 6 Å in the plane of the membrane have been published (Baldwin et al., 1997). This level of resolution has been insufficient to provide a detailed model for binding pocket interactions. Furthermore, these electron density maps represent the structure of bovine and frog rhodopsin in the inactive state. Thus, although these models provide the only available scaffolds for the development of rat 5-HT_2A receptor models, there will no doubt be significant structural and functional species differences in both the inactive and activated states.

As described below, our accumulated data suggest that minor changes in ligand structure have variable effects on the apparent orientations of bound ligands. These results could not have been predicted based on the assumptions made with previous models using similar drug structures. The resulting apparent differences in ligand-receptor interactions and the changes in ligand binding and receptor activation caused by substituting drugs differing by as little as one methyl group are likely to be of tremendous importance for structure-based drug design.

Structure of Transmembrane Region 5. Because our prior rat 5-HT_2A receptor model (Kristiansen et al., 2000) could not account for all effects of the present mutation data in helix 5, an updated model was constructed. In this new model, F243[5.47] and F244[5.48] are pointing into the binding site, F240[5.44] is pointing toward the membrane, and S239[5.43] and A242[5.46] are pointing more in the direction of helix 4. In a regular -helix, there is a 200° arc between the F244[5.48] and A242[5.46] side chains, making it impossible to place both simultaneously into the binding pocket. Theoretically, the presence of a highly conserved proline in transmembrane helix 5 (P246[5.50]) might lead to a partial unwinding of the helix in the region containing S239[5.43] and A242[5.46]. A very extreme unwinding for the corresponding region of helix 5 (F189-F198) in the human dopamine D₂ receptor has been proposed from experiments in which the substituted cysteine accessibility method has been used (Javitch et al., 1995). Alternatively, binding of ligands may influence the rotational orientation of helix 5 by specifically interacting with residues on different faces of the helix as supported by data from the human _2A adrenergic receptor (Marjamäki et al., 1999). The finding that A242[5.46] was inaccessible for ligand binding in the present models was supported by the lack of effect of the A242S and A242T mutations in the rat 5-HT_2A receptor on binding of ketanserin, DOI, and serotonin (Johnson et al., 1994). However, the Ser/Ala difference between human 5-HT_2A and 5-HT_2C receptor (Almaula et al., 1996a) and between rat and human 5-HT_2A receptors (Kao et al., 1992; Johnson et al., 1994) have been identified as a major determinant for the subtype specificity of N-1-substituted ergolines, such as mesulergine.

Role of Aromatic Residues in Receptor Folding, Ligand Binding, and Signal Transduction. In the absence of a crystal structure for the receptor, structure activity relationships, coupled with molecular modeling, have been used to define the three-dimensional binding interactions between receptor and ligand (Kristiansen et al., 1993; Westkaemper and Glennon, 1993; Choudhary et al., 1995; Holtje and Jendretzki, 1995; Kristiansen and Dahl, 1996). Many of these models suggest the presence of key aromatic residues, including W200[4.50], F340[6.52], W336[6.48], W367[7.40], and Y370[7.43], which account for much of the binding affinity and specificity of the receptor. Mutation of these positions caused a markedly reduced agonist affinity and efficacy at 5-HT_2A receptors (Choudhary et al., 1995; Roth et al., 1997b), whereas mutation of other aromatic residues (e.g., F365[7.38]), predicted to be near the binding pocket, had little or no effect on agonist affinity (but did diminish agonist efficacy; Roth et al., 1997b). Mutation of other, nonconserved residues (F125[2.55], M132[2.62], and T134[2.64]) had no significant effect on agonist- or antagonist-binding affinity.

The Rat 5HT_2A Receptor Interacts Differently with Phenylisopropylamines than with Tryptamines. According to the present modeling, (R)DOM was located closer than the tryptamines to helix 6, and the hydrophobic part of the protonated side chain had vdW contacts with F339[6.51] and W336[6.48] after simulation 1. The F243[5.47] residue was located close to the 2-methoxy group of DOM and may, therefore, sterically interfere with formation of a hydrogen bond between 2-methoxy in DOI/DOM and the hydroxyl group of S159[3.36]. This hypothesis is in agreement with the 40-fold increase in DOI binding observed for the F243A mutation. The 5-fold increase in affinity of DOI observed for the S239A mutation could be due to a direct vdW interaction between the A239 side chain and the 5-methoxy substituent (simulation 2) or indirect structural effects on the binding pocket.

Distinct Binding Modes for -Methyl-5HT and the N1-Unsubstituted Tryptamines. Based on prior molecular dynamics simulations, the agonist-binding site was suggested to occupy the same general location inside the helix bundle for structurally diverse agonists. However, results from the most recent molecular dynamics simulations and mutagenesis experiments suggest the existence of different binding modes for N1-unsubstituted tryptamines and -methyl-5HT.