Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist.

Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein–coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the...

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.

Evidence for antagonist activity of the dopamine D3 receptor partial agonist, BP 897, at human dopamine D3 receptor

Restructuring G-Protein- Coupled Receptor Activation

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.

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the AT(1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. Here, we investigated the role of the first and fourth transmembrane domains (TMDs) in the formation of the binding pocket of the human AT(1) receptor using the substituted-cysteine accessibility method. Each residue within the Phe-28((1.32))-Ile-53((1.57)) fragment of TMD1 and Leu-143((4.40))-Phe-170((4.67)) fragment of TMD4 was mutated, one at a time, to a cysteine. The resulting mutant receptors were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydryl-specific alkylating agent methanethiosulfonate ethylammonium (MTSEA). This treatment led to a significant reduction in the binding affinity of TMD1 mutants M30C((1.34))-AT(1) and T33C((1.37))-AT(1) and TMD4 mutant V169C((4.66))-AT(1). Although this reduction in binding of the TMD1 mutants was maintained when examined in a constitutively active receptor (N111G-AT(1)) background, we found that V169C((4.66))-AT(1) remained unaffected when treated with MTSEA compared with untreated in this context. Moreover, the complete loss of binding observed for R167C((4.64))-AT(1) was restored upon treatment with MTSEA. Our results suggest that the extracellular portion of TMD1, particularly residues Met-30((1.34)) and Thr-33((1.37)), as well as residues Arg-167((4.64)) and Val-169((4.66)) at the junction of TMD4 and the second extracellular loop, are important binding determinants within the AT(1) receptor binding pocket but that these TMDs undergo very little movement, if at all, during the activation process.

Characterisation of G s activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine

Grand opening of structure-guided design for novel opioids

Activation of G protein-coupled receptors like the beta(1)-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human beta(1)-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for beta-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at beta(2)- and beta(3)-receptors; however, they act as antagonists at the beta(1)-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the beta(1)-adrenergic receptor for residues that were identical in the beta(2)- and beta(3)-subtypes but different in the beta(1)-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human beta(1)-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation.

The focal distribution of the dopamine (DA) D(3) receptor in brain regions implicated in emotional and cognitive functions has made this target a main focus of drug discovery efforts. This paper will review the most recent lines of research in support of the use of selective DA D(3) receptor antagonists for the pharmacotherapeutic management of drug addiction: (1) expression of the DA D(3) receptor in the rodent and human brain; (2) changes in expression of the DA D(3) receptor following exposure to drugs of abuse, and (3) efficacy of selective DA D(3) receptor antagonists in preclinical paradigms assessing the behavioral effects of drugs such as cocaine, nicotine, alcohol, methamphetamine, and heroin. This manuscript, however, will not review the effects of nonselective DA D(2)/D(3) receptor antagonists or partial D(3) receptor agonists. Growing evidence suggests that selective DA D(3) receptor antagonists do not affect the primary reinforcing effects of drugs of abuse, but rather seem to regulate the motivation to self-administer drugs under schedules of reinforcement that require an increase in work demand. In addition, selective antagonism at DA D(3) receptors appears to disrupt significantly the responsiveness to drug-associated stimuli that play a key role in reinstatement of drug-seeking behavior. These preclinical findings will be discussed in the context of translational research relevant to the design of early clinical trials and hypothesis testing in humans.

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

In addition to the serotonin 5-HT2A receptor (5-HT2AR), the dopamine D2 receptor (D2R) is a key therapeutic target of antipsychotics for the treatment of schizophrenia. The inactive state structures of D2R have been described in complex with the inverse agonists risperidone (D2Rris) and haloperidol (D2Rhal). Here we describe the structure of human D2R in complex with spiperone (D2Rspi). In D2Rspi, the conformation of the extracellular loop (ECL) 2, which composes the ligand-binding pocket, was substantially different from those in D2Rris and D2Rhal, demonstrating that ECL2 in D2R is highly dynamic. Moreover, D2Rspi exhibited an extended binding pocket to accommodate spiperone’s phenyl ring, which probably contributes to the selectivity of spiperone to D2R and 5-HT2AR. Together with D2Rris and D2Rhal, the structural information of D2Rspi should be of value for designing novel antipsychotics with improved safety and efficacy.

G protein-coupled receptors occur as dimers within arrays of oligomers. We visualized ensembles of dopamine receptor oligomers in living cells and evaluated the contributions of receptor conformation to the dynamics of oligomer association and dissociation, using a strategy of trafficking a receptor to another cellular compartment. We incorporated a nuclear localization sequence into the D1 dopamine receptor, which translocated from the cell surface to the nucleus. Receptor inverse agonists blocked this translocation, retaining the modified receptor, D1-nuclear localization signal (NLS), at the cell surface. D1 co-translocated with D1-NLS to the nucleus, indicating formation of homooligomers. (+)-Butaclamol retained both receptors at the cell surface, and removal of the drug allowed translocation of both receptors to the nucleus. Agonist-nonbinding D1(S198A/S199A)-NLS, containing two substituted serine residues in transmembrane 5 also oligomerized with D1, and both were retained on the cell surface by (+)-butaclamol. Drug removal disrupted these oligomerized receptors so that D1 remained at the cell surface while D1(S198A/S199A)-NLS trafficked to the nucleus. Thus, receptor conformational differences permitted oligomer disruption and showed that ligand-binding pocket occupancy by the inverse agonist induced a conformational change. We demonstrated robust heterooligomerization between the D2 dopamine receptor and the D1 receptor. The heterooligomers could not be disrupted by inverse agonists targeting either one of the receptor constituents. However, D2 did not heterooligomerize with the structurally modified D1(S198A/S199A), indicating an impaired interface for their interaction. Thus, we describe a novel method showing that a homogeneous receptor conformation maintains the structural integrity of oligomers, whereas conformational heterogeneity disrupts it.

The determination of G protein-coupled receptor (GPCR) structures at atomic resolution has improved understanding of cellular signaling and will accelerate the development of new drug candidates. However, experimental structures still remain unavailable for a majority of the GPCR family. GPCR structures and their interactions with ligands can also be modelled computationally, but such predictions have limited accuracy. In this work, we explored if molecular dynamics (MD) simulations could be used to refine the accuracy of in silico models of receptor-ligand complexes that were submitted to a community-wide assessment of GPCR structure prediction (GPCR Dock). Two simulation protocols were used to refine 30 models of the D3 dopamine receptor (D3R) in complex with an antagonist. Close to 60 μs of simulation time was generated and the resulting MD refined models were compared to a D3R crystal structure. In the MD simulations, the receptor models generally drifted further away from the crystal structure conformation. However, MD refinement was able to improve the accuracy of the ligand binding mode. The best refinement protocol improved agreement with the experimentally observed ligand binding mode for a majority of the models. Receptor structures with improved virtual screening performance, which was assessed by molecular docking of ligands and decoys, could also be identified among the MD refined models. Application of weak restraints to the transmembrane helixes in the MD simulations further improved predictions of the ligand binding mode and second extracellular loop. These results provide guidelines for application of MD refinement in prediction of GPCR-ligand complexes and directions for further method development.

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.