Three amino acids in the D2 dopamine receptor regulate selective ligand function and affinity
The D2 dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype-selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120-fold) for the D4 subtype of dopamine receptor were tested on wild-type and mutant D2 receptors. Five of the selective ligands were observed to have 21-fold to 293-fold increases in D2 receptor affinity when three non-conserved amino acids in TM2 and TM3 were mutated to the corresponding D4 amino acids. The two ligands with the greatest improvement in affinity for the D2 mutant receptor [i.e., 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine (L-750,667) and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild-type receptor, concentrations of L-750,667 or RBI-257 that produced large reductions in the potency of quinpirole\xe2\x80\x99s functional response in the mutant did not significantly reduce quinpirole\xe2\x80\x99s functional response in the wild-type D2 receptor. In contrast to RBI-257 which is an antagonist at all receptors, L-750,667 is a partial agonist at the wild-type D2 but an antagonist at both the mutant D2 and wild-type D4 receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D2 dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non-conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype-selective ligands and the wild-type D2, mutant D2, or wild-type D4 receptors.
<!>Reagents<!>Site-directed mutagenesis and transfections<!>Membrane preparation and radioligand binding assays<!>cAMP functional assays<!>Calculations and data analysis<!>Construction of receptor homology models<!>Building ensembles of receptor conformers<!>Docking ligands into receptor conformers<!>Results<!>Discussion
<p>Modulation of dopaminergic pathways is a principal aim of numerous pharmacological interventions because dopamine receptors play an integral role in movement, emotional cognition, memory, and attention. Parkinson's disease and schizophrenia are but two examples of severely debilitating disorders mitigated by dopaminergic treatments (Ustun et al. 1999) that prolong life and improve its quality. Still there is much to be learned concerning the molecular basis of selective ligand actions on dopamine receptors and analogous heterotrimeric G-protein-coupled receptor systems.</p><p>Dopamine receptors are biogenic amine heterotrimeric G protein-coupled receptors sharing structural homology with rhodopsin. Five genes coding for five subtypes of the dopamine receptor (D1–D5) are known to exist in mammals. With the exception of the D2 receptor partial agonist, aripiprazole, the most commonly prescribed neuroleptics are thought to attenuate psychosis by antagonizing the D2 receptor subtype (Lawler et al. 1999; Shapiro et al. 2003; Schetz and Sibley 2007). Although the D4 receptor was previously considered a drug target for the treatment of psychosis (Sanyal and Van Tol 1997), the failure of three clinical trials suggests that this is not the case (Kramer et al. 1997; Truffinet et al. 1999; Corrigan et al. 2004). However, the D4 receptor has remained a potentially important clinical target because of its roles in regulating hyperactivity and CNS-mediated penile tumescence (Schetz and Sibley 2007; Schetz 2009).</p><p>Previous work on the D4 receptor revealed that the non-conserved amino acids at positions 2.60, 2.61, 3.28, and 3.29 provided key structural determinants for drug selectivity between D2 and D4 receptor subtypes (Schetz et al. 2000). This conclusion was reached by analyzing reciprocal mutations, i.e., mutations where one or more amino acids in the D4 sequence were substituted with the corresponding amino acid of the D2 sequence. A more comprehensive study focusing on a large number of D4-selective ligands from the 1,4-disubstituted aromatic piperazine/piperidine (1,4-DAP) structural class revealed that a majority of 1,4-DAPs have a pattern of molecular recognition (mode-1) dominated by amino acids occupying positions 2.61, 3.28, and 3.29 (Kortagere et al. 2004). Related studies in a D2 receptor background have corroborated the importance of amino acids at position 2.61, 3.28, and 3.29 in the D2 receptor as binding determinants for several D4-selective ligands (Simpson et al. 1999); however, the functional properties of D2 mutant receptors with improved affinity for specific D4-selective drugs have never been investigated.</p><p>We report here for the first time the functional properties of a combined TM2/TM3 reciprocal mutant D2 receptor (D2-V2.61F + FV3.28-3.29LM) and demonstrate that it not only has increased affinity and improved functional sensitivity for D4-selective ligands, but that such ligands [L-750,667 and 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine (RBI-257)] exert the same functional effects on the D2 mutant as they do on the wild-type D4 receptor. These findings suggest that a D2 receptor microdomain which controls a ligand's selectivity can also control a ligand's function.</p><!><p>Cell culture media was purchased from Hyclone (Logan, UT, USA). [3H]Methylspiperone,8-[4-(4-fluorophenyl)-4-oxobutyl]-(3-methyl-1-phenyl)-1,3,8-triazaspiro[4,5]decan-4-one hydrochloride ([3H] MSP; NET-856, 70–80 Ci/mmol) was purchased from Perkin Elmer (St Louis, MO, USA). Other drugs were purchased from Sigma (St Louis, MO, USA) or Tocris (Ellisville, MO, USA).</p><!><p>Mutagenesis was accomplished using a QwikChange™ kit (Stratagene, CA, USA) and confirmed by full-length sequencing. Mutant receptors were named by slight modification of the receptor nomenclature system as described previously (Ballesteros and Weinstein 1995; Ericksen et al. 2009): each amino acid in a transmembrane segment (TMS) is described by coordinates based on its position relative to the most conserved amino acid of that TMS (position 50). For example, D2-V2.61F denotes a valine to phenylalanine mutation at position 61 of TMS 2 in the D2 receptor. Plasmid DNA was transfected into cells by calcium phosphate precipitation as described previously (Ericksen et al. 2009). Stable receptor expression was maintained under 100 µg/mL G418 drug selection pressure.</p><!><p>Cell membranes were prepared as described previously (Ericksen et al. 2009) by suspension in lysis buffer (5 mM Tris, 5 mM MgCl2, pH 7.4, 4°C) for 5–10 min followed by homogenization with eight strokes of a Dounce homogenizer and centrifugation at 28 000 g for 45 min. The resulting pellet was resuspended in cold binding buffer and recentrifuged. The final membrane pellet was resuspended in cold binding buffer, homogenized with four strokes, and stored on ice until use. Radioligand binding buffer consisted of 50 mM Tris adjusted to pH 7.4 at 25°C with 1 N KOH or 1 N HCl (Ericksen et al. 2009). For saturation isotherms, cell membranes were equilibrated (90 min) with increasing concentrations of [3H]MSP in the presence or absence of 5 µM (+)-butaclamol, a drug used to define non-specific binding. For competition binding experiments, a fixed concentration of 0.5 nM [3H]MSP was equilibrated (90 min) with increasing concentrations of non-radiolabeled competing ligand. Equilibrated samples were rapidly filtered through Whatman GF/C filters (Brandel, Gaithersburg, MD, USA) pre-treated with 0.3% poly(ethyleneimine) (10 min) and washed three times with 3 mL of ice-cold binding buffer (50 mM Tris, pH 7.4, 0°C). Radioactivity was quantified in a scintillation counter. Membrane protein concentration was determined by bicinchoninic acid assay (Pierce, IL, USA).</p><!><p>Intracellular cAMP concentrations were determined using a cAMP Alphascreen™ detection kit (Perkin Elmer, Waltham, MA, USA) as described previously (Ericksen et al. 2009). Briefly, human embryonic kidney (HEK293) cells were seeded at 50 000 cells/well in a poly-l-lysine-coated 96-well microtiter plate. Stimulation buffer (Dulbecco's modified Eagle's medium, 20 mM HEPES, 100 µM sodium metabisulfite, 30 µM Ro 20-1724), cell lysis buffer (0.3% Tween 20, 20 mM HEPES, 1 µg/µL bovine serum albumin), and bead buffer (20 mM HEPES, 30 µM Ro 20-1724, 1 µg/µL bovine serum albumin, 1× Hank's Balanced Salt Solution) were prepared the next morning and adjusted to pH 7.4 with 1 N cell culture tested NaOH (Sigma-Aldrich, St Louis, MO, USA). Drug dilutions prepared in stimulation buffer containing 6 µM forskolin were equilibrated (37°C, 5% CO2) for 30 min. Cells were challenged with the equilibrated dilutions for 20 min (37°C, 5% CO2) followed by 5-min centrifugation at 1500 g. Cells were lysed by 90 min of shaking in 100 µL of cell lysis buffer. Prior to quantification, half units of acceptor and donor beads pre-incubated with biotinylated cAMP (3.76 nM) were equilibrated (1 h) with 30 µL of each sample in an opaque 96-well Costar plate (Corning, NY, USA) protected from light. Samples were read on a Perkin-Elmer Alpha Fusion™ plate reader in alpha mode using a 2-sec window (680 nm excitation at 0.6 s/sample and 520–620 emission 1.4 s/sample).</p><!><p>All data points for each experiment were sampled in triplicate and each experiment was repeated three times, unless noted otherwise. All data were analyzed using Prism version 4.0 (Graphpad Software Inc., San Diego, CA, USA). Geometric means and standard deviations are reported in the tables and SEM are plotted in the graphs. For saturation isotherm analysis, specifically bound [3H]MSP was calculated by subtracting non-specifically bound [3H]MSP (defined as binding in the presence of 5 µM (+)-butaclamol) from the total bound [3H]MSP at each concentration of [3H]MSP. The equilibrium dissociation constant (KD) for [3H]MSP and the receptor density (Bmax) were determined by best-fitting the specific binding data versus the concentration for free radioligand to a single-site square hyperbola curve. Inhibition constants (Ki) for unlabeled ligands were calculated using the Cheng-Prusoff equation: Ki = IC50/(1 + [radioligand]/KD). For cases where drug solubility was limited, IC50 values were generated by extrapolating concentration–response curves to zero. All cAMP values were converted to the amount of cAMP generated per milligram of sample protein (cAMP/mg) then normalized relative to the amount of cAMP generated by 6 µM forskolin in the absence of agonist. Efficacy was determined by subtracting the best-fit values for the lowest horizontal asymptote from the highest horizontal asymptote of the fitted sigmoidal semi-log dose–response curves. Statistical analyses of the curve-fitting procedures included the run test, F-test, and Pearson's correlation coefficient. Values were analyzed by one-way anova and Dunnett's post hoc analysis with significance established at the 95% confidence level (p ≤ 0.05).</p><!><p>D2 and D4 receptor structures were constructed with Modeller 9v1 (San Francisco, CA, USA) (Šali and Blundell 1993) using both the bovine rhodopsin (Li et al. 2004) (1GZM) and β2 adrenergic (Cherezov et al. 2007) (2RH1) structures as templates. For each dopamine receptor model, 1000 structures were generated and ranked using Modeller's objective function. The models were then aligned and clustered using the GROningen Machine for Chemical Simulations (GROMACS, v3.3; Uppsala, Sweden) (Van Der Spoel et al. 2005) gcluster utility (cutoff = 0.20 Å). The most representative (central) structure from the first (wild-type D2 receptor) and second (wild-type D4 receptor) most populated clusters were used for minimization, normal mode analysis (NMA), and docking. These structures were minimized in vacuo using the L-BGFS (low-memory Brouden–Fletcher–Goldfarb–Shanno, quasi-Newtonian algorithm for energy minimization) method in two stages, each applied to convergence (Fmax < 10 kJ/mol/nm) using gromacs v3.3 (Van Der Spoel et al. 2005) with the molecular systems parameterized according to the Optimized Potentials for Liquid Simulations (OPLS) all atom forcefield (Jorgensen et al. 1996). Electrostatic interactions were treated by the particle-mesh Ewald method. In the first stage all backbone protein atoms were restrained with half-harmonic force restraints (k = 1000 kJ/mol/nm). In the second minimization stage, only Cα atoms were restrained.</p><!><p>To obtain receptor conformers based on perturbations along low-frequency normal modes, each minimized receptor model was reduced to a Cα-only elastic network model and submitted to NMA deformation and refinement web server for NMA and automated decoy generation using default parameters with the exception of the distance weighting parameter. For the distance weighting parameter a non-default value of 3.0 Å was applied as recommended for Cα-only models and the approximate average Cα root mean square deviation (Å) of the decoy value was set to 1 Å to focus on smaller backbone motions near the initial conformation. (http://lorentz.immstr.pasteur.fr/decoys/submission.php) (Lindahl et al. 2006). Based on the motion of the first five, non-trivial (non-zero eigenvalue), normal modes, 1000 decoy structures were generated from random linear combinations of movements along the five mode eigenvectors. These five modes embody TMS movements that appeared to influence binding crevice geometry. Vibrational modes beyond the lowest five frequencies were excluded because they involved motion in the non-binding cleft regions or relatively low amplitude motions in the TM core region. For the resulting 1000 decoys generated for each receptor we rebuilt all-atom structures on the Cα frames. Backbone and side chain atoms were built onto the fixed Cα template with Modeller 9v1, followed by minimization, a short 15 ps MD run, and minimization with Cα atoms fixed for each procedure. The structure was then minimized to convergence with positional restraints on the Cα carbons in gromacs, as described above. For use in docking, the 1000 rebuilt and minimized decoys for each receptor were clustered, as described above, into a more manageable subset of 24–25 representative conformers.</p><!><p>The ligands were constructed in Discovery Studio (Accelrys, Inc., San Diego, CA, USA) and geometry optimized in Gaussian03 with ab initio quantum mechanical calculations using the Hartree-Fock 6-31G** basis set (Frisch et al. 2004). The partial charges were set according to the automated Gasteiger partial charge assignment of AutoDockTools 1.4.5 (The Scripps Research Institute, La Jolla, CA, USA). Each ligand was docked 25 times to each receptor conformation (24–25 conformers) using the default Lamarckian genetic algorithm search routine in AutoDock 4.0 (Morris et al. 1998) with non-default parameters for the genetic algorithm population size (increased to 500) and maximum number of energy evaluations (ramped up to 2.5 × 106). Selective side chain flexibility was allowed within the docking routine: the rotation of dihedrals in the side chains of 2.61, 3.28, 3.29, 3.32, and 6.51 were explored because of their apparently critical positions in the ligand binding site and influences on cleft shape. After docking with each receptor conformation, poses were clustered using a Root Mean Square Deviation (RMSD) tolerance of 3.0 Å and then ranked by mean energy. Poses with reinforced ionic interaction between the ligand's protonatable amine and the conserved aspartate at position 3.32 (D3.32) were visually screened for mode-1 (Kortagere et al. 2004) orientations in the binding cleft (see Results for a description). Complexes with poses meeting these criteria were included in the subset used to obtain the contact-likelihood matrices (CLMs) from the intermolecular contacts extracted with Ligplot v4.4.2 (London, UK) (Wallace et al. 1995). PyMOL v0.99 (DeLano Scientific LLC, San Carlos, CA, USA) was used for rendering figures.</p><!><p>To characterize the subtype-selective binding domain in a D2 receptor background, we tested an extensive panel of D4-selective 1,4-DAPs that were previously examined in the D4 receptor background (Kortagere et al. 2004). When mutant D2-FV3.28-3.29LM and D2-V2.61F + FV3.28-3.29LM receptors were transiently expressed in COS-7 cells, the levels of expression and the affinities for the moderately D2-selective 1,4,-DAP [3H]MSP were comparable with the wild-type D2 receptor (1.1-fold to 1.3-fold and 1.7-fold to 2.3-fold, respectively, Floresca et al. 2005). The affinities of the mutant D2 receptors for the agonist (−)-quinpirole, which lacks a 1,4-DAP structural motif, were similar to those of the wild-type D2 receptor (1.2-fold to 3.8-fold, Fig. 1 and Table 1). The small to moderately improved affinities (4.8-fold to 22-fold) observed for the mutant D2-FV3.28-3.29LM receptor for five of the 1,4-DAPs [3-{[4-(4-chlorophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine] (L-745,870, also known as CPPMA, which stands for chlorophenylpiperazinyl methylazaindole), L-750,667, 2-[4-(4-chlorophenyl)piperazin-1-ylmethyl1]pyrazolo[1,5-a]pyridine (FAUC213), 2-amino-benzoic acid 1-benzyl-piperidin-4-yl ester (Ro 61-6270), RBI-257] improved further (21-fold to 292-fold) for the mutant D2-V2.61F + FV3.28-3.29LM receptor (Table 1). The two remaining 1,4-DAPs 2-phenyl-4(5)-[4-(2-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazole (NGD 94-1) and N-[[4-(2-cyanophenyl)-1-piperazinyl] methyl]-3-methylbenzamide (PD168,077)] had higher affinities for the mutant D2-FV3.28-3.29LM receptor (13-fold and 34-fold, respectively), than for the mutant D2-V2.61F + FV3.28-3.29LM receptor (4.8-fold to 6.4-fold, respectively) whose affinities were closer to the wild-type D2 receptor (Table 1). RBI-257 and L-750,667, the compounds with the largest improvements in affinity for the D2-V2.61F + FV3.28-3.29LM receptor relative to the wild-type D2, were selected for further characterization in functional assays.</p><p>Although identification of binding site residues that could impart 1,4-DAP recognition and D2/D4 preference was important, we were more interested in characterizing changes in subtype-specific function related to the subtype-selective binding domain of the D2 receptor. COS-7 cells, while appropriate for binding experiments, were unable to inhibit cAMP formation through activation of D2-like dopamine receptors (data not shown). Therefore the HEK293 line, which lacks endogenous dopamine receptors but contains G proteins capable of mediating the cAMP responses of transfected D2 or D4 dopamine receptors (Gazi et al. 1998; Chemel et al. 2006) was selected for functional assessments. Wild-type D2 and mutant D2-V2.61F + FV3.28-3.29LM receptors were stably expressed in HEK293 cells and characterized by receptor binding studies. The cell surface density of dopamine receptors expressed in several stable HEK293 clones was determined by [3H]MSP saturation isotherm analysis. The corresponding [3H]MSP affinity values (KD) were 0.78 ± 0.39 nM for the wild-type D2 receptor and 1.6 ± 0.76 nM for the D2-V2.61F + FV3.28-3.29LM receptor. Note that while absolute affinity values for [3H]MSP were quite different for wild-type D2 and mutant D2-V2.61F + FV3.28-3.29LM receptors expressed in COS-7 and HEK293 cells, the relative difference between wild-type and mutant D2 receptors within a cell type were essentially the same (2.3-fold vs. 2.1-fold, respectively). To limit any ambiguity caused by unequal receptor expression, clones expressing wild-type or mutant receptors were matched by receptor density. HEK293 cell lines expressing 4.6 ± 1.6 pmole/mg protein of wild-type D2-WT receptor or 4.6 ± 2.5 pmole/mg protein of mutant D2-V2.61F + FV3.28-3.29LM receptor were analyzed for their ability to inhibit cAMP formation.</p><p>Because RBI-257 had strikingly different affinity for the wild-type and mutant D2 receptors, preliminary tests to determine the most appropriate concentration range for investigating the antagonism of (−)-quinpirole function at the wild-type D2 and mutant D2-V2.61F + FV3.28-3.29LM receptors were performed (data not shown). Based on these results, the following concentrations of RBI-257 were selected: 0.2 and 2 µM for the D2-V2.61F + FV3.28-3.29LM receptor and 2 and 20 µM for the D2-WT receptor. Similar tests performed for L-750,667 resulted in the selection of 0.5 µM L-750,667 as an appropriate single concentration for comparing functional antagonism of (−)-quinpirole at the wild-type D2 and mutant D2-V2.61F + FV3.28-3.29LM receptors. Dose–response curves demonstrated that the mutant receptor had 4.3-fold lower potency and 18% less efficacy for the agonist (−)-quinpirole than did the wild-type D2 receptor (Table 2). Further, the right shift in (−)-quinpirole potency induced by RBI-257 occurred at a much lower RBI-257 concentration in the mutant D2-V2.61F + FV3.28-3.29LM than in the wild-type D2 receptor: a >5000-fold potency shift was observed in the presence of 2 µM RBI-257 for the mutant D2-V2.61F + FV3.28-3.29LM receptor with no significant shift for the wild-type D2 receptor (Fig. 2a and b and Table 2). However, a 10-fold higher concentration of RBI-257 was able to significantly shift the potency (120-fold) of (−)-quinpirole for the wild-type receptor (Table 2 and Fig. 2a). A similar pattern of shifted (−)-quinpirole potency was observed for L-750,667 (Table 2 and Fig. 2d). Neither RBI-257 nor L-750,667 had statistically significant effects on the efficacy of (−)-quinpirole (≤ 20% effect, Table 2 and Fig. 2). No attempt was made to quantify the functional antagonism of these ligands as pKb values (Schild analysis) because D4 selective ligands and (−)-quinpirole do not compete for the same binding site (Table 1) which is an assumption that must be met to yield meaningful values (Kenakin 1997).</p><p>Assays of cAMP accumulation designed to differentiate the functional properties of D4-selective 1,4-DAPs revealed that RBI-257 was an antagonist at the wild-type D2, wild-type D4, and the mutant D2-V2.61F + FV3.28-3.29LM receptors (Fig. 3b and d). In contrast, L-750,667, which acted as an antagonist at the wild-type D4 and mutant D2-V2.61F + FV3.28-3.29LM receptors, exhibited partial agonism at the wild-type D2 receptor (Fig. 3b–d). Neither (−)-quinpirole nor L-750,667 was able to reduce forskolin-stimulated cAMP accumulation in untransfected HEK293 cells (Fig. 3a), i.e., those lacking dopamine receptors. Further confirmation of the partial agonist properties of L-750,667 at the wild-type D2, but not the mutant D2-V2.61F + FV3.28-3.29LM receptor, was established by evaluating the effect of increasing concentrations of L-750,667 at a fixed 60 nM concentration of (−)-quinpirole (Fig. 4). At higher doses in the mutant D2-V2.61F + FV3.28-3.29LM receptor, L-750,667 completely reversed the (−)-quinpirole-stimulated functional response confirming functional antagonism of the mutant. In contrast, the highest concentrations of L-750,667 were only able to partly decrease (−)-quinpirole-stimulated wild-type D2 receptor inhibition of cAMP formation. This reduced maximal functional response confirmed that L-750,667 was a partial agonist of the wild-type D2 receptor. The EC50 for antagonism of the (−)-quinpirole functional response by L-750,667 was 3.2-fold higher for the mutant than the wild-type receptor (0.57 ± 0.27 and 1.9 ± 2.3 µM respectively, Fig. 4).</p><p>To examine the role that residues conferring D4/D2 selectivity have on the positioning of 1,4-DAPs in the binding cleft, several 1,4-DAPs (L-750,667, RBI-257, NGD 94-1, and PD168,077) were docked into wild-type D2, mutant D2-V2.61F + FV3.28-3.29LM, and wild-type D4 homology models. The resulting complexes (600–625) were screened using two geometric criteria: (i) an H-bond reinforced ionic interface formed by the protonatable amine group of the ligand interacting with the conserved aspartate at position 3.32 of biogenic amine receptors (for review, see Floresca and Schetz 2004) and (ii) pose orientations where the short arm of the 1,4-DAP (Arm-B, Kortagere et al. 2004) was directed, in accordance with prior experimental evidence from our lab, at the region of TMS 2 and 3 rather than TMS 5 and 6 (Kortagere et al. 2004). These criteria focused our investigations to the ligand poses that were most suitable for residue contact analysis.</p><p>When ligand poses that met our criteria were examined, consistent patterns of intermolecular contacts emerged that reconciled some of the observed selectivity features attributed to positions 2.61, 3.28, and 3.29. The poses observed for L-750,667 in both the mutant D2-V2.61F + FV3.28-3.29LM and wild-type D4 receptors directed the p-iodophenyl ring of Arm-B toward F2.61, a resident of the cleft formed by TMS 2, 3, and 7 (Fig. 5a top and middle panels). A similar orientation was observed for L-745,870, the p-chloro analog of L-750,667 in the D2-V2.61F receptor (Ericksen et al. 2009). However, despite similarities between the ligand orientation and contact residues in wild-type D4 and mutant D2-V2.61F + FV3.28-3.29LM receptors in the wild-type D2 receptor, L-750,667 was rotated counter-clockwise in the binding pocket (from an extracellular perspective) which allowed the p-iodophenyl ring to make significant contact with the phenylalanine of 3.28 within the TM2/3 cleft (Fig. 5a). These changes in the preferred binding configuration for L-750,667 and RBI-257 (5B, right panel; wire model not shown) suggest that positions 2.61, 3.28, and 3.29 provide recognition features integral to positioning 1,4-DAPs with p-halogenated Arm-B moieties in the binding pocket.</p><p>To quantitatively compare the observed intermolecular contact distributions for 1,4-DAPs docked to the receptor models, tendencies within a ligand–receptor pairing for ligand–residue interactions were plotted as CLMs (Fig. 5b). Each CLM was constructed from the sum of specific ligand–residue contacts (< 3.9 Å) normalized against the total amount of residue contacts (< 3.9 Å) achieved within the entire subset of ligand–receptor poses that passed the screening criteria (see details above). For L-750,667 and RBI-257, the distribution of CLM index values was better correlated between the wild-type D4 and mutant D2-V2.61F + FV3.28-3.29LM receptors (correlation = 0.867), than the wild-type D2 and mutant D2-V2.61F + FV3.28-3.29LM receptors (correlation = 0.723). This showed quantitatively that the distribution of contacts achieved for poses of L-750,667 in the mutant D2-V2.61F + FV3.28-3.29LM receptor were more like the poses observed at wild-type D4 receptor and less like the poses observed at the wild-type D2 receptor. A similar trend (0.863 vs. 0.806) in the CLM was observed for RBI-257. While this trend was consistent for p-halogenated 1,4-DAPs (L-750,667 and RBI-257) the results for o-electronegative 1,4-DAPs (NGD 94-1 and PD168,077) exhibited complex patterning of the CLM in which mutant D2-V2.61F + FV3.28-3.29LM receptor poses were dissimilar to poses in both wild-type D2 and wild-type D4 receptors. These findings for NGD 94-1 and PD168,077 suggested that 1,4-DAPs with an ortho-electronegative Arm-B group did not adopt D4-like binding modes in the mutant D2-V2.61F + FV3.28-3.29LM receptor and that mutations of the TM2/3 microdomain were not sufficient for conferring ortho-electronegative 1,4-DAP selectivity to the mutant receptor. Our observation was supported by the revertant affinities of NGD 94-1 and PD168,077 for the mutant D2-V2.61F + FV3.28-3.29LM receptor, meaning that this mutant's affinity profile looked more like the wild-type D2 receptor than did the mutant D2-FV3.28-3.29LM (Table 1).</p><p>Despite specific differences in ligand interaction between receptor subtypes, Fig. 5b revealed a general qualitative similarity within the CLMs. For 1,4-DAPs, the contact residues with the strongest ligand–residue interactions were typically the following positions (in order of general prominence in contact index value): V/F2.61, V/M3.29, F6.51, D3.32, F/L3.28, V3.33, T7.39, V5.39, L4.61, Y/V7.35, H6.55, F/Y5.38, S7.36, S5.42, Y7.43, C3.36, W7.40, V2.57, C3.25, and L/S2.64. Interestingly, 2.61, 3.28, and 3.29 represented three of the top five contact sites in terms of CLM index values and comprised three of the six contact positions that varied in residue identity between the wild-type D2 and wild-type D4 receptors.</p><!><p>The three amino acid TM2/3 microdomain that forms the extracellular portion of the binding site crevice has been suggested, on the basis of radioligand binding studies, to be a site of interaction for ligands with high selectivity for the D4 subtype of dopamine receptor (Simpson et al. 1999; Schetz et al. 2000; Kortagere et al. 2004). Recognition that the receptor background (i.e., D2 vs. D4), ligand structures, and binding reaction conditions (Floresca et al. 2005; Ericksen et al. 2009) greatly influenced the experimental outcome of previous TM2/3 microdomain studies (Simpson et al. 1999; Schetz et al. 2000; Kortagere et al. 2004) lead us to ask whether the difference in the observed binding affinities for D4 selective ligands were the result of localized changes in the TM2/3 microdomain or global changes in the receptor complex. It was reasoned that rigorous assessment of the overall state of the mutant receptor should include a measure of its functional properties relative to the wild-type receptor, as has only been carried out here. Assessment of the functional properties of a combined TM2/3 reciprocal D2 mutant receptor (D2-V2.61F + FV3.28-3.29LM) revealed improved sensitivity of the mutant receptor for the D4-selective ligands L-750,667 and RBI-257. Further, these ligands produced the same functional outcome at both the D2 mutant and wild-type D4 receptors. In contrast, non-selective ligands, such as MSP and (−)-quinpirole, were insensitive to these TM2/3 microdomain alterations. These findings demonstrated that the three amino acids exerted localized effects while regulating the affinity and function of D4-selective ligands. A molecular docking technique that permits flexibility in the receptor backbone was used to enhance the accommodation of 1,4-DAPs into representations of the receptor structure. Consistent with the empirical results, the docking studies suggested that the three amino acid TM2/3 microdomain accounts not only for a large portion of the differences in receptor phenotypes, but also for a large portion of the differences in steric contacts between D4-selective ligands, L-750,667 and RBI-257, and wild-type or mutant dopamine receptors.</p><p>Similar to D2 and D4 dopamine receptors, positions 2.61, 3.28, and 3.29 are implicated in various biogenic amine receptor–ligand interaction domains. For example, in muscarinic receptors positions 2.61, 3.28, and 3.29 are significant mediators of muscarinic ligand selectivity and function (Drubbisch et al. 1992; Matsui et al. 1995; Lu and Hulme 1999). However, the pattern of recognition between ligands with selectivity for D1 versus D2 receptor subtypes appears to be more complex. In the macaque D1 dopamine receptor, reciprocal mutations at position 3.28 with residues of the rat D2L receptor revealed moderate to large increases in affinity for the D2-like selective ligands spiperone, domperidone, and YM-09151-02 (224-fold, 45-fold, and 24-fold, respectively), but less than 4-fold changes in affinity for D2-like selective ligands haloperidol, piquindone, raclopride, sulpiride, and tropapride, as well as the D1-like selective ligand SCH23390 (Lan et al. 2006). However, the reciprocal mutant D2 receptor, D2-F3.28W, did not replicate the substantial changes in affinity observed for the mutant D1 receptor, i.e., less than a 2-fold change for any of the listed ligands.</p><p>Positions 2.61, 3.28, and 3.29 play an important role in rhodopsin folding and conformational stability. For instance, a T2.61I mutation causes congenital night blindness (al-Jandal et al. 1999), while a G3.29D mutation results in retinitis pigmentosa (Millan et al. 1995). The increased reactivity of both the T2.61I and E3.28A mutants to hydroxylamine in the dark state combined with decreased meta II state decays suggests that these mutant receptors exhibit pathological instability of the rhodopsin resting-state (Sakmar et al. 1991; Han et al. 1996; Ramon et al. 2003). The poorly expressed mutants E3.28V, E3.28I, E3.28M, E3.28W, and T2.61K did not reconstitute to form pigments in the presence of 11-cis-retinal (Han et al. 1996; Ramon et al. 2003). These findings demonstrated that positions 2.61, 3.28, and 3.29 were located in conformationally sensitive regions of rhodopsin. In the case of 2.61 and 3.28, the proximity of the retinal Schiff base allows for potential proton exchange with an appropriate amino acid residue. This releases 11-cis-retinal from the binding pocket in a manner analogous to the dissociation of a ligand from a biogenic amine receptor. As a whole, these experiments demonstrate the importance of these three positions for understanding ligand interactions with certain biogenic amine receptors.</p><p>While a great deal had been revealed in previous studies concerning the discriminant structural features of 1,4-DAP ligands and the receptor residues governing the strength of ligand interactions with specific D2 or D4 receptor microdomains, virtually nothing was known in this context about receptor activation states (Floresca and Schetz 2004). Thus, we measured the functional properties of a combined TM2/3 reciprocal mutation D2 receptor and report here for the first time that the D2-V2.61F + FV3.28-3.29LM mutant not only has increased affinity for the D4-selective ligands L-750,667 and RBI-257, but also requires a much lower concentration of these antagonists to produce a rightward shift of the agonist functional response. We observed that both of these ligands exert the same functional effects on the D2-V2.61F + FV3.28-3.29LM mutant as they do on the wild-type D4 receptor. In the case of L-750,667, this amounts to conversion of the D2-V2.61F + FV3.28-3.29LM mutant from a D2 receptor background that was activated by this ligand to one like the wild-type D4 receptor that was not. The switch in the functional effect of L-750,667 for the D2-V2.61F + FV3.28-3.29LM mutant was not because of a concentration effect, because L-750,667 had higher affinity and potency for this mutant than it did for the wild-type D2 receptor. Further, the switch in functional effect for the mutant persisted at dose ranges well beyond that needed to produce an observable reversal of (−)-quinpirole's functional effect for the mutant or the wild-type D2 receptor. In contrast to L-750,667, the functional profile of RBI-257 was not changed in the mutant receptor; however, since RBI-257 is an antagonist at both the D2 and D4 receptor subtypes, no change in functional properties was expected for the mutant receptor.</p><p>To gain a molecular perspective on the interactions of 1,4-DAPs with the dopamine receptor subtypes, we docked several 1,4-DAPs into our initial wild-type D4, wild-type D2, and mutant D2-V2.61F + FV3.28-3.29LM receptor homology models. However, we initially failed to observe any 1,4-DAP poses satisfying our experimental interaction criteria. This failure was attributed to the limitations of our initial docking strategy. In general, ligand docking strategies have been plagued by two major limitations that hinder their applicability and accuracy (Perola et al. 2004; Sousa et al. 2006). One problem is the poor performance of scoring functions which rank the likelihood of ligand–receptor complex geometries by attempting to estimate the relative pose energies. The other major problem (one we appeared to encounter here) is the static receptor approximation as currently implemented in grid-based docking approaches. Lately, some progress has been made in addressing this latter issue by expanding the ligand docking search over multiple receptor conformations based on NMA (Cavasotto et al. 2005; Lindahl and Delarue 2005). In a strategy similar to that used by Cavasotto et al. we explored relatively small backbone conformational changes by docking not into merely one receptor conformer, but by docking into an ensemble of receptor conformers generated by movements along a combination of the lowest frequency normal mode vectors. By incorporating receptor backbone flexibility into our docking study, we were able to identify 1,4-DAP binding poses that produced the expected ionic interaction with D3.32, and furthermore, were able to utilize this well-established interaction as a selection criterion (Floresca and Schetz 2004) rather than only relying on the scoring function.</p><p>The poses that resulted from the enhanced strategy, in agreement with the current data and related studies of D2 and D4 dopamine receptors (Simpson et al. 1999; Schetz et al. 2000; Kortagere et al. 2004), suggested that positions 2.61, 3.28, and 3.29 are primary determinants of 1,4-DAP recognition in the D2 dopamine receptor. These three positions accounted for a large proportion of the intermolecular contacts made by docked 1,4-DAPs. For L-750,667 and RBI-257, positions 2.61, 3.28, and 3.29 appeared to be responsible for mimicry of wild-type D4 binding in the D2-V2.61F + FV3.28-3.29LM receptor. For Ro 61-6270, L-745,870, and 2-[4-(4-chlorophenyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine (FAUC213), a similar pattern holds: the increase of affinity with the addition of a phenylalanine mutation at 2.61 suggested a similar process of interaction for all five of these ligands at the mutant D2-V2.61F + FV3.29-3.29LM receptor (Table 1). However, this trend did not hold true for NGD 94-1 and PD168,077. These ligands lost affinity when the valine at 2.61 was mutated in the mutant D2-FV3.28-3.29LM receptor to create the mutant D2-V2.61F + FV3.29-3.29LM receptor. Careful examination of ligand molecular structure revealed that ligands possessing para-halogenated or unhalogenated Arm-B aryl rings exhibited a trend of increased affinity when more subtype selective amino acids were mutated. This trend did not hold true for ligands with ortho-electronegative groups (ring nitrogens or nitrile groups).</p><p>From close inspection of the CLMs (Fig. 5b), it is interesting to note that the differences in ligand positioning (Fig. 5a) could potentially explain functional differences for certain 1,4-DAPs. For example, contact between the D2 receptor and the pyrrolopyridine ring of L-750,667 yielded the index values observed for residues F5.47, W6.48, F6.51, and F6.52 (comprising 19.6% of total intermolecular contacts). These contact positions are thought to influence the rotamer state of W6.48 and thus play a role in triggering early steps in the cascade of events leading to receptor activation (Lin and Sakmar 1996; Ebersole and Sealfon 2002). The CLM index values for these four residues decreased for the D2-V2.61F + FV3.28-3.29LM mutant (12.2%) and wild-type D4 (12.4%) in agreement with the finding that L-750,667 lost partial agonism in the mutant D2 receptor (Fig. 3c). While it is speculative to suggest a role between docking contacts and receptor efficacy, it remains quite likely that ligand positioning plays a key role in receptor activation. More informed conclusions may be drawn from assessing the dynamic nature of ligand–receptor interactions to show how the 'trigger' positions are specifically affected by a given ligand's occupancy. In summary, substitution of amino acids in the TM2/3 microdomain of the D2 receptor for the corresponding amino acids in the D4 receptor yielded a mutant D2-V2.61F + FV3.28-3.29LM receptor with pharmacology and function that reflected that of the wild-type D4 receptor.</p>