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NEUROPHARMACOLOGY
Program in Genetics, Sackler School of Graduate Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts (M.A.A., A.S.K.); and Molecular Pharmacology Research Center, Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, Massachusetts (Y.R., M.B., A.S.K.)
Received November 1, 2006; accepted January 2, 2007.
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Abstract |
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-adrenergic receptor. Dopamine receptors have been divided into two subgroups, D1- and D2-like receptors, based on amino acid homology as well as biochemical and pharmacological properties. The D1 and D5 subtypes (classified as D1-like receptors) signal primarily through Gs, whereas the D2, D3, and D4 subtypes (classified as D2-like receptors) signal predominantly through Gi/o (Missale et al., 1998
; Emilien et al., 1999; Vallone et al., 2000).
Because of the inherent difficulties in crystallizing membrane proteins, including GPCRs high-resolution structural information is only available for one class A family member, rhodopsin (Gether, 2000). As an alternative approach to understanding D2 receptor function, a variety of molecular and pharmacologic approaches have been utilized. Several laboratories have used the substituted cysteine accessibility method (SCAM) to map residues that project into the hD2R ligand pocket. By systematically substituting amino acids in transmembrane domains 2, 3, 4, 5, 6, and 7 with cysteines and reacting the altered receptors with charged sulfhydryl-specific methanethiosulfonate derivatives, the accessibility of these mutated residues in the predicted binding crevice was determined (Javitch et al., 1994, 1995a,b, 1998, 1999, 2000; Fu et al., 1996; Shi et al., 2001). SCAM analysis along with a series of complementary approaches (e.g., studies of chimeric receptors and point mutants, as well as computer-based modeling using the known rhodopsin structure as a template) enabled the identification of amino acids that line the putative binding pocket of the hD2R (Neve et al., 1991; Mansour et al., 1992; Javitch et al., 1994, 1995a,b, 1996, 1998, 1999, 2000; Kozell et al., 1994; Naylor et al., 1995; Fu et al., 1996; Wilcox et al., 1998, 2000; Simpson et al., 1999; Coley et al., 2000; Ballesteros et al., 2001; Shi et al., 2001). These studies identified single residues and multiple amino acid combinations that play a role in determining ligand affinity (Javitch et al., 1996; Alberts et al., 1998; Simpson et al., 1999; Schetz and Sibley, 2000). In contrast, the contribution of hD2R amino acids as potential determinants of hD2R agonist function has been less extensively studied.
Our laboratory cloned and pharmacologically characterized the Drosophila D2-like receptor (DD2R), the first known invertebrate homolog of the hD2R. Like its mammalian counterpart, the DD2R signals through the inhibitory G protein, Gi, shows highest potency for dopamine (versus other biogenic amines) and is fully activated by bromocriptine with nanomolar potency (Hearn et al., 2002). Bromocriptine is among a group of synthetic agonists that are used clinically for the treatment of Parkinson's disease. In contrast to this ligand, other drugs in this group, including piribedil, pergolide, and ropinirole, have little or no activity at the fly DD2R. The differential response to synthetic hD2R agonists, in combination with the relatively high conservation of the predicted fly binding pocket when aligned with its human counterpart (48/73 identical amino acids), suggests a limited number of candidate residues that confer synthetic ligand potency/efficacy. Consistent with this prediction, we show by exchanging homologous residues between the human and fly D2-like receptors that a combination of amino acids in the ligand pocket that are not conserved between the two receptors underlie the species-specific differences in pharmacology. Comparison between a mammalian versus a corresponding non-mammalian GPCR thus enabled the identification of a novel combination of residues that are molecular determinants of ligand activity at both the human and fly D2 receptors.
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Materials and Methods |
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-[(methylthio)methyl]-6-propylergoline methanesulfonate salt), ropinirole hydrochloride (diethyl[2-(2-oxo-2,3-dihydro-1H-indol-4-yl)ethyl]ammonium chloride), dopamine hydrochloride (2-(3,4-dihydroxyphenyl)ethylamine hydrochloride), and forskolin (7-acetoxy-8,13-epoxy-1
,6,9-trihydroxylabd-14-en-11-one). All the stock solutions of ligands were prepared in deionized water with the following exceptions: bromocriptine, which was prepared in ethanol, and pergolide and forskolin, which were prepared in dimethyl sulfoxide. Aliquots of ligands were stored frozen at –80°C and were diluted in serum-free media immediately before use. Comparison of Amino Acid Sequences. An amino acid alignment of the DD2R with the hD2R was performed using ALIGN X with the blosum62mt2 scoring matrix (Vector NTI Suite, version 5.5, InforMax, North Bethesda, MD). An alignment comparing the hD2R with mammalian and invertebrate species orthologs was performed using ClustalW version 3.2 (http://workbench.sdsc.edu).
Generation of Mutant Receptors. The fly and human D2 receptors (506 and 443 amino acids, respectively) (Grandy et al., 1989; Hearn et al., 2002) were each subcloned into the expression vector pcDNA1.1 (Invitrogen, Carlsbad, CA). Mutants were generated by modifying the coding region of either the wild-type human or fly D2 receptors. Amino acid substitutions were introduced via oligonucleotide-directed, site-specific mutagenesis, as described previously (Beinborn et al., 1993). Oligonucleotides of interest were synthesized at the Tufts University DNA Synthesis Core Facility (Boston, MA). Introduction of desired mutations was confirmed by restriction enzyme analyses followed by dideoxynucleotide sequencing of the entire protein-coding region using an automated ABI 37X DNA sequencer (Applied Biosystems, Foster City, CA).
Cell Culture. Human embryonic kidney (HEK) 293 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Biotechnics Research Inc., Lake Forest, CA) and 100 units/ml penicillin-streptomycin (Invitrogen) at 37°C in a humidified environment containing 5% CO2.
Transfection and Luciferase Assay. HEK293 cells were plated (5000–7000/well) onto 96-well Primaria plates (BD Biosciences, Bedford, MA). Cells were transiently transfected using Lipofectamine reagent (Invitrogen) with cDNA encoding 1) either wild-type or mutant receptors (hD2R or DD2R), 2) a reporter gene construct consisting of five tandem repeats of the serum-response element (SRE5x) ligated upstream from a reporter gene encoding firefly luciferase (Feuerbach et al., 2000), and 3) Gq5i, a chimeric G protein consisting of Gq with substitution of the five corresponding carboxyl-terminal amino acids of Gi (Conklin et al., 1993; Elshourbagy et al., 2000). The five amino acids at the carboxyl terminus of Gq5i are sufficient to enable interaction with Gi-coupled receptors (Conklin et al., 1993; Coward et al., 1999). The Gq domains of Gq5i enable receptor-mediated signaling to the Gq pathway, which can in turn be detected using the SRE5x-luciferase reporter gene construct.
As a complementary approach to examine Gi-mediated signaling, cells were stimulated with forskolin, and dopamine receptor-mediated inhibition of cAMP-induced transcription was examined. For these studies, cells were transiently transfected with cDNA encoding 1) either wild-type or mutant receptors (hD2R or DD2R) and 2) a reporter gene construct consisting of six tandem repeats of the cAMP-response element (CRE6x) (George et al., 1998; Kemp et al., 1999) ligated upstream from a reporter gene encoding firefly luciferase (Feuerbach et al., 2000).
Twenty-four hours after transfection, cells were exposed to ligand for 3 h in serum-free medium either in the presence or absence of forskolin (as indicated in the figure legends). The cells were then lysed, and luciferase activity was quantified using LucLite reagents (PerkinElmer Life and Analytical Sciences, Wellesley, MA). All measurements were repeated in at least three separate experiments, each performed in triplicate. Ligand potencies were assessed by stimulating receptor-expressing cells with increasing agonist concentrations. pEC50 values were calculated from concentration-response curves using computerized nonlinear curve fitting (Prism 3.0; GraphPad Software, Inc., San Diego, CA). Percent efficacies were calculated by dividing the maximal light unit value of an agonist by the maximal value for bromocriptine at the corresponding receptor.
Statistical Analysis. All the statistical analyses were performed using one-way analysis of variance (ANOVA) and Dunnett's post-test (INSTAT; GraphPad Software, Inc.).
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Results |
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Comparison of hD2R and DD2R Reveals Candidate Molecular Determinants of Agonist Efficacy/Potency. As a first step toward identifying candidate residues that may underlie the pharmacological differences between the DD2R and hD2R, an amino acid alignment of the two GPCR was made. Residues that are conserved or different between the two receptors are indicated in Fig. 3. Seventy-three residues comprise the human D2 receptor pocket (Javitch et al., 1994, 1995a,b, 1998, 1999, 2000; Fu et al., 1996; Ballesteros et al., 2001; Shi et al., 2001). Within this pocket, only 25 amino acids are not conserved between the fly and human homologs. Among these, the 24 residues that are found in the outer two-thirds of the transmembrane domain pocket were examined in this study and are highlighted in Fig. 3. We hypothesize that species divergence in these amino acids is the most likely explanation for the distinct pharmacologies of the fly versus the human D2 receptor.
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). To assess all the candidate amino acids (24 residues), 13 mutants were generated; each included one to five substitutions. The mutants were each expressed in HEK293 cells and stimulated with increasing concentrations of agonist. Full efficacy of bromocriptine or dopamine and conserved potency for at least one of these agonists (within 3-fold of either the wild-type hD2R or DD2R values) was used as an index of intact tertiary structure of the mutant receptors. The pharmacologic profile of each of the variants satisfied these criteria. Two mutants (hD2R-IFV109FYI and hD2R-C118S) exhibited decreased potency for bromocriptine (Table 1) but displayed EC50 values for dopamine that were indistinguishable from the wild-type hD2R value (mean pEC50 ± S.E.M. for hD2R is 6.08 ± 0.24, for hD2R-IFV109FYI is 6.05 ± 0.27, and for hD2R-C118S is 5.84 ± 0.26; no significant differences by ANOVA). These findings suggest that the overall structure of the hD2R-IFV109FYI and hD2R-C118S mutants was conserved despite a slight decrease in bromocriptine potency.
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When the hD2R mutants were stimulated with pergolide, six of these constructs displayed a significant decrease in potency compared with the wild-type receptor (shown in boldface in Table 1). Thus, corresponding substituted residues were considered potential contributors to the pharmacological species differences between the fly and human receptors.
Selected Mutant DD2Rs Show an Increase in Agonist Potency/Efficacy. Further investigation focused on the six mutant human receptors that displayed a significant decrease in pergolide potency (Table 1). To explore whether converse substitutions confer increased agonist potency, hD2R residues were introduced in place of the corresponding fly amino acids. Each of the generated receptors maintained conserved potency for bromocriptine and/or dopamine, suggesting that the DD2R mutants retained intact tertiary structure (Table 2). Two mutants (DD2R-FA132WV and DD2R-FL136LE) displayed decreased bromocriptine potency (Table 2) but retained dopamine potency within 3-fold of the wild-type DD2R value (mean pEC50 ± S.E.M. for DD2R, 6.07 ± 0.26; for DD2R-FA132WV, 6.05 ± 0.24; and for DD2R-FL136LE, 6.68 ± 0.30; no significant differences by ANOVA). Four mutants (DD2R-S160C, DD2R-FA132WV, DD2R-IVl211LLF, and DD2R-LS239IV) showed an increase in pergolide potency; however, the change did not quite reach statistical significance (Table 2). Among these mutants with a tendency toward gain of function, the DD2R-S160C variant was chosen for further analysis because the complementary hD2R construct (hD2R-C118S) showed the most pronounced loss of function phenotype with regard to pergolide potency (Table 1). Taken together, the findings with the C118S and S160C substitution mutants provided preliminary evidence that the respective residues in DD2R and hD2R are important determinants of agonist potency/efficacy. To further explore this possibility, a series of additional Drosophila mutants was generated. Substitutions in each of these constructs included the S160C alteration in combination with one of the other putative gain of function mutations shown in Table 2. Of the five different constructs generated, two (DD2R-S160C/FA132WV and DD2R-S160C/IVL211LLF) showed a significant increase in both pergolide potency and efficacy versus the wild-type DD2R (Table 3). Findings with the two combination mutants suggested that a subset of the six substituted residues act in concert to define pergolide activity at the hD2R.
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A Triple Mutant Markedly Enhances the Potency of Pergolide at the DD2R. To further define the residues that are important determinants of pergolide activity, different combinations of amino acids were selected from S160C, FA132WV, and IVL211LLF. A series of six additional mutants (DD2R-A133V/S160C/I211L, DD2R-F132W/S160C/I211L, DD2R-A133V/S160C/V212L, DD2R-F132W/S160C/V212L, DD2R-A133V/S160C, and DD2R-F132W/S160C) was generated and assessed sequentially with bromocriptine, pergolide, piribedil, and ropinirole (data not shown). We focused on two of these constructs that conferred the most pronounced gain of function to compounds with either minor or no detectable activity at the wild-type fly DD2R. A triple amino acid substitution in the DD2R (DD2R-A133V/S160C/I211L) markedly increased pergolide efficacy and enhanced the potency of this compound to within 10-fold of the wild-type hD2R (Fig. 4). In addition, a double mutant (DD2R-A133V/S160C) increased the efficacy and potency of pergolide, albeit to a lesser degree than the triple substitution. Whereas piribedil and ropinirole fail to stimulate the wild-type fly DD2R, introduction of either the double or triple combination of human amino acids into the wild-type fly DD2R enabled these agonists to induce a limited degree of signaling (Fig. 4).
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Taken together, two complementary readouts of signal transduction (which rely on either recombinant Gq5i or endogenous Gi-mediated signaling) provide consistent results. The findings suggest that Val91, Cys118, and Leu170 (i.e., the residues that are represented by the double and triple amino acid substitutions discussed above) are important determinants of activity for selected synthetic agonists at the hD2R.
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Discussion |
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; Oksenberg et al., 1992; Beinborn et al., 1993; Cascieri et al., 1994) and provided an experimental basis for exploring the molecular determinants of ligand function. In contrast, known D2 receptor agonists seem to act comparably on mammalian D2 receptor orthologs (Grandy et al., 1989). Therefore, the similarity in the pharmacological profiles of mammalian D2 receptors does not enable these isoforms to be used as molecular probes to identify functionally important residues. Consistent with these observations, a comparison of mammalian D2 receptors (human versus mouse versus rat) reveals a fully conserved (100% identical) putative ligand pocket.
The cloning and the characterization of the DD2R (Hearn et al., 2002) presented a novel opportunity to identify synthetic agonist efficacy/potency determinants. Both the fly and human D2 receptors signal through Gi, and both are activated by dopamine and bromocriptine with relatively high potency. At the same time, these two receptors showed marked differences in sensitivity to piribedil, pergolide, and ropinirole (Fig. 2). The receptor ligand pockets show 66% identity and 77% similarity at the amino acid level, leaving a finite number of residues that may underlie the distinct pharmacological profiles.
Systematic exchange of residues between the fly and human D2 receptors ultimately led to the identification of a three amino acid combination (A133V/S160C/I211L) that when transferred from the hD2R into the fly receptor resulted in a marked increase in pergolide efficacy (converting this compound into a full DD2R agonist) and potency (to within 10-fold of the wild-type human value) (Fig. 4). These residue substitutions in the DD2R also increased the function of the two other species-selective synthetic agonists that were tested (i.e., piribedil and ropinirole), suggesting that these amino acids are functionally important to multiple structurally diverse ligands. The observed sequential increase in drug function with single versus double versus triple amino acid substitutions in the DD2R is consistent with current models of synthetic agonist-receptor interaction in which combinations of selected amino acids within a receptor's binding pocket are postulated to interact with ligands to define potency and/or efficacy (Javitch et al., 1996).
The most pronounced mutation-induced potency and efficacy increases in this study were observed with the agonist pergolide (Fig. 4). The observation that limited alterations in the DD2R binding pocket were sufficient to enhance pergolide function close to that of bromocriptine (which acts as a potent agonist at the fly receptor) suggests that the mode of action of these two ligands may be similar. Consistent with this hypothesis, an inspection of the structure of the ligands used in this study reveals that pergolide and bromocriptine are structurally related compounds and on this basis are classified as ergoline derivatives. In contrast, the two ligands that were less affected by the receptor mutations that were studied, piribedil and ropinirole, are both nonergots. Future studies will be needed to identify the additional pharmacological determinants that further contribute to potency/activity differences of piribedil and ropinirole at the fly versus human D2 receptor.
Our studies suggest that Val91, Cys118, and Leu170 are critical to synthetic agonist function at the hD2R. A model of the hD2R was proposed by Kalani et al. (2004) using computational techniques to predict the binding sites and relative binding energies of various dopaminergic ligands. Based on in silico analysis, a model was thus proposed in which Cys118 is among the residues that form a predominantly hydrophobic pocket where dopamine and class I (exemplified by clozapine) and class II (exemplified by haloperidol) antagonists bind to the receptor. Using the same approach, Val91 is modeled to lie in a largely hydrophobic pocket for class II antagonists (Kalani et al., 2004). It is of note that the predicted relevance of the Cys118 and Val91 for receptor-ligand interactions is in line with our experimental findings. However, according to the in silico model, Leu170 (which we found to be pharmacologically relevant) is not predicted to interact with any of the ligands that were studied. It is well established that only some of the residues forming the surface of a GPCR-binding crevice are in direct contact with agonists or antagonists, whereas others primarily play a structural role in stabilizing the overall pocket configuration (Javitch et al., 1996). Leu170 may fall into the latter class of amino acids.
In addition to modeling approaches, our conclusion that residues Cys118 and Val91 are important determinants of synthetic agonist potency/efficacy at the hD2R is also supported by complementary biochemical/pharmacological investigations. Javitch et al. (1994) have shown by SCAM analysis that covalently linking Cys118 to sulfhydryl-reactive reagents results in an inhibition of ligand affinity. Therefore, this transmembrane domain 3 amino acid was postulated to reside within the binding site crevice of the hD2R. In addition, serial substitutions in place of Cys118 (i.e., Lys, Ser, Met, and Ala) resulted in mutant receptors exhibiting variable reductions in the binding affinities of dopamine and a synthetic D2 receptor ligand, sulpiride. These observations led the authors to conclude that the residue occupying position 118 in the hD2R plays an important role in ligand binding affinity, an effect that depends on both the size and the charge of the corresponding side chain (Javitch et al., 1996). Our study extends these findings by showing that Cys118 is an important determinant not only of affinity but also of potency and efficacy for multiple structurally diverse drugs that are commonly used for the treatment of Parkinson's disease (i.e., pergolide, piribedil, and ropinirole).
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; Kortagere et al., 2004; Floresca et al., 2005). Valine 91 in the hD2R was found to be in the water-accessible surface of the receptor by SCAM analysis (Javitch et al., 1999). The potential relevance of the residue occupying position 91 was also inferred from studies on the D4 receptor. When the corresponding residue in the rat D4 receptor (F88V) was substituted with its hD2R homolog (Val91), there was a 100-fold decrease in affinity for the D4R-selective ligand L-750, 667 (Schetz et al., 2000). Conversely, when Val91 in the hD2R was substituted with phenylalanine (the corresponding residue in the hD4R), there was 
100-fold increase in affinity for the highly selective D4R compound CPPMA compared with the wild-type hD2R (Simpson et al., 1999). The latter observation, although focused on the binding properties of a D4R-selective compound, supports our conclusion that Val91 in the hD2R is an important determinant of receptor-ligand interaction.
An amino acid alignment comparing the hD2R with mammalian and invertebrate species orthologs (Fig. 7) highlights the residues that were found to be determinants of synthetic agonist activity (Cys118/Val91/Leu170). Of note, residue Cys118 in the hD2R receptor is conserved among mammalian D2 receptors, whereas its corresponding amino acid is a serine in both the fly DD2R and Caenorhabditis elegans D2 receptors. Residue Val91 is also highly conserved among mammalian receptors, whereas the corresponding residue in both the fly and C. elegans D2 receptors is an alanine. Leucine 170 is also conserved among the mammalian D2 receptors; however, in the fly and C. elegans D2-like receptors, it is an isoleucine and methionine, respectively. Variability in the amino acid sequence of a given mammalian GPCR subtype (e.g., the cholecystokinin type 2 and neurokinin type 1 receptors) has been previously used to identify residues underlying ligand affinity and/or efficacy differences between species (e.g., rat versus human, dog versus human) (Fong et al., 1992; Beinborn et al., 1993). The conservation of critical amino acids (i.e., Cys118, Val91, and Leu170) among mammalian D2 receptors precluded the use of these receptors in mapping the efficacy/potency determinants of synthetic ligands. In contrast, comparison of hD2R with the DD2R homolog provided a greater degree of divergence between the corresponding binding pockets. Thus, this strategy enabled the identification of functionally important amino acids that could not have been appreciated by comparison of only mammalian receptor isoforms.
In summary, our study shows that pharmacologic differences that occur between vertebrate and invertebrate receptor homologs may be used to map residues that are relevant to synthetic agonist binding and function. This approach revealed three amino acids in the hD2R that together serve as important potency/efficacy determinants for selected antiparkinsonian drugs. We suggest that parallel strategies utilizing other invertebrate orthologous GPCRs may have utility in exploring the interplay between receptor-selective ligands and their cognate GPCRs.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: hD2R, human dopamine D2 receptor; GPCR, G protein-coupled receptor; SCAM, substituted cysteine accessibility method; DD2R, Drosophila D2-like receptor; HEK, human embryonic kidney; SRE, serum-response element; CRE, cAMP response element; ANOVA, analysis of variance; L-750, 667, 3-{[4-(4-iodophenyl)piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine; CPPMA, (3-[4-(4-chlorophenyl)piperazin-1-yl]methyl-1H-pyrrolo[2,3-b]pyridine.
Address correspondence to: Alan S. Kopin, 750 Washington Street, Box 7703, Boston, MA 02111. E-mail: akopin{at}tufts-nemc.org
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