Advances and challenges in the search for D2 and D3 dopamine receptor-selective compounds

Compounds that target D2-like dopamine receptors (DRs) are currently used as therapeutics for several neuropsychiatric disorders including schizophrenia (antagonists) and Parkinson's disease (agonists). However, as the D₂R and D₃R subtypes are highly homologous, creating compounds with sufficient subtype-selectivity as well as drug-like properties for therapeutic use has proved challenging. This review summarizes the progress that has been made in developing D₂R- or D₃R-selective antagonists and agonists, and also describes the experimental conditions that need to be considered when determining the selectivity of a given compound, as apparent selectivity can vary widely depending on assay conditions. Future advances in this field may take advantage of currently available structural data to target alternative secondary binding sites through creating bivalent or bitopic chemical structures. Alternatively, the use of high-throughput screening techniques to identify novel scaffolds that might bind to the D₂R or D₃R in areas other than the highly conserved orthosteric site, such as allosteric sites, followed by iterative medicinal chemistry will likely lead to exceptionally selective compounds in the future. More selective compounds will provide a better understanding of the normal and pathological functioning of each receptor subtype, as well as offer the potential for improved therapeutics.     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

Comparative MD Simulations Indicate a Dual Role for Arg1323.50 in Dopamine-Dependent D2R Activation

Residue Arg^3.50 belongs to the highly conserved DRY-motif of class A GPCRs, which is located at the bottom of TM3. On the one hand, Arg^3.50 has been reported to help stabilize the inactive state of GPCRs, but on the other hand has also been shown to be crucial for stabilizing active receptor conformations and mediating receptor-G protein coupling. The combined results of these studies suggest that the exact function of Arg^3.50 is likely to be receptor-dependent and must be characterized independently for every GPCR. Consequently, we now present comparative molecular-dynamics simulations that use our recently described inactive-state and Gα-bound active-state homology models of the dopamine D2 receptor (D2R), which are either bound to dopamine or ligand-free, performed to identify the function of Arg132^3.50 in D2R. Our results are consistent with a dynamic model of D2R activation in which Arg132^3.50 adopts a dual role, both by stabilizing the inactive-state receptor conformation and enhancing dopamine-dependent D2R-G protein coupling.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Evidence for D2 Receptor Regulation of Dopamine Release in the Goldfish Retina

The possible existence of a dopamine D₂ receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [³H]dopamine and superfused with D₂ dopamine receptor agonists or antagonists to determine if there was an effect on [³H]dopamine release. The D₂ receptor antagonist sulpiride increased both baseline [³H]- dopamine release and [³H]dopamine release induced by an increase in extracellular potassium concentration. The D₂ receptor agonists LY-171555 and RU-24213 did not reduce baseline [³H]dopamine release but completely inhibited [³H]dopamine release induced by an increase in [K^±]_o. This action of the D₂ agonists was blocked by sulpiride. These studies demonstrate the existence of D₂ receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.     

CODA-RET reveals functional selectivity as a result of GPCR heteromerization

Here we present a new method that combines protein complementation with resonance energy transfer to study conformational changes in response to activation of a defined G protein-coupled receptor heteromer, and we apply the approach to the putative dopamine D1-D2 receptor heteromer. Remarkably, the potency of the D2 dopamine receptor (D2R) agonist R-(-)-10,11-dihydroxy-N-n-propylnoraporphine (NPA) to change the Gα(i) conformation via the D2R protomer in the D1-D2 heteromer was enhanced ten-fold relative to its potency in the D2R homomer. In contrast, the potencies of the D2R agonists dopamine and quinpirole were the same in the homomer and heteromer. Thus, we have uncovered a molecular mechanism for functional selectivity in which a drug acts differently at a G protein-coupled receptor (GPCR) protomer depending on the identity of the second protomer participating in the formation of the signaling unit--opening the door to enhancing pharmacological specificity by targeting differences between homomeric and heteromeric signaling.     

Receptor,Ligand and Transducer Contributions to Dopamine D2 Receptor Functional Selectivity

Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor’s signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D₂R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and β-arrestins. D₂R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D₂R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D₂R mutants in which G protein-dependent (^[Gprot]D₂R) and β-arrestin-dependent signaling (^[βarr]D₂R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D₂R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating ^[Gprot]D₂R and ^[βarr]D₂R conferred control of partial agonism at G protein and/or β-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and β-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D₂R functional selectivity and point to means by which novel in vivo therapies could be modeled.     

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary 1. Our aim was to test the hypothesis that selectivity for D₃ dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with³H-emonapride, using human D₃ receptors in mouse fibroblasts and D₂ receptors in rat striatal tissue.3. D₃ receptors showed a picomolar affinity for³H-emonapride, Na⁺ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D₃/D₂ pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (–)-PPHT and its fluorescein derivative], (–)-N-propylnorapomorphine, (–)-3-PPP, (–)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D₃-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.     

The Role of Water in Activation Mechanism of Human N-Formyl Peptide Receptor 1 (FPR1) Based on Molecular Dynamics Simulations

The Formyl Peptide Receptor 1 (FPR1) is an important chemotaxis receptor involved in various aspects of host defense and inflammatory processes. We constructed a model of FPR1 using as a novel template the chemokine receptor CXCR4 from the same branch of the phylogenetic tree of G-protein-coupled receptors. The previously employed template of rhodopsin contained a bulge at the extracellular part of TM2 which directly influenced binding of ligands. We also conducted molecular dynamics (MD) simulations of FPR1 in the apo form as well as in a form complexed with the agonist fMLF and the antagonist tBocMLF in the model membrane. During all MD simulation of the fMLF-FPR1 complex a water molecule transiently bridged the hydrogen bond between W254^6.48 and N108^3.35 in the middle of the receptor. We also observed a change in the cytoplasmic part of FPR1 of a rotamer of the Y301^7.53 residue (tyrosine rotamer switch). This effect facilitated movement of more water molecules toward the receptor center. Such rotamer of Y301^7.53 was not observed in any crystal structures of GPCRs which can suggest that this state is temporarily formed to pass the water molecules during the activation process. The presence of a distance between agonist and residues R201^5.38 and R205^5.42 on helix TM5 may suggest that the activation of FPR1 is similar to the activation of β-adrenergic receptors since their agonists are separated from serine residues on helix TM5. The removal of water molecules bridging these interactions in FPR1 can result in shrinking of the binding site during activation similarly to the shrinking observed in β-ARs. The number of GPCR crystal structures with agonists is still scarce so the designing of new ligands with agonistic properties is hampered, therefore homology modeling and docking can provide suitable models. Additionally, the MD simulations can be beneficial to outline the mechanisms of receptor activation and the agonist/antagonist sensing.     

Dopamine D2 and 5-hydroxytryptamine 5-HT(₂A) receptors assemble into functionally interacting heteromers

In view of the co-distribution of dopamine D_2LR and 5-hydroxytryptamine 5-HT_2A receptors (D_2LR and 5-HT_2AR, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D_2LR-5-HT_2AR heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D_2LR and the 5-HT_2AR form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D_2LR-5-HT_2AR heteromeric signaling was analyzed we found that the 5-HT_2AR-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D_2LR as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D_2LR-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D_2LR and 5-HT_2AR within the heteromer led to inhibition of the D_2LR functioning, thus suggesting the existence of a 5-HT_2AR-mediated D_2LR trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D₂R-5-HT_2AR may be involved in the receptor interface. Overall, the presence of the D_2LR-5-HT_2AR heteromer in discrete brain regions is postulated based on the existence of D_2LR-5-HT_2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.     

Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and beta-arrestin interaction

β-arrestin mediates the desensitization of GPCRs and acts as an adaptor molecule to recruit the receptor complex to clathrin-rich regions. Class-A GPCRs subsequently dissociate from β-arrestin but class-B GPCRs internalize with β-arrestin in the endocytic vesicles. Here the dopamine D₂ and D₃ receptors, which have similar structural features but different intracellular trafficking properties, were used in an attempt to better understand the structural requirements for the classification of GPCRs. The C-terminus tail of the vasopressin type-2 receptor was added to the ends of D₂R and D₃R to increase their affinity to β-arrestin. A point mutation was introduced into the DRY motif to change their basal activation levels. Among a battery of constructs in which the C-terminus tail and/or DRY motif was altered, class-B behavior was observed with the constructs whose affinities for β-arrestin were increased complementarily and their signaling was either maintained or regained. In conclusion, the DRY motif and C-terminal tail of the GPCRs determine complementarily their intracellular trafficking behavior by regulating the affinity to β-arrestin and G protein coupling.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

Functional expression of single-chain heterodimeric G-protein-coupled receptors for adenosine and dopamine

The direct homo- and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A(2A)R) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A(2A)R/A(2A)R vs. A(2A)R/D2R). In order to stimulate the heteromerization of A(2A)R and D2R, we have designed a single-polypeptide-chain heterodimeric A(2A)R/D2R complex by fusing the C-terminus of the A(2A)R via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A(2A)R/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.     

Can molecular dynamics simulations improve the structural accuracy and virtual screening performance of GPCR models?

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.     

Three amino acids in the D2 dopamine receptor regulate selective ligand function and affinity

The D₂ dopamine receptor is an important therapeutic target for the treatment of psychotic, agitated, and abnormal behavioral states. To better understand the specific interactions of subtype‐selective ligands with dopamine receptor subtypes, seven ligands with high selectivity (>120‐fold) for the D₄ subtype of dopamine receptor were tested on wild‐type and mutant D₂ receptors. Five of the selective ligands were observed to have 21‐fold to 293‐fold increases in D₂ receptor affinity when three non‐conserved amino acids in TM2 and TM3 were mutated to the corresponding D₄ amino acids. The two ligands with the greatest improvement in affinity for the D₂ mutant receptor [i.e., 3‐{[4‐(4‐iodophenyl) piperazin‐1‐yl]methyl}‐1H‐pyrrolo[2,3‐b]pyridine (L‐750,667) and 1‐[4‐iodobenzyl]‐4‐[N‐(3‐isopropoxy‐2‐pyridinyl)‐N‐methyl]‐aminopiperidine (RBI‐257)] were investigated in functional assays. Consistent with their higher affinity for the mutant than for the wild‐type receptor, concentrations of L‐750,667 or RBI‐257 that produced large reductions in the potency of quinpirole’s functional response in the mutant did not significantly reduce quinpirole’s functional response in the wild‐type D₂ receptor. In contrast to RBI‐257 which is an antagonist at all receptors, L‐750,667 is a partial agonist at the wild‐type D₂ but an antagonist at both the mutant D₂ and wild‐type D₄ receptors. Our study demonstrates for the first time that the TM2/3 microdomain of the D₂ dopamine receptor not only regulates the selective affinity of ligands, but in selected cases can also regulate their function. Utilizing a new docking technique that incorporates receptor backbone flexibility, the three non‐conserved amino acids that encompass the TM2/3 microdomain were found to account in large part for the differences in intermolecular steric contacts between the ligands and receptors. Consistent with the experimental data, this model illustrates the interactions between a variety of subtype‐selective ligands and the wild‐type D₂, mutant D₂, or wild‐type D₄ receptors.     

Molecular mechanisms involved in epidermal growth factor receptor-mediated inhibition of dopamine D3 receptor signaling

The phenomenon wherein the signaling by a given receptor is regulated by a different class of receptors is termed transactivation or crosstalk. Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) is highly diverse and has unique functional implications because of the distinct structural features of the receptors and the signaling pathways involved. The present study used the epidermal growth factor receptor (EGFR) and dopamine D₃ receptor (D₃R), which are both associated with schizophrenia, as the model system to study crosstalk between RTKs and GPCRs. Loss-of-function approaches were used to identify the cellular components involved in the tyrosine phosphorylation of G protein-coupled receptor kinase 2 (GRK2), which is responsible for EGFR-induced regulation of the functions of D₃R. SRC proto-oncogene (Src, non-receptor tyrosine kinase), heterotrimeric G protein Gβγ subunit, and endocytosis of EGFR were involved in the tyrosine phosphorylation of GRK2. In response to EGF treatment, Src interacted with EGFR in a Gβγ-dependent manner, resulting in the endocytosis of EGFR. Internalized EGFR in the cytosol mediated Src/Gβγ-dependent tyrosine phosphorylation of GRK2. The binding of tyrosine-phosphorylated GRK2 to the T142 residue of D₃R resulted in uncoupling from G proteins, endocytosis, and lysosomal downregulation. This study identified the molecular mechanisms involved in the EGFR-mediated regulation of the functions of D₃R, which can be extended to the crosstalk between other RTKs and GPCRs.     

Modeling GPCR active state conformations: the β(2)-adrenergic receptor

The recent publication of several G protein-coupled receptor (GPCR) structures has increased the information available for homology modeling inactive class A GPCRs. Moreover, the opsin crystal structure shows some active features. We have therefore combined information from these two sources to generate an extensively validated model of the active conformation of the β(2)-adrenergic receptor. Experimental information on fully active GPCRs from zinc binding studies, site-directed spin labeling, and other spectroscopic techniques has been used in molecular dynamics simulations. The observed conformational changes reside mainly in transmembrane helix 6 (TM6), with additional small but significant changes in TM5 and TM7. The active model has been validated by manual docking and is in agreement with a large amount of experimental work, including site-directed mutagenesis information. Virtual screening experiments show that the models are selective for β-adrenergic agonists over other GPCR ligands, for (R)- over (S)-β-hydroxy agonists and for β(2)-selective agonists over β(1)-selective agonists. The virtual screens reproduce interactions similar to those generated by manual docking. The C-terminal peptide from a model of the stimulatory G protein, readily docks into the active model in a similar manner to which the C-terminal peptide from transducin, docks into opsin, as shown in a recent opsin crystal structure. This GPCR-G protein model has been used to explain site-directed mutagenesis data on activation. The agreement with experiment suggests a robust model of an active state of the β(2)-adrenergic receptor has been produced. The methodology used here should be transferable to modeling the active state of other GPCRs.     

Vasopressin V2 Receptor/Bioligand Interactions

We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3–TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.