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Subtype-selective agents for the dopamine D3 receptor (D3R) have been considered as potential medications for drug addiction and other neuropsychiatric disorders. Medicinal chemistry efforts have led to the discovery of 4-phenylpiperazine derivatives that are >100-fold selective for the dopamine D3 receptor over dopamine D2 receptor (D2R), despite high sequence identity (78% in the transmembrane domain). Based on the recent crystal structure of D3R, we demonstrated that the 4-phenylpiperazine moiety in this class of D3R-selective compounds binds to the conserved orthosteric binding site, whereas the extended aryl amide moiety is oriented toward a divergent secondary binding pocket (SBP). In an effort to further characterize molecular determinants of the selectivity of these compounds, we modeled their binding modes in D3R and D2R by comparative ligand docking and molecular dynamics simulations. We found that the aryl amide moiety in the SBP differentially induces conformational changes in transmembrane segment 2 and extracellular loop 1 (EL1), which amplify the divergence of the SBP in D3R and D2R. Receptor chimera and site-directed mutagenesis studies were used to validate these binding modes and to identify a divergent glycine in EL1 as critical to D3R over D2R subtype selectivity. A better understanding of drug-dependent receptor conformations such as these is key to the rational design of compounds targeting a specific receptor among closely related homologs, and may also lead to discovery of novel chemotypes that exploit subtle differences in protein conformations.

Biased agonism offers an opportunity for the medicinal chemist to discover pathway-selective ligands for GPCRs. A number of studies have suggested that biased agonism at the dopamine D2 receptor (D2R) may be advantageous for the treatment of neuropsychiatric disorders, including schizophrenia. As such, it is of great importance to gain insight into the SAR of biased agonism at this receptor. We have generated SAR based on a novel D2R partial agonist, tert-butyl (trans-4-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)carbamate (4). This ligand shares structural similarity to cariprazine (2), a drug awaiting FDA approval for the treatment of schizophrenia, yet displays a distinct bias toward two different signaling end points. We synthesized a number of derivatives of 4 with subtle structural modifications, including incorporation of cariprazine fragments. By combining pharmacological profiling with analytical methodology to identify and to quantify bias, we have demonstrated that efficacy and biased agonism can be finely tuned by minor structural modifications to the head group containing the tertiary amine, a tail group that extends away from this moiety, and the orientation and length of a spacer region between these two moieties.

Novel analogs of L-prolyl-L-leucylglycinamide (PLG) were synthesized wherein the prolyl residue was replaced with other amino acids based on a 3,5-disubstituted proline scaffold. In some examples, the L-leucyl residue was also replaced by L-valine. These analogs were tested for their ability to enhance the binding of [(3)H]-N-propylnorapomorphine to short isoform of human dopamine D₂ receptors. Compounds 18b and 19b, increased [(3)H] NPA binding at concentrations between 10(-12) and 10(-9) M, which is similar to the effect of PLG in this assay and, provides evidences that these compounds are acting as allosteric modulators of dopamine D₂ receptors., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

It is known that schizophrenia patients are behaviorally supersensitive to dopamine-like drugs (amphetamine, methylphenidate). There is evidence for an increased release of dopamine, a slight increase of dopamine D2 receptors and an increase of dopamine D2High receptors in schizophrenia, all possibly explaining the clinical supersensitivity to dopamine. The elevation in apparent D2High receptors in vivo in schizophrenia matches the elevation in D2High receptors in many animal models of psychosis. The increased amounts of D2High receptors in psychotic-like behavior in animals may result from a loss of control of D2 by various factors. These factors include the rate of phosphorylation and desensitization of D2 receptors by kinases, the attachment of arrestin to D2 receptors, internalization of D2 receptors, the rate of receptor de-phosphorylation, formation of D2 receptor dimers, and GTP regulation by various GTPases. While at present there are no statistically significant associations of any of these controlling factors and their genes with schizophrenia, investigation of D2High receptors in schizophrenia will require a new radioligand in order to selectively label D2High receptors in vivo in patients. Finally, haloperidol reduces the number of D2High receptors that are elevated by amphetamine, indicating that this therapeutic effect may occur clinically., (© 2013.)

Dopamine-mediated neurotransmission plays an important role in relevant psychiatric and neurological disorders. Nowadays, there is an enormous interest in the development of new dopamine receptors (DR) acting drugs as potential new targets for the treatment of schizophrenia or Parkinson's disease. Previous studies have revealed that isoquinoline compounds such as tetrahydroisoquinolines (THIQs) and tetrahydroprotoberberines (THPBs) can behave as selective D2 dopaminergic alkaloids since they share structural similarities with dopamine. In the present study we have synthesized eleven 2,3,9- and 2,3,11-trisubstituted THPB compounds (six of them are described for the first time) and evaluated their potential dopaminergic activity. Binding studies on rat striatal membranes were used to evaluate their affinity and selectivity towards D1 and D2 DR and establish the structure-activity relationship (SAR) as dopaminergic agents. In general, all the tested THPBs with protected phenolic hydroxyls showed a lower affinity for D1 and D2 DR than their corresponding homologues with free hydroxyl groups. In previous studies in which dopaminergic affinity of 1-benzyl-THIQs (BTHIQs) was evaluated, the presence of a Cl into the A-ring resulted in increased affinity and selectivity towards D2 DR. This is in contrast with the current study since the existence of a chlorine atom into the A-ring of the THPBs caused increased affinity for D1 DR but dramatically reduced the selectivity for D2 DR. An OH group in position 9 of the THPB (9f) resulted in a higher affinity for DR than its homologue with an OH group in position 11 (9e) (250 fold for D2 DR). None of the compounds showed any cytotoxicity in freshly isolated human neutrophils. A molecular modelling study of three representative THPBs was carried out. The combination of MD simulations with DFT calculations provided a clear picture of the ligand binding interactions from a structural and energetic point of view. Therefore, it is likely that compound 9d (2,3,9-trihydroxy-THPB) behave as D2 DR agonist since serine residues cluster are crucial for agonist binding and receptor activation., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Magnetic resonance imaging (MRI) and positron emission tomography (PET) are two extremely important imaging modalities with unlimited tissue penetration. Molecular imaging is a field by which specific targets or biological processes are imaged. MRI, which is used for functional imaging and for the diagnosis of a broad range of pathologic conditions, suffers from limited specificity and intrinsically low sensitivity. One possibility to alleviate partially these limitations is to use contrast agents (CAs) and more importantly target-specific CAs. We have developed a modular synthesis of novel ligands and gadolinium(III)-based target-specific MRI CAs with high relaxivity and high binding affinity toward the dopamine receptors. The prepared ligands and MRI CAs are based on spiperone as targeting moiety. The prepared target-specific CAs can potentially be used for in vitro and possibly in vivo MR imaging of dopaminergic receptors. Importantly the ligands prepared using the modular approach presented in this paper may also be useful for other imaging modalities such as PET (or SPECT) by just replacing, at the last stage of the synthesis, the gadolinium cation by other metal cations having relatively long half-lives, such as (64)Cu, (89)Zr, (11)In, and more.

Dopaminergics of types 1 and 2 incorporating a conjugated enyne as an atypical catechol-simulating moiety were synthesized in enantiomerically pure form and investigated for their metabolic stability. Radioligand binding studies indicated high affinity to D2-like receptors. The test compounds were evaluated for their ability to differentially activate distinct signaling pathways. Measurement of D(2L)- and D(2S)-mediated [(35)S]GTPγS incorporation in the presence of coexpressed Gα(o) and Gα(i) subunits showed significantly biased receptor activation for several test compounds. Thus, the 2-azaindolylcarboxamide (S)-2a exhibited substantial functional selectivity for D(2S)-promoted G(o) activation over G(i) coupling. The most significant bias was determined for the triazolylalkoxy-substituted benzamide (S)-2c that displayed higher potency for G(o) activation than for G(i) coupling at the D(2L) subtype. Functional selectivity for β-arrestin recruitment over G(i) activation was observed for the biphenylcarboxamide (R)-1 and the 2-benzothiophenylcarboxamide (S)-2d, whereas the 2-substituted azaindole (S)-2a preferred β-arrestin recruitment compared to G(o) coupling.

A series of N-(2-methoxyphenyl)homopiperazine analogs was prepared and their affinities for dopamine D2, D3, and D4 receptors were measured using competitive radioligand binding assays. Several ligands exhibited high binding affinity and selectivity for the D3 dopamine receptor compared to the D2 receptor subtype. Compounds 11a, 11b, 11c, 11f, 11j and 11k had K(i) values ranging from 0.7 to 3.9 nM for the D3 receptor with 30- to 170-fold selectivity for the D3 versus D2 receptor. Calculated logP values (logP=2.6-3.6) are within the desired range for passive transport across the blood-brain barrier. When the binding and the intrinsic efficacy of these phenylhomopiperazines was compared to those of previously published phenylpiperazine analogues, it was found that (a) affinity at D2 and D3 dopamine receptors generally decreased, (b) the D3 receptor binding selectivity (D2:D3 K(i) value ratio) decreased and, (c) the intrinsic efficacy, measured using a forskolin-dependent adenylyl cyclase inhibition assay, generally increased., (Copyright © 2013. Published by Elsevier Ltd.)

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (K(i)) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPγS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Biochemical, histochemical and coimmunoprecipitation experiments have indicated the existence of antagonistic dopamine D2 (D2R) and neurotensin 1 (NTS1R) receptor-receptor interactions in the dorsal and ventral striatum indicating a potential role of these receptor-receptor interactions in Parkinson's disease and schizophrenia. By means of Bioluminiscence Resonance energy transfer (BRET(2)) evidence has for the first time been obtained in the current study for the existence of both D2LR/NTS1R and D2SR/NTS1R heteromers in living HEK293T cells. Through confocal laser microscopy the NTS1R(GFP2) and D2R(YFP) were also shown to be colocated in the plasma membrane of these cells. A bioinformatic analysis suggests the existence of a basic set of three homology protriplets (TVM, DLL and/or LRA) in the two participating receptors which may contribute to the formation of the D2R/NTS1R heteromers by participating in guide-clasp interactions in the receptor interface. The CREB reporter gene assay indicated that the neurotensin receptor agonist JMV 449 markedly reduced the potency of the D2R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the neurotensin agonist was found to markedly increase the quinpirole potency to activate the MAPK pathway as also studied with luciferase reporter gene assay measuring the degree of SRE activity as well as with ERK1/2 phosphorylation assays. These dynamic changes in D2R signaling produced by the neurotensin receptor agonist may involve antagonistic allosteric receptor-receptor interactions in the D2LR-NTS1R heteromers at the plasma membrane level (CREB pathway) and synergistic interactions in PKC activation at the cytoplasmatic level (MAPK pathway)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100/D-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor., (© 2013 American Institute of Chemical Engineers.)

Protein domains involved in receptor heteromer formation are disordered and rich in the amino acids necessary for the formation of noncovalent complexes (NCX). We present mass spectral NCX data from proteins and protein receptors' epitopes obtained by combining ion mobility (IM) and MALDI. We focus on NCX involved in heteromer formation occurring between epitopes of the Dopamine D2 (D2R) and Adenosine A2A receptors (A2AR) as well as D2R and the α2 nicotinic (NR) receptor's subunit. The IM data yield information on the gas phase conformation of the singly charged NCX which are observed either directly from MALDI or as codesorbed neutrals that are subsequently postionized by a time-delayed excimer laser pulse directed onto a portion of the neutral plume created by the MALDI desorption laser. Imaging mass spectrometry of the matrix/epitope dried droplet surface shows that the acidic and basic epitopes and their NCX are found to be spatially collocated within regions as small as 25 × 50 μm(2). Subtle differences in the relative abundance of protonated and cationized NCX and epitopes are measured in spatial regions near the sodium-rich outer border of the droplet.

Interest in structure-based G-protein-coupled receptor (GPCR) ligand discovery is huge, given that almost 30 % of all approved drugs belong to this category of active compounds. The GPCR family includes the dopamine receptor subtype D2 (D2DR), but unfortunately--as is true of most GPCRs--no experimental structures are available for these receptors. In this publication, we present the molecular model of D2DR based on the previously published crystal structure of the dopamine D3 receptor (D3DR). A molecular modeling study using homology modeling and docking simulation provided a rational explanation for the behavior of the arylpiperazine ligand. The observed binding modes and receptor-ligand interactions provided us with fresh clues about how to optimize selectivity for D2DR receptors.

Recent breakthroughs in crystallographic studies of G protein-coupled receptors (GPCRs), together with continuous progress in molecular modeling methods, have opened new perspectives for structure-based drug discovery. A crucial enhancement in this area was development of induced fit docking procedures that allow optimization of binding pocket conformation guided by the features of its active ligands. In the course of our research program aimed at discovery of novel antipsychotic agents, our attention focused on dopaminergic D2 and D1 receptors (D2R and D1R). Thus, we decided to investigate whether the availability of a novel structure of the closely related D3 receptor and application of induced fit docking procedures for binding pocket refinement would permit the building of models of D2R and D1R that facilitate a successful virtual screening (VS). Here, we provide an in-depth description of the modeling procedure and the discussion of the results of a VS benchmark we performed to compare efficiency of the ligand-optimized receptors in comparison with the regular homology models. We observed that application of the ligand-optimized models significantly improved the VS performance both in terms of BEDROC (0.325 vs 0.182 for D1R and 0.383 vs 0.301 for D2R) as well as EF1% (20 vs 11 for D1R and 18 vs 10 for D2R). In contrast, no improvement was observed for the performance of a D2R model built on the D3R template, when compared with that derived from the structure of the previously published and more evolutionary distant β2 adrenergic receptor. The comparison of results for receptors built according to various protocols and templates revealed that the most significant factor for the receptor performance was a proper selection of "tool ligand" used in induced fit docking procedure. Taken together, our results suggest that the described homology modeling procedure could be a viable tool for structure-based GPCR ligand design, even for the targets for which only a relatively distant structural template is available.

Background and Purpose: GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D₂ receptor were investigated., Experimental Approach: All of the S/T residues located within the intracellular loops of D₂ receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D₂ receptors were investigated in the transfected cells., Key Results: T134, T225/S228/S229 and S325 were involved in PKC-mediated D₂ receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D₂ receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D₂ receptors, which induced receptor resensitization. ARF6 mediated the recycling of D₂ receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D₂ receptors internalized in a PKC-dependent manner., Conclusions and Implications: GRK- and PKC-mediated internalizations of D₂ receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D₂ receptors and different sorting proteins are involved in the dissimilar regulation of D₂ receptors by GRK2 and PKC., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β1 adrenergic receptor model in complex with (S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D2 receptor were produced with the selective and rigid agonist (R)-N-propylapomorphine ((R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D2 receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.

We have demonstrated that D(5) and D(2) dopamine receptors exist as heteromers in cells, and determined these receptor interact through amino acids in the cytoplasmic regions of each receptor. Specifically involved in heteromer formation we identified in the carboxyl tail of the D(5) receptor three adjacent glutamic acid residues, and in intracellular loop 3 of the D(2) receptor two adjacent arginine residues. Any pairing of these three D(5) receptor glutamic acids were sufficient for heteromer formation. These identified residues in D(5) and D(2) receptors are oppositely charged and likely interact by electrostatic interactions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In the CNS, an antagonistic interaction has been shown between adenosine A(2A) and dopamine D(2) receptors (A(2A)Rs and D(2)Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor-receptor interaction have recently been explored, as the fine tuning of this target (namely the A(2A)R/D(2)R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer-based approach to examine the allosteric modulation of the D(2)R within the A(2A)R/D(2)R oligomer and the dependence of this receptor-receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D(2)R. Interestingly, we observed a negative allosteric effect of the D(2)R agonist quinpirole on A(2A)R ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217-222 and 267-269) on intracellular loop 3 of the D(2)R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor-receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A(2A)R/D(2)R oligomer in living cells., (© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

G-protein-coupled receptors (GPCRs) are cell surface receptors. The dynamic property of receptor-receptor interactions in GPCRs modulates the kinetics of G-protein signaling and stability. In the present work, the structural and dynamic study of A(2A)R-D(2)R interactions was carried to acquire the understanding of the A(2A)R-D(2)R receptor activation and deactivation process, facilitating the design of novel drugs and therapeutic target for Parkinson's disease. The structure-based features (Alpha, Beta, SurfAlpha, and SurfBeta; GapIndex, Leakiness and Gap Volume) and slow mode model (ENM) facilitated the prediction of kinetics (K (off), K (on), and K (d)) of A(2A)R-D(2)R interactions. The results demonstrated the correlation coefficient 0.294 for K (d) and K (on) and the correlation coefficient 0.635 for K (d) and K (off), and indicated stable interfacial contacts in the formation of heterodimer. The coulombic interaction involving the C-terminal tails of the A(2A)R and intracellular loops (ICLs) of D(2)R led to the formation of interfacial contacts between A(2A)R-D(2)R. The properties of structural dynamics, ENM and KFC server-based hot-spot analysis illustrated the stoichiometry of A(2A)R-D(2)R contact interfaces as dimer. The propensity of amino acid residues involved in A(2A)R-D(2)R interaction revealed the presence of positively (R, H and K) and negatively (E and D) charged structural motif of TMs and ICL3 of A(2A)R and D(2)R at interface of dimer contact. Essentially, in silico structural and dynamic study of A(2A)R-D(2)R interactions will provide the basic understanding of the A(2A)R-D(2)R interfacial contact surface for activation and deactivation processes, and could be used as constructive model to recognize the protein-protein interactions in receptor assimilations.

The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46, and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51, show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic-aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur-π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.

The dopamine D3 receptor (D3R) has been implicated in substance abuse and other neuropsychiatric disorders. The high sequence homology between the D3R and D2R, especially within the orthosteric binding site (OBS) that binds dopamine, has made the development of D3R-selective compounds challenging. Here, we deconstruct into pharmacophoric elements a series of D3R-selective substituted-4-phenylpiperazine compounds and use computational simulations and binding and activation studies to dissect the structural bases for D3R selectivity and efficacy. We find that selectivity arises from divergent interactions within a second binding pocket (SBP) separate from the OBS, whereas efficacy depends on the binding mode in the OBS. Our findings reveal structural features of the receptor that are critical to selectivity and efficacy that can be used to design highly D3R-selective ligands with targeted efficacies. These findings are generalizable to other GPCRs in which the SBP can be targeted by bitopic or allosteric ligands.

5-HT(1A) serotonin and D1 dopamine receptor agonists have been postulated to be able to improve negative and cognitive impairment symptoms of schizophrenia, while partial agonists and antagonists of the D2 and 5-HT(2A) receptors have been reported to be effective in reducing positive symptoms. There is therefore a need for well-defined homology models for the design of more selective antipsychotic agents, since no three-dimensional (3D) crystal structures of these receptors are currently available. In this study, homology models were built based on the high-resolution crystal structure of the β(2)-adrenergic receptor (2RH1) and further refined via molecular dynamics simulations in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer system with the GROMOS96 53A6 united atom force field. Docking evaluations with representative agonists and antagonists using AutoDock 4.2 revealed binding modes in agreement with experimentally determined site-directed mutagenesis data and significant correlations between the computed and experimental pK (i) values. The models are also able to distinguish between antipsychotic agents with different selectivities and binding affinities for the four receptors, as well as to differentiate active compounds from decoys. Hence, these human 5-HT(1A), 5-HT(2A), D1 and D2 receptor homology models are capable of predicting the activities of novel ligands, and can be used as 3D templates for antipsychotic drug design and discovery.

The evidence for the existence of receptor heteromers opens up a new field for a better understanding of neural transmission. Based on our theory, we have discovered main triplets of amino acid residues in cell-adhesion receptors of marine sponges, which appear also as homologies in several dopamine D2 receptor heteromers of human brain. The obtained results probably mean a general molecular mechanism for receptor-receptor interactions in heteromers originated from the lowest animals (marine sponges).

In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 receptor, specifically for substituted benzamides (SBAs) having polar 4- (para) and/or 5- (meta) benzamide ring substituents. D4-T7.39A was fully functional, and the mutation did not alter the sodium-mediated positive and negative allostery observed with SBAs and agonists, respectively. With the exception of the non-SBA ligand (+)-butaclamol, which, in contrast to certain SBAs, had decreased affinity for the D4-T7.39A mutant, the interactions of numerous other ligands were unaffected by this mutation. SBAs were docked into D4 models in the same mode as observed for eticlopride in the D3 crystal structure. In this mode, interactions with TM5 and TM6 residues constrain the SBA ring position that produces distal steric crowding between pyrrolidinyl/diethylamine moieties and D4-Thr7.39. Ligand-residue interaction energy profiles suggest this crowding is mitigated by substitution with a smaller alanine. The profiles indicate sites that contribute to the SBA binding interaction and site-specific energy changes imparted by the D4-T7.39A mutation. Substantial interaction energy changes are observed at only a few positions, some of which are not conserved among the dopamine receptor subtypes and thus seem to account for this D4 subtype-specific structure-activity relationship.

It is suggested that the ratio of dopamine D(2) to 5-hydroxytryptamine 5-HT(1A) activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D(2) and 5-hydrohytryptamine 5-HT(1A) receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT(1A) receptors., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although functional selectivity is now widely accepted, the molecular basis is poorly understood. We have studied how aspects of transmembrane region 5 (TM5) of the dopamine D(2L) receptor interacts with three rationally selected rigid ligands (dihydrexidine, dinapsoline, and dinoxyline) and the reference compounds dopamine and quinpirole. As was expected from homology modeling, mutation of three TM5 serine residues to alanine (S5.42A, S5.43A, S5.46A) had little effect on antagonist affinity. All three mutations decreased the affinity of the agonist ligands to different degrees, S5.46A being somewhat less affected. Four functions [adenylate cyclase (AC), extracellular signal-regulated kinase 1/2 phosphorylation (MAPK), arachidonic acid release (AA), and guanosine 5'-O-(3-thio)triphosphate binding (GTPγS)] were assessed. The intrinsic activity (IA) of quinpirole was unaffected by any of the mutations, whereas S5.42A and S5.46A mutations abolished the activity of dopamine and the three rigid ligands, although dihydrexidine retained IA at MAPK function only with S5.42A. Remarkably, S5.43A did not markedly affect IA for AC and MAPK for any of the ligands and eliminated AA activity for dinapsoline and dihydrexidine but not dinoxyline. These data suggest that this mutation did not disrupt the overall conformation or signaling ability of the mutant receptors but differentially affected ligand activation. Computational studies indicate that these D(2) agonists stabilize multiple receptor conformations. This has led to models showing the stabilized conformations and interhelical and receptor-ligand contacts corresponding to the different activation pathways stabilized by various agonists. These data provide a basis for understanding D(2L) functional selectivity and rationally discovering functionally selective D(2) drugs.

Background and Purpose: Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D(2) and D(3) receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region., Experimental Approach: The N-terminal region of the D(2) receptor was gradually shortened or switched with that of the D(3) receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined., Key Results: Shortening the N-terminal region of the D(2) receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D(2) and D(3) receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D(2) receptors., Conclusions and Implications: Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D(2) receptors. The N-terminal region of the D(2) receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

Human dopamine D(2long) and D(3) receptors were modified by N-terminal addition of SNAP or CLIP forms of O(6)-alkylguanine-DNA-alkyltransferase plus a peptide epitope tag. Cells able to express each of these four constructs only upon addition of an antibiotic were established and used to confirm regulated and inducible control of expression, the specificity of SNAP and CLIP tag covalent labeling reagents, and based on homogenous time-resolved fluorescence resonance energy transfer, the presence of cell surface D(2long) and D(3) receptor homomers. Following constitutive expression of reciprocal constructs, potentially capable of forming and reporting the presence of cell surface D(2long)-D(3) heteromers, individual clones were assessed for levels of expression of the constitutively expressed protomer. This was unaffected by induction of the partner protomer and the level of expression of the partner required to generate detectable cell surface D(2long)-D(3) heteromers was defined. Such homomers and heteromers were found to co-exist and using a reconstitution of function approach both homomers and heteromers of D(2long) and D(3) receptors were shown to be functional, potentially via trans-activation of associated G protein. These studies demonstrate the ability of dopamine D(2long) and D(3) receptors to form both homomers and heteromers, and show that in cells expressing each subtype a complex mixture of homomers and heteromers co-exists at steady state. These data are of potential importance both to disorders in which D(2long) and D(3) receptors are implicated, like schizophrenia and Parkinson disease, and also to drugs exerting their actions via these sites.

The aim of this study was to use a combined structure and pharmacophore modeling approach to extract information regarding dopamine D₁ receptor agonism and D₁/D₂ agonist selectivity. A 3D structure model of the D₁ receptor in its agonist-bound state was constructed with a full D₁ agonist present in the binding site. Two different binding modes were identified using (+)-doxanthrine or SKF89626 in the modeling procedure. The 3D model was further compared with a selective D₁ agonist pharmacophore model. The pharmacophore feature arrangement was found to be in good agreement with the binding site composition of the receptor model, but the excluded volumes did not fully reflect the shape of the agonist binding pocket. A new receptor-based pharmacophore model was developed with forbidden volumes centered on atom positions of amino acids in the binding site. The new pharmacophore model showed a similar ability to discriminate as the previous model. A comparison of the 3D structures and pharmacophore models of D₁ and D₂ receptors revealed differences in shape and ligand-interacting features that determine selectivity of D₁ and D₂ receptor agonists. A hydrogen bond pharmacophoric feature (Ser-TM5) was shown to contribute most to the selectivity. Non-conserved residues in the binding pocket that strongly contribute to D₁/D₂ receptor agonist selectivity were also identified; those were Ser/Cys³·³⁶, Tyr/Phe⁵·³⁸, Ser/Tyr⁵·⁴¹, and Asn/His⁶·⁵⁵ in the transmembrane (TM) helix region, together with Ser/Ile and Leu/Asn in the second extracellular loop (EC2). This work provides useful information for the design of new selective D₁ and D₂ agonists. The combined receptor structure and pharmacophore modeling approach is considered to be general, and could therefore be applied to other ligand-protein interactions for which experimental information is limited., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A combined modeling approach was used to identify structural factors that underlie the structure-activity relationships (SARs) of full dopamine D₂ receptor agonists and structurally similar inactive compounds. A 3D structural model of the dopamine D₂ receptor was constructed, with the agonist (-)-(R)-2-OH-NPA present in the binding site during the modeling procedure. The 3D model was evaluated and compared with our previously published D₂ agonist pharmacophore model. The comparison revealed an inconsistency between the projected hydrogen bonding feature (Ser-TM5) in the pharmacophore model and the TM5 region in the structure model. A new refined pharmacophore model was developed, guided by the shape of the binding site in the receptor model and with less emphasis on TM5 interactions. The combination of receptor and pharmacophore modeling also identified the importance of His393⁶·⁵⁵ for agonist binding. This convergent 3D pharmacophore and protein structure modeling strategy is considered to be general and can be highly useful in less well-characterized systems to explore ligand-receptor interactions. The strategy has the potential to identify weaknesses in the individual models and thereby provides an opportunity to improve the discriminating predictivity of both pharmacophore searches and structure-based virtual screens., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ligand bias is a recently introduced concept in the receptor signaling field that underlies innovative strategies for targeted drug design. Ligands, as a consequence of conformational selectivity, produce signaling bias in which some downstream biochemical pathways are favored over others, and this contributes to variability in physiological responsiveness. Though the concept of bias and its implications for receptor signaling have become more important, its working definition in biochemical signaling is sufficiently imprecise as to impede the use of bias as an analytical tool. In this work, we provide a precise mathematical definition for receptor signaling bias using a formalism expressly applied to logistic response functions, models of most physiological behaviors. We show that signaling-response bias of biological processes may be represented by hyperbolae, or more generally as families of bias coordinates that index hyperbolae. Furthermore, we show bias is a property of a parametric mapping of these indexes into vertical strings that reside within a cylinder of stacked Poincare disks and that bias factors representing signaling probabilities are the radial distance of the strings from the cylinder axis. The utility of the formalism is demonstrated with logistic hyperbolic plots, by transducer ratio modeling, and with novel examples of Poincare disk plots of Gi and β-arrestin biased dopamine 2 receptor signaling. Our results provide a platform for categorizing compounds using distance relationships in the Poincare disk, indicate that signaling bias is a relatively common phenomenon at low ligand concentrations, and suggest that potent partial agonists and signaling pathway modulators may be preferred leads for signal bias-based therapies.

A molecular modeling study on 16 1-benzyl tetrahydroisoquinolines (BTHIQs) acting as dopaminergic ligands was carried out. By combining molecular dynamics simulations with ab initio and density functional theory (DFT) calculations, a simple and generally applicable procedure to evaluate the binding energies of BTHIQs interacting with the human dopamine D2 receptor (D2 DR) is reported here, providing a clear picture of the binding interactions of BTHIQs from both structural and energetic viewpoints. Molecular aspects of the binding interactions between BTHIQs and the D2 DR are discussed in detail. A significant correlation between binding energies obtained from DFT calculations and experimental pKi values was obtained, predicting the potential dopaminergic effect of non-synthesized BTHIQs.

The diverse activities of dopamine D2-like receptors, including D2, D3, and D4 receptors, are mediated by proteins that interact with the third cytoplasmic loop and regulate receptor signaling, receptor trafficking, and apoptosis. Such interacting proteins include calmodulin, the N-methyl-D: -aspartate receptor 2B subunit, calcium/calmodulin-dependent protein kinase II, prostate apoptosis response-4, and β-arrestins, which regulate receptor signaling and the pharmacological action through D2 receptor. The gene encoding the D2 receptor gives rise to two isoforms, termed the dopamine D2 receptor long isoform (D2L) and the dopamine D2 receptor short isoform; the latter lacks 29 amino acids of the D2L receptor within the third cytoplasmic loop. In this review, we first focus on novel functions of the hetero-oligomeric D1/D2 and D2/adenosine A(2A) receptors. We next discuss novel signaling through proteins interacting with the D2 receptor third cytoplasmic loop and define the function of a novel binding protein, heart-type fatty acid binding protein, which interacts with the D2L third cytoplasmic loop.

We report here an exhaustive and complete conformational study on the conformational potential energy hypersurface (PEHS) of dopamine (DA) interacting with the dopamine D2 receptor (D2-DR). A reduced 3D model for the binding pocket of the human D2-DR was constructed on the basis of the theoretical model structure of bacteriorhodopsin. In our reduced model system, only 13 amino acids were included to perform the quantum mechanics calculations. To obtain the different complexes of DA/D2-DR, we combined semiempirical (PM6), DFT (B3LYP/6-31G(d)), and QTAIM calculations. The molecular flexibility of DA interacting with the D2-DR was evaluated from potential energy surfaces and potential energy curves. A comparative study between the molecular flexibility of DA in the gas phase and at D2-DR was carried out. In addition, several molecular dynamics simulations were carried out to evaluate the molecular flexibility of the different complexes obtained. Our results allow us to postulate the complexes of type A as the "biologically relevant conformations" of DA. In addition, the theoretical calculations reported here suggested that a mechanistic stepwise process takes place for DA in which the protonated nitrogen group (in any conformation) acts as the anchoring portion, and this process is followed by a rapid rearrangement of the conformation allowing the interaction of the catecholic OH groups.

Positron Emission Tomography (PET) has been used for more than three decades to image and quantify dopamine D2 receptors (D2R) in vivo with antagonist radioligands but in the recent years agonist radioligands have also been employed. In vitro competition studies have demonstrated that agonists bind to both a high and a low-affinity state of the D2Rs, of which the high affinity state reflects receptors that are coupled to G-proteins and the low-affinity state reflects receptors uncoupled from G-proteins. In contrast, antagonists bind with uniform affinity to the total pool of receptors. Results of these studies led to the proposal that D2Rs exist in high and low-affinity states for agonists in vivo and sparked the development and use of agonist radioligands for PET imaging with the primary purpose of measuring the proportion of receptors in the high-affinity (activating) state. Although several lines of research support the presence of high and low-affinity states of D2Rs and their detection by in vivo imaging paradigms, a growing body of controversial data has now called this into question. These include both in vivo and ex vivo studies of anesthesia effects, rodent models with increased proportions of high-affinity state D2Rs as well as the molecular evidence for stable receptor-G-protein complexes. In this commentary we review these data and discuss the evidence for the in vivo existence of D2Rs configured in high and low-affinity states and whether or not the high-affinity state of the D2R can, in fact, be imaged in vivo with agonist radioligands., (Copyright © 2011 Elsevier Inc. All rights reserved.)

D(1) and D(2) dopamine receptors exist as heteromers in cells and brain tissue and are dynamically regulated and separated by agonist concentrations at the cell surface. We determined that these receptor pairs interact primarily through discrete amino acids in the cytoplasmic regions of each receptor, with no evidence of any D(1)-D(2) receptor transmembrane interaction found. Specifically involved in heteromer formation we identified, in intracellular loop 3 of the D(2) receptor, two adjacent arginine residues. Substitution of one of the arginine pair prevented heteromer formation. Also involved in heteromer formation we identified, in the carboxyl tail of the D(1) receptor, two adjacent glutamic acid residues. Substitution of one of the glutamic acid pair prevented heteromer formation. These amino acid pairs in D(1) and D(2) receptors are oppositely charged, and presumably interact directly by electrostatic interactions., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies for combating drug addiction.

Dopamine (DA) receptors, a class of G-protein coupled receptors (GPCRs), have been targeted for drug development for the treatment of neurological, psychiatric and ocular disorders. The lack of structural information about GPCRs and their ligand complexes has prompted the development of homology models of these proteins aimed at structure-based drug design. Crystal structure of human dopamine D(3) (hD(3)) receptor has been recently solved. Based on the hD(3) receptor crystal structure we generated dopamine D(2) and D(3) receptor models and refined them with molecular dynamics (MD) protocol. Refined structures, obtained from the MD simulations in membrane environment, were subsequently used in molecular docking studies in order to investigate potential sites of interaction. The structure of hD(3) and hD(2L) receptors was differentiated by means of MD simulations and D(3) selective ligands were discriminated, in terms of binding energy, by docking calculation. Robust correlation of computed and experimental K(i) was obtained for hD(3) and hD(2L) receptor ligands. In conclusion, the present computational approach seems suitable to build and refine structure models of homologous dopamine receptors that may be of value for structure-based drug discovery of selective dopaminergic ligands.

Background: The interaction between the dopaminergic and somatostatinergic systems and their role in mood regulation have been well-documented. Therefore, we decided to investigate the effect of antidepressant drugs on the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors., Methods: The human receptor proteins were tagged with fluorescent proteins, expressed in the HEK 293 cells and incubated with antidepressant drugs: desipramine and citalopram. To determine the FRET efficiency, the fluorescence resonance energy transfer (FRET) and photobleaching confocal microscopy techniques were used., Results: We found that the efficiency of FRET is markedly increased in cells coexpressing the somatostatin Sst5 and dopamine D2 receptors after 48 h of incubation with desipramine and citalopram., Conclusions: In the present study we provide physical evidence, based on FRET analysis, that antidepressants increase Sst5 and D2 receptors heterodimerization. The effect is specific because desipramine in the incubation medium uncouples other pairs of receptors, such as the dopamine D1-D2 receptors.

The dopamine D2 receptor (DRD2) is a crucial mediator for normal physiological processes. We cloned the pig DRD2 gene, investigated its distribution in tissues and identified polymorphisms by RT-PCR, quantitative real-time PCR and direct sequencing. Two Yorkshire pigs from Guangdong Academy of Agricultural Sciences (Guangzhou, China) were selected to clone the gene and investigate its expression; 16 individuals from four pig breeds (Yorkshire, Landrace, small-ear spotted, and Xinchang) were used to scan the variations. The two transcripts (DRD2L and DRD2S), obtained through insertion or deletion of exon 5 and part of 3'UTR, were found to encode 444- and 415-amino acid proteins, respectively. The 574-bp indel in 3'UTR comprises five miRNA targeting sites, based on bioinformatics predictions. The pig DRD2 gene expresses predominantly in the pituitary gland, and then in oviducts and the hypothalamus. Both DRD2L and DRD2S mRNA were detected in cerebrum, cerebellum, hypothalamus, pituitary gland, back muscle, oviduct, uterus, and testis tissues; DRD2L was more abundant than DRD2S. The DRD2 gene is located on chromosome 9 and contains seven exons. Sixty-one different sequences were identified in this gene; among seven in the coding region, only one altered the encoded amino acid. These findings will help us understand the functions of the DRD2 gene in pigs.

S100B is a dimeric EF-hand protein that undergoes a calcium-induced conformational change and exposes a hydrophobic protein-binding surface. Recently S100B was identified as a binding partner of the dopamine D2 receptor in a bacterial two-hybrid screen involving the third intracellular loop (IC3). The low in vivo calcium concentration in bacteria (100-300 nM) suggests this interaction may occur in the absence of calcium. In this work the calcium-sensitive ability for S100B to recruit the IC3 of the dopamine D2 receptor was examined, and regions in both proteins required for complex formation were identified. Peptide array experiments identified the C-terminal 58 residues of the IC3 (IC3-C58) as the major interacting site for S100B. These experiments along with pull-down assays showed the IC3 interacts with S100B in the absence and presence of calcium. (1)H-(15)N HSQC experiments were used to identify residues, primarily in helices III and IV, utilized in the IC3-C58 interaction. NMR titration data indicated that although an interaction between apo-S100B and IC3-C58 occurs without calcium, the binding was enhanced more than 100-fold upon calcium binding. Further, it was established that shorter regions within IC3-C58 comprising its N- and C-terminal halves had diminished binding to Ca(2+)-S100B and did not display any observable affinity in the absence of calcium. This indicates that residue or structural components within both regions are required for optimal interaction with Ca(2+)-S100B. This work represents the first example of an S100B target that interacts with both the apo- and calcium-saturated forms of S100B.

G protein-coupled receptors (GPCRs) contain a number of conserved proline residues in their transmembrane helices, and it is generally assumed these play important functional and/or structural roles. Here we use unnatural amino acid mutagenesis, employing α-hydroxy acids and proline analogues, to examine the functional roles of five proline residues in the transmembrane helices of the D2 dopamine receptor. The well-known tendency of proline to disrupt helical structure is important at all sites, while we find no evidence for a functional role for backbone amide cis-trans isomerization, another feature associated with proline. At most proline sites, the loss of the backbone NH is sufficient to explain the role of the proline. However, at one site, P210(5.50), a substituent on the backbone N appears to be essential for proper function. Interestingly, the pattern in functional consequences that we see is mirrored in the pattern of structural distortions seen in recent GPCR crystal structures.

Neuronal calcium sensor-1 (NCS-1) is a small calcium binding protein that plays a key role in the internalization and desensitization of activated D2 dopamine receptors (D2Rs). Here, we have used fluorescence anisotropy (FA) and a panel of NCS-1 EF-hand variants to interrogate the interaction between the D2R and NCS-1. Our data are consistent with the following conclusions. (1) FA titration experiments indicate that at low D2R peptide concentrations calcium-loaded NCS-1 binds to the D2R peptide in a monomeric form. At high D2R peptide concentrations, the FA titration data are best fit by a model in which the D2R peptide binds two NCS-1 monomers sequentially in a cooperative fashion. (2) Competition FA experiments in which unlabeled D2R peptide was used to compete with labeled peptide for binding to NCS-1 shifted titration curves to higher NCS-1 concentrations, suggesting that the binding of NCS-1 to the D2R is highly specific and that binding occurs in a cooperative fashion. (3) N-Terminally myristoylated NCS-1 dimerizes in a calcium-dependent manner. (4) Co-immunoprecipitation experiments in HEK-293 confirm that NCS-1 can oligomerize in cell lysates and that oligomerization is dependent on calcium binding and requires functionally intact EF-hand domains. (5) Ca(2+)/Mg(2+) FA titration experiments revealed that NCS-1 EF-hands 2-4 (EF2-4) contributed to binding with the D2R peptide. EF2 appears to have the highest affinity for Ca(2+), and occupancy of this site is sufficient to promote high-affinity binding of the NCS-1 monomer to the D2R peptide. Magnesium ions may serve as a physiological cofactor with calcium for NCS-1-D2R binding. Finally, we propose a structural model that predicts that the D2R peptide binds to the first 60 residues of NCS-1. Together, our results support the possibility of using FA to screen for small molecule drugs that can specifically block the interaction between the D2R and NCS-1., (© 2011 American Chemical Society)

The synthesis, radiolabeling and in vitro evaluation of new silicon-fluoride acceptor (SiFA) derivatized D(2)-receptor ligands is reported. The SiFA-technology simplifies the introduction of fluorine-18 into target specific biomolecules for Positron-Emission-Tomography (PET). However, one of the remaining challenges, especially for small molecules such as receptor-ligands, is the bulkiness of the SiFA-moiety. We therefore synthesized four Fallypride SiFA-conjugates derivatized either directly at the benzoic acid ring system (SiFA-DMFP, SiFA-FP, SiFA-DDMFP) or at the butyl-side chain (SiFA-M-FP) and tested their receptor affinities. We found D(2)-receptor affinities for all compounds in the nanomolar range (K(i(SiFA-DMFP)) = 13.6 nM, K(i(SiFA-FP)) = 33.0 nM, K(i(SiFA-DDMFP)) = 62.7 nM and K(i(SiFA-M-FP)) = 4.21 nM). The radiofluorination showed highest yields when 10 nmol of the precursors were reacted with [(18)F]fluoride/TBAHCO(3) in acetonitrile. After a reversed phased cartridge purification the desired products could be isolated as an injectable solution after only 10 min synthesis time with radiochemical yields (RCY) of more than 40% in the case of SiFA-DMFP resulting in specific activities >41 GBq/µmol (>1,100 Ci/mmol). Furthermore, the radiolabeled products were shown to be stable in the injectable solutions, as well as in human plasma, for at least 90 min.

G-protein-coupled receptors (GPCRs) represent a diverse protein family of receptors that transduce signals from the extracellular surrounding to intracellular signaling molecules evoking various cellular responses. It is now widely accepted that GPCRs are expressed and function as dimers or most probably as oligomers of more than two receptor protomers. The heteromer has different biochemical and pharmacological characteristics from the monomers, which increases the functional responses of GPCRs. GPCRs are involved in many diseases, and are also the target of around half of all modern medicinal drugs. In the case of Parkinson's disease, a degenerative process caused by gradual disappearance of dopaminergic nigrostriatal neurons, it is suspected that the targets for treatment should be dopamine-receptor-containing heteromers. Technologies based on the use of fluorescent- or luminescent-fused receptors and adaptations of resonance energy transfer (RET) techniques have been useful in investigating the functional inter-relationships between receptors in a heteromer. In this study functional recombinant adenosine A(2A)-Rluc, dopamine D(2)-GFP(2) and histamine H(3)-YFP receptor fusion proteins were successfully cloned and characterized, producing the essential basis for heteromerization studies between these receptors. This might provide a better insight into their pharmacological and functional inter-relationships in the brain and enable the design and evaluation of new therapeutic strategies for Parkinson's disease., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The existence of G protein-coupled receptor (GPCR) dimers and/or oligomers has been demonstrated in heterologous systems using a variety of biochemical and biophysical assays. While these interactions are the subject of intense research because of their potential role in modulating signaling and altering pharmacology, evidence for the existence of receptor interactions in vivo is still elusive because of a lack of appropriate methods to detect them. Here, we adapted and optimized a proximity ligation assay (PLA) for the detection in brain slices of molecular proximity of two antigens located on either the same or two different GPCRs. Using this approach, we were able to confirm the existence of dopamine D2 and adenosine A2A receptor complexes in the striatum of mice ex vivo.

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In many fishes, dopamine (DA) strongly inhibits luteinizing hormone (LH) release by direct action at the pituitary level. In this study, the effect of DA on LH synthesis was examined by detecting its β-subunit mRNA level in immature grass carp pituitary cells. Results showed that DA inhibited LHβ mRNA expression and its inhibition was antagonized by a DA D2 receptor (DRD2) antagonist, sulpiride, suggesting that DA inhibited LH synthesis via DRD2. This notion was further supported by the finding that the grass carp DRD2 (gcDRD2) immunoreactivity was observed in the proximal pars distalis of the pituitary in which gonadotrophs are distributed. Accordingly, a full-length cDNA for DRD2 was cloned from grass carp pituitary and it showed closer phylogenetic relationships to the DA D2 receptors compared with the D3 and D4 or D1-like receptors in other vertebrates. Besides brain, the expression of this receptor in pituitary was revealed by tissue distribution assay, implying the pituitary function of gcDRD2 in immature grass carp. In grass carp pituitary cells, gcDRD2 transcript level was stimulated by DA and this stimulation was blocked by sulpiride. However, hCG, a functional homolog of grass carp LH, was found to inhibit gcDRD2 mRNA expression, indicating an intrapituitary negative feedback of LH on gcDRD2 expression. In view of our observation that the DRD2 mediated the dopaminergic inhibition of LH synthesis, we speculate that the DA stimulation and LH suppression on gcDRD2 may reinforce or attenuate the DA inhibition on LH synthesis, respectively and this regulation of gcDRD2 may at least partially contribute to the steady state levels of LH mRNA in prepubertal grass carp., (Copyright © 2011 Elsevier Inc. All rights reserved.)