Human Dopamine D2 Receptor Research Articles

Synthesis, pharmacological evaluation and molecular modeling studies of triazole containing dopamine D3 receptor ligands

A series of 2-methoxyphenyl piperazine analogues containing a triazole ring were synthesized and their in vitro binding affinities at human dopamine D2 and D3 receptors were evaluated. Compounds 5b, 5c, 5d, and 4g, demonstrate high affinity for dopamine D3 receptors and moderate selectivity for the dopamine D3 versus D2 receptor subtypes. To further examine their potential as therapeutic agents, their intrinsic efficacy at both D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay. Affinity at dopamine D4 and serotonin 5-HT1A receptors was also determined. In addition, information from previous molecular modeling studies of the binding of a panel of 163 structurally-related benzamide analogues at dopamine D2 and D3 receptors was applied to this series of compounds. The results of the modeling studies were consistent with our previous experimental data. More importantly, the modeling study results explained why the replacement of the amide linkage with the hetero-aromatic ring leads to a reduction in the affinity of these compounds at D3 receptors.

The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes

G-protein coupled receptors (GPCRs) share a common seven-transmembrane topology and mediate cellular responses to a variety of extracellular signals. However, structural and functional approaches to GPCRs have often been limited by the difficulty of producing a sufficient amount of receptor protein using conventional expression systems. We synthesized human dopamine D1 receptors using a wheat cell-free protein synthesis system with liposomes and then analyzed their receptor binding ability. We determined the specific binding of [3H]SCH23390 to the synthesized receptors generated from a cell-free protein synthesis system or rat striatal membranes. From Scatchard plot analysis, the dissociation constant (Kd) and the maximum density (Bmax) of the synthesized receptors were 6.61±0.06nM and 1.85±0.05pmol/mg protein, respectively. The same analysis for rat striatal membrane gave a Kd of 2.67±0.05nM and Bmax of 0.70±0.10pmol/mg protein. Using a competition binding assay, Ki values of antagonists, SCH23390, LE300 and SKF83566, for the synthetic receptors were the same as those for rat striatal membranes, but Ki values of agonists, A68930, SKF38393 and dopamine, were 5–17 fold higher than those for rat striatal membranes. These results suggest that the dopamine D1 receptors synthesized in liposomes have a functional binding capacity. The different patterns of binding of antagonists and agonists to the synthetic receptors and rat striatal membranes indicate that G proteins are involved in agonist binding to dopamine D1 receptors. The cell-free protein synthesis method with liposomes will be invaluable for the functional analysis of GPCRs.

Abstract 257: miR-217 Mediates the Protective Effects of the Dopamine D2 Receptor on Renal Inflammation and Fibrosis.

Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the human dopamine D2 receptor (DRD2) gene are associated with decreased receptor expression and function, as well as high blood pressure. We have reported that human renal proximal tubule cells (hRPTCs) from subjects carrying D2R SNPs had decreased D2R expression and function and increased expression of the pro-fibrotic factor TGFβ1 and its signaling targets Smad3 and Snail1. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins fibronectin-1 (FN-1), Collagen 1a (Col 1a), and vimentin. To determine the mechanisms underlying the effects of the D2R SNPs, we studied the expression of several microRNAs (miRs) that are associated with the regulation of chemokines, anti-fibrosis, extracellular matrix remodeling, and cell adhesion through post-transcriptional repression of their target mRNAs. The expression of 4 miRs (miR-217, miR-224, miR-335, miR-1265) was downregulated (-10.3 to -6.0 fold) and that of one miRNA (miR-1290; 5.3 fold) was upregulated in hRPTCs with D2R SNPs. The expression of miR-217 and miR-224 is regulated by the D2R; the expression is decreased by D2R downregulation in cells not carrying D2R SNPs (WT) and normalized by treating cells carrying D2R SNPs with a Drd2 plasmid. Treatment of WT cells with a miR-217 mimic significantly decreased the mRNA expression of TGFβ1 (43±2%), MMP3 (65±4%), and FN-1 (30±4%) while treatment with a miR-217 inhibitor increased TGFβ1 (94± 9%), MMP3 (96±5%), and FN-1 (212±30%). miR-217 mimic treatment of D2R SNPs cells also significantly decreased the expression of TGFβ1, MMP3, and FN-1. However, treatment of these cells with a miR-217 inhibitor had non-significant effects probably because miR-217 level was already reduced. In contrast treatment of either WT or D2R SNPs cells with miR-224 mimic or inhibitor had no significant effects on the expression of TGFβ1, MMP3, or FN-1. These results indicate that the protective effects of D2R on inflammation and fibrosis are at least, in part, mediated by regulation of miR-217 expression. Dysregulation of miR-217 expression may be responsible for the profibrotic phenotype of hRPTCs in individuals with these D2R SNPs.

Characterization of [(3) H]LS-3-134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand.

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit: (i) high-affinity binding (Ki value 0.2nM) at human D3 dopamine receptors, (ii) >100-fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low-affinity binding (Ki >5000nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [(3) H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15min at 37°C) and binds with high affinity to both human (Kd =0.06±0.01nM) and rat (Kd =0.2±0.02nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [(3) H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [(3) H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.

Multiple fragment docking and linking in primary and secondary pockets of dopamine receptors.

A sequential docking methodology was applied to computationally predict starting points for fragment linking using the human dopamine D3 receptor crystal structure and a human dopamine D2 receptor homology model. Two focused fragment libraries were docked in the primary and secondary binding sites, and best fragment combinations were enumerated. Similar top scoring fragments were found for the primary site, while secondary site fragments were predicted to convey selectivity. Three linked compounds were synthesized that had 9-, 39-, and 55-fold selectivity in favor of D3 and the subtype selectivity of the compounds was assessed on a structural basis.

Tranylcypromine substituted cis-hydroxycyclobutylnaphthamides as potent and selective dopamine D₃ receptor antagonists.

We report a class of potent and selective dopamine D3 receptor antagonists based upon tranylcypromine. Although tranylcypromine has a low affinity for the rat D3 receptor (Ki = 12.8 μM), our efforts have yielded (1R,2S)-11 (CJ-1882), which has Ki values of 2.7 and 2.8 nM at the rat and human dopamine D3 receptors, respectively, and displays respective selectivities of >10000-fold and 223-fold over the rat and human D2 receptors. Evaluation in a β-arrestin functional assay showed that (1R,2S)-11 is a potent and competitive antagonist at the human D3 receptor.

Polypharmacology profiles and phylogenetic analysis of three-finger toxins from mamba venom: case of aminergic toxins.

Composition of mamba's venom is quite atypical and characterized by the presence of a large diversity of three-finger fold toxins (3FTx) interacting with various enzymes, receptors and ion channels. In particular, 3FTx from mambas display the unique property to interact with class A GPCRs, sometimes with a high affinity and selectivity. A screening of five of these toxins (MT1, MT3, MT7, ρ-Da1a and ρ-Da1b) on 29 different subtypes of bioaminergic receptors, using competition binding experiments, highlights the diversity of their pharmacological profiles. These toxins may display either absolute selectivity for one receptor subtype or a polypharmacological property for various bioaminergic receptors. Nevertheless, adrenoceptor is the main receptor family targeted by these toxins. Furthermore, a new receptor target was identified for 3FTx and toxins in general, the ρ-Da1b interacting competitively with the human dopamine D3 receptor in the micromolar range. This result expands the diversity of GPCRs targeted by toxins and more generally highlights the multipotent interacting property of 3FTx. Phylogenic analyzes of these toxins show that muscarinic, adrenergic and dopaminergic toxins may be pooled in one family called aminergic toxins, this family coming probably from a specific radiation of ligands present in mamba venoms.

MicroRNA-9 and MicroRNA-326 Regulate Human Dopamine D2 Receptor Expression, and the MicroRNA-mediated Expression Regulation Is Altered by a Genetic Variant

The human dopamine receptor D2 (DRD2) has been implicated in the pathophysiology of schizophrenia and other neuropsychiatric disorders. Most antipsychotic drugs influence dopaminergic transmission through blocking dopamine receptors, primarily DRD2. We report here the post-transcriptional regulation of DRD2 expression by two brain-expressed microRNAs (miRs), miR-326 and miR-9, in an ex vivo mode, and show the relevance of miR-mediated DRD2 expression regulation in human dopaminergic neurons and in developing human brains. Both miRs targeted the 3'-UTR (untranslated region) of DRD2 in NT2 (neuron-committed teratocarcinoma, which endogenously expresses DRD2) and CHO (Chinese hamster ovary) cell lines, decreasing luciferase activity measured by a luciferase reporter gene assay. miR-326 overexpression reduced DRD2 mRNA and DRD2 receptor synthesis. Both antisense miR-326 and antisense miR-9 increased DRD2 protein abundance, suggesting an endogenous repression of DRD2 expression by both miRs. Furthermore, a genetic variant (rs1130354) within the DRD2 3'-UTR miR-targeting site interferes with miR-326-mediated repression of DRD2 expression. Finally, co-expression analysis identified an inverse correlation of DRD2 expression with both miR-326 and miR-9 in differentiating dopaminergic neurons derived from human induced pluripotent stem cells (iPSCs) and in developing human brain regions implicated in schizophrenia. Our study provides empirical evidence suggesting that miR-326 and miR-9 may regulate dopaminergic signaling, and miR-326 and miR-9 may be considered as potential drug targets for the treatment of disorders involving abnormal DRD2 function, such as schizophrenia.

Functional characterization of rare variants in human dopamine receptor D4 gene by genotype–phenotype correlations

Next generation sequencing technologies have facilitated a notable shift from common disease common variant hypothesis to common disease rare variant, as also witnessed in recent literature on schizophrenia. Dopamine receptor D4 (DRD4), a G-protein-coupled receptor is associated with psychiatric disorders and has high affinity for atypical antipsychotic clozapine. We investigated the functional role of rare genetic variants in DRD4 which may have implications for translational medicine. CHO-K1 cells independently expressing four rare non-synonymous variants of DRD4 namely R237L, A281P, S284G located in the third cytosolic loop and V194G, located in the fifth transmembrane domain were generated. Their genotype–phenotype correlations were evaluated using [3H]spiperone binding, G-protein activation and molecular dynamics-simulation studies. A281P and S284G were functionally similar to wildtype (WT). With R237L, potency of dopamine and quinpirole reduced ∼sixfold and threefold respectively compared to WT; [3H]spiperone binding studies showed a reduction in total number of binding sites (∼40%) but not binding affinity, in silico docking studies revealed that binding of both dopamine and spiperone to R237L was structurally similar to WT. Of note, V194G variant failed to inhibit forskolin-stimulated adenylate cyclase activity and phosphorylate extracellular signal-regulated kinase; showed significant reduction in binding affinity (Kd=2.16nM) and total number of binding sites (∼66%) compared to WT in [3H]spiperone binding studies; and ligand docking studies showed that binding of dopamine and spiperone is superficial due to probable structural alteration. Transmembrane variant V194G in DRD4.4 results in functional alteration warranting continuing functional analysis of rare variants.

Modeling of ligand binding to dopamine D2 receptor

The dopaminic receptors have been for long time the major targets for developing new small molecules with high affinity and selectivity to treat psychiatric disorders, neurodegeneration, drug abuse, and other therapeutic areas. In the absence of a 3D structure for the human D2 dopamine (HDD2) receptor, the efforts for discovery and design of new potential drugs rely on comparative models generation, docking and pharmacophore development studies. To get a better understanding of the HDD2 receptor binding site and the ligand-receptor interactions a homology model of HDD2 receptor based on the X-ray structure of ?2-adrenergic receptor has been built and used to dock a set of partial agonists of HDD2 receptor. The main characteristics of the binding mode for the HDD2 partial agonists set are given by the ligand particular folding and a complex network of contacts represented by stacking interactions, salt bridge and hydrogen bond formation. The characterization of the partial agonist binding mode at HDD2 receptor provide the needed information to generate pharmacophore models which represent essential information in the future virtual screening studies in order to identify new potential HDD2 partial agonists.

Brain human monoclonal autoantibody from sydenham chorea targets dopaminergic neurons in transgenic mice and signals dopamine D2 receptor: implications in human disease.

How autoantibodies target the brain and lead to disease in disorders such as Sydenham chorea (SC) is not known. SC is characterized by autoantibodies against the brain and is the main neurologic manifestation of streptococcal-induced rheumatic fever. Previously, our novel SC-derived mAb 24.3.1 was found to recognize streptococcal and brain Ags. To investigate in vivo targets of human mAb 24.3.1, VH/VL genes were expressed in B cells of transgenic (Tg) mice as functional chimeric human VH 24.3.1-mouse C-region IgG1(a) autoantibody. Chimeric human-mouse IgG1(a) autoantibody colocalized with tyrosine hydroxylase in the basal ganglia within dopaminergic neurons in vivo in VH 24.3.1 Tg mice. Both human mAb 24.3.1 and IgG1(a) in Tg sera were found to react with human dopamine D2 receptor (D2R). Reactivity of chorea-derived mAb 24.3.1 or SC IgG with D2R was confirmed by dose-dependent inhibitory signaling of D2R as a potential consequence of targeting dopaminergic neurons, reaction with surface-exposed FLAG epitope-tagged D2R, and blocking of Ab reactivity by an extracellular D2R peptide. IgG from SC and a related subset of streptococcal-associated behavioral disorders called "pediatric autoimmune neuropsychiatric disorder associated with streptococci" (PANDAS) with small choreiform movements reacted in ELISA with D2R. Reaction with FLAG-tagged D2R distinguished SC from PANDAS, whereas sera from both SC and PANDAS induced inhibitory signaling of D2R on transfected cells comparably to dopamine. In this study, we define a mechanism by which the brain may be altered by Ab in movement and behavioral disorders.

An historical perspective on “The world-wide distribution of allele frequencies at the human dopamine D4 receptor locus”

Human population genetics is a completely different science today compared to two decades ago, at least at the empiric level. Our paper [Chang (Hum Genet 98:91-101, 1996a)] demonstrated that three different alleles were common when one considered many populations although other low frequency alleles occurred. Because previous work had been largely done on European subjects, our findings involved 36 distinct populations and showed that East Asian populations had nearly lost the 7-repeat allele, and that Native American populations had the highest frequencies of that allele globally, was a significant early empiric demonstration of the potential magnitude of population variation at important genes. There are thousands of loci tested on many of the same populations and the gene frequency pattern seen for the DRD4 7-repeat allele is seen at other loci, arguing that this pattern commonly reflects the pattern of divergence of populations and accumulated random genetic drift.

Mothers' prenatal stress and their children's antisocial outcomes – a moderating role for the Dopamine D4 Receptor (DRD4) gene

Maternal distress during pregnancy has been linked to aggressive behavior in offspring. This effect has been interpreted in terms of 'fetal programming'. The 7-repeat (7r) allele of a VNTR polymorphism in exon III of the human dopamine receptor D4 (DRD4) has consistently been associated with externalizing behavior problems, especially in the presence of adverse environmental factors. So far, it is not known whether the DRD4 genotype moderates the effect of prenatal maternal stress on the development of childhood antisocial behavior. As part of an ongoing epidemiological cohort study, prenatal maternal stress was assessed using self-report 3 months following child birth. When children were 8, 11, and 15 years old, mothers rated their children's externalizing behavior, and diagnoses of conduct disorder and/or oppositional defiant disorder (CD/ODD) according to DSM-IV were obtained. In a sample of N = 308 participants, the effects of the DRD4 genotype, prenatal maternal stress, and the interaction thereof on antisocial outcome were tested. Under conditions of elevated prenatal maternal stress, children carrying one or two DRD4 7r alleles were at increased risk of a diagnosis of CD/ODD. Moreover, homozygous carriers of the DRD4 7r allele displayed more externalizing behavior following exposure to higher levels of prenatal maternal stress, while homozygous carriers of the DRD4 4r allele turned out to be insensitive to the effects of prenatal stress. This study is the first to report a gene-environment interaction related to DRD4 and prenatal maternal stress using data from a prospective study, which extends earlier findings on the impact of prenatal maternal stress with respect to childhood antisocial behavior.

Structure-Based Ligand Discovery Targeting Orthosteric and Allosteric Pockets of Dopamine Receptors

Small molecules targeting allosteric pockets of G protein-coupled receptors (GPCRs) have a great therapeutic potential for the treatment of neurologic and other chronic disorders. Here we performed virtual screening for orthosteric and putative allosteric ligands of the human dopamine D3 receptor (D3R) using two optimized crystal-structure-based models: the receptor with an empty binding pocket (D3R(APO)), and the receptor complex with dopamine (D3R(Dopa)). Subsequent biochemical and functional characterization revealed 14 novel ligands with a binding affinity of better than 10 μM in the D3R(APO) candidate list (56% hit rate), and 8 novel ligands in the D3R(Dopa) list (32% hit rate). Most ligands in the D3R(APO) model span both orthosteric and extended pockets and behave as antagonists at D3R, with compound 7 showing the highest potency of dopamine inhibition (IC₅₀ = 7 nM). In contrast, compounds identified by the D3R(Dopa) model are predicted to occupy an allosteric site at the extracellular extension of the pocket, and they all lack the anchoring amino group. Compounds targeting the allosteric site display a variety of functional activity profiles, where behavior of at least two compounds (23 and 26) is consistent with noncompetitive allosteric modulation of dopamine signaling in the extracellular signal-regulated kinase 1 and 2 phosphorylation and β-arrestin recruitment assays. The high affinity and ligand efficiency of the chemically diverse hits identified in this study suggest utility of structure-based screening targeting allosteric sites of GPCRs.

Restoration of Glutamatergic Transmission by Dopamine D4 Receptors in Stressed Animals

The prefrontal cortex (PFC), a key brain region for cognitive and emotional processes, is highly regulated by dopaminergic inputs. The dopamine D4 receptor, which is enriched in PFC, has been implicated in mental disorders, such as attention deficit-hyperactivity disorder and schizophrenia. Recently we have found homeostatic regulation of AMPA receptor-mediated synaptic transmission in PFC pyramidal neurons by the D4 receptor, providing a potential mechanism for D4 in stabilizing cortical excitability. Because stress is tightly linked to adaptive and maladaptive changes associated with mental health and disorders, we examined the synaptic actions of D4 in stressed rats. We found that neural excitability was elevated by acute stress and dampened by repeated stress. D4 activation produced a potent reduction of excitatory transmission in acutely stressed animals and a marked increase of excitatory transmission in repeatedly stressed animals. These effects of D4 targeted GluA2-lacking AMPA receptors and relied on the bi-directional regulation of calcium/calmodulin kinase II activity. The restoration of PFC glutamatergic transmission in stress conditions may enable D4 receptors to serve as a synaptic stabilizer in normal and pathological conditions.

Cell‐free protein synthesis and purification of human dopamine D2 receptor long isoform

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.

Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10822-013-9640-z) contains supplementary material, which is available to authorized users.

Synthesis and in vitro pharmacological evaluation of indolyl carboxylic amide analogues as D3 dopamine receptor selective ligands.

A series of substituted 1H-indolyl carboxylic acid amides that contain a N-(2-methoxyphenyl)piperazine or N-(2-fluoroethoxy)piperazine group were synthesized and their affinities for human dopamine D2, D3, and D4 receptors were determined. Two of these compounds, 14a and 14b, displayed high binding affinity at D3 (Ki = 0.18 and 0.4 nM, respectively), and selectivity for D3vs. D2 receptors (87-fold and 60-fold, respectively). These two compounds had low binding affinity at D4 receptors and σ receptor sites. The intrinsic activity of these compounds at D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay; both 14a and 14b were found to be partial agonists. Furthermore, for compound 14a, the log D value of 2.85 suggested it has suitable lipophilicity for crossing the blood-brain-barrier.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Dopamine D4 receptor deficiency in mice alters behavioral responses to anxiogenic stimuli and the psychostimulant methylphenidate

An allele of the human dopamine D4 receptor (D4R) gene (DRD4), containing seven tandem repeats of a 48-base nucleotide sequence (DRD4.7), has been reproducibly found in novelty seekers, substance abusers, and individuals with attention-deficit hyperactivity disorder. One hypothesis predicts the resultant protein product of the DRD4.7 polymorphism is deficient in G protein-coupled signaling. If attenuated D4R signaling contributes to these complex behaviors, then wild-type (WT) mice and mice completely lacking D4Rs (D4R KO) might be expected to display significantly different behavioral responses to stimuli known to affect dopamine signaling, such as novelty or psychostimulants. Adolescent male D4R KO mice exhibited greater locomotor activity and spent less time in the anxiogenic center of a novel open field environment than WT littermates. The presence of D4Rs had no effect on emergence into a novel environment from a sheltered space or exploration of a novel object. Low doses of acute methylphenidate (MP) had no effect on the exploration of a novel object, but dose-dependently increased the latency to emerge into a novel environment from a sheltered space. WT and D4R KO mice responded differently to high doses of acute MP, displaying significantly elevated locomotor activity and reduced stereotypy relative to WT mice. Chronic MP treatment produced enhanced locomotor sensitization in D4R KO mice, however this effect could not be fully recapitulated using the putative D4R antagonist L-745-870. These studies suggest that the roles of D4R signaling in novelty-seeking behaviors and the response to psychostimulants are not as clear as previously reported, and that some of these effects may be due to developmental compensatory effects as a consequence of lost D4R expression. If the DRD4.7 variant results in deficient D4R signaling in vivo, this may contribute to an elevated risk of sensitization to drugs of abuse including psychostimulants used to treat ADHD.