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The D1- and D2-dopamine receptors (D1R and D2R), which signal through G s and G i , respectively, represent the principal stimulatory and inhibitory dopamine receptors in the central nervous system. D1R and D2R also represent the main therapeutic targets for Parkinson's disease, schizophrenia, and many other neuropsychiatric disorders, and insight into their signaling is essential for understanding both therapeutic and side effects of dopaminergic drugs. Here, we report four cryoelectron microscopy (cryo-EM) structures of D1R-G s and D2R-G i signaling complexes with selective and non-selective dopamine agonists, including two currently used anti-Parkinson's disease drugs, apomorphine and bromocriptine. These structures, together with mutagenesis studies, reveal the conserved binding mode of dopamine agonists, the unique pocket topology underlying ligand selectivity, the conformational changes in receptor activation, and potential structural determinants for G protein-coupling selectivity. These results provide both a molecular understanding of dopamine signaling and multiple structural templates for drug design targeting the dopaminergic system., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Depression and stress-related disorders are leading causes of death worldwide. Standard treatments elevating serotonin or noradrenaline levels are not sufficiently effective and cause adverse side effects. A connection between dopamine pathways and stress-related disorders has been suggested. Compounds derived from herbal medicine could be a promising alternative. We examined the neuroprotective effects of ursolic acid (UA) by focusing on dopamine signalling., Methods: Trolox equivalent capacity assay was used to determine the antioxidant activities of UA in vitro. C. elegans N2 wildtype and dopamine receptor-knockout mutants (dop1-deficient RB665 and dop3-deficient LX703 strains) were used as in vivo models. H 2 DCFDA and acute juglone assays were applied to determine the antioxidant activity in dependency of dopamine pathways in vivo. Stress was assessed by heat and acute osmotic stress assays. The influence of UA on overall survival was analyzed by a life span assay. The dop1 and dop3 mRNA expression was determined by real time RT-PCR. We also examined the binding affinity of UA towards C. elegans Dop1 and Dop3 receptors as well as human dopamine receptors D1 and D3 by molecular docking., Results: Antioxidant activity assays showed that UA exerts strong antioxidant activity. UA increased resistance towards oxidative, osmotic and heat stress. Additionally, UA increased life span of nematodes. Moreover, dop1 and dop3 gene expression was significantly enhanced upon UA treatment. Docking analysis revealed stronger binding affinity of UA to C. elegans and human dopamine receptors than the natural ligand, dopamine. Binding to Dop1 was stronger than to Dop3., Conclusion: UA reduced stress-dependent ROS generation and acted through Dop1 and to a lesser extent through Dop3 to reduce stress and prolong life span in C. elegans. These results indicate that UA could be a promising lead compound for the development of new antidepressant medications., (Copyright © 2020. Published by Elsevier GmbH.)

The high functional plasticity of the glutamatergic synapse of the vestibular epithelium is supported by a delicate balance of excitatory and inhibitory interactions. One of the peptides co-localized with acetylcholine (ACh) in the efferent fibers is dopamine. Using external perfusion of the synaptic zone and multiunit recording of an afferent fibers activity the effect of dopamine antagonists on the background firing rate of the semicircular canal sensory fibers was studied on the isolated frog vestibular. The present research revealed that the dopamine receptor antagonist (D1) SCH-23390 significantly reduced the level of background activity at high concentrations. In contrast, D2 antagonist eticlopride caused positive-negative answer of the background activity of the sensory fibers in dose-depending manner. The data confirm the hypothesis that dopamine, being tonic released from the efferent fibers, realizes neuroprotective inhibitory control over the afferent glutamatergic synapse activity in the vestibular epithelium via D1 and D2 receptors.

The dorsolateral striatum (DLS) is essential for motor and procedure learning, but the role of DLS spiny projection neurons (SPNs) of direct and indirect pathways, as marked, respectively, by D1 and D2 receptor (D1R and D2R) expression, remains to be clarified. Long-term two-photon calcium imaging of the same neuronal population during mouse learning of a cued lever-pushing task revealed a gradual emergence of distinct D1R and D2R neuronal ensembles that reproducibly fired in a sequential manner, with more D1R and D2R neurons fired during the lever-pushing period and intertrial intervals (ITIs), respectively. This sequential firing pattern was specifically associated with the learned motor behavior, because it changed markedly when the trained mice performed other cued motor tasks. Selective chemogenetic silencing of D1R and D2R neurons impaired the initiation of learned motor action and suppression of erroneous lever pushing during ITIs, respectively. Thus, motor learning involves reorganization of DLS neuronal activity, forming stable D1R and D2R neuronal ensembles that fired sequentially to regulate different aspects of the learned behavior., Competing Interests: The authors declare no conflict of interest.

Methiopropamine (MPA) is structurally categorized as a thiophene ring-based methamphetamine (MA) derivative. Although abusive potential of MPA was recognized, little is known about the neurotoxic potential of MPA up to now. We investigated whether MPA induces dopaminergic neurotoxicity, and whether MPA activates a specific dopamine receptor. Here, we observed that treatment with MPA resulted in dopaminergic neurotoxicity in a dose-dependent manner. MPA treatment potentiated oxidative parameters (i.e., increases in the level of reactive oxygen species, 4-hydroxynonenal, and protein carbonyl), M1 phenotype-related microglial activity, and pro-apoptotic property (i.e., increases in Bax- and cleaved caspase-3-expressions, while a decrease in Bcl-2-expression). Moreover, treatment with MPA resulted in significant impairments in dopaminergic parameters [i.e., changes in dopamine level, dopamine turnover rate, tyrosine hydroxylase (TH) levels, dopamine transporter (DAT) expression, and vesicular monoamine transporter-2 (VMAT-2) expression], and in behavioral deficits. Both dopamine D1 receptor antagonist SCH23390 and D2 receptor antagonist sulpiride protected from these neurotoxic consequences. Therefore, our results suggest that dopamine D1 and D2 receptors simultaneously mediate MPA-induced dopaminergic neurodegeneration in mice via oxidative burdens, microgliosis, and pro-apoptosis., (Copyright © 2019. Published by Elsevier B.V.)

Dopamine D1/D2 receptors are important targets for drug discovery in the treatment of central nervous system diseases. To discover new and potential D1/D2 ligands, 17 derivatives of tetrahydroprotoberberine (THPB) with various substituents were prepared by chemical synthesis or microbial transformation using Streptomyces griseus ATCC 13273. Their functional activities on D1 and D2 receptors were determined by cAMP assay and calcium flux assay. Seven compounds showed high activity on D1/D2 receptor with low IC 50 values less than 1 µM. Especially, top compound 5 showed strong antagonistic activity on both D1 and D2 receptor with an IC 50 of 0.391 and 0.0757 µM, respectively. Five compounds displayed selective antagonistic activity on D1 and D2 receptor. The SAR studies revealed that (1) the hydroxyl group at C-9 position plays an important role in keeping a good activity and small or fewer substituents on ring D of THPBs may also stimulate their effects, (2) the absence of substituents at C-9 position tends to be more selective for D2 receptor, and (3) hydroxyl substitution at C-2 position and the substitution at C-9 position may facilitate the conversion of D1 receptor from antagonist to agonist. Molecular docking simulations found that Asp 103/Asp 114, Ser 107/Cys 118, and Trp 285/ Trp 386 of D1/ D2 receptors are the key residues, which have strong interactions with the active D1/D2 compounds and may influence their functional profiles., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Dysfunction of the dopaminergic pathway is linked to numerous diseases of the nervous system. The D1-D2 receptor heteromer is known to play a role in certain neuropsychiatric disorders, such as depression. Here, we synthesized an eight amino acid residue peptide, EAARRAQE, derived from the third intracellular loop of the D2 receptor and show that the peptide binds to the D1 receptor with comparable efficiency as that of the full-length D2 receptor protein. Moreover, immunoprecipitation studies show the existence of a heteromeric complex formed both in vitro and in total protein derived from temporal and frontal lobe tissue from normal and depressed subjects. The efficiency of the peptide to block the D1-D2 heteromeric complex was comparable in all the samples tested., (© 2019 Federation of European Biochemical Societies.)

We tested the interactions with four different G protein-coupled receptors (GPCRs) of arrestin-3 mutants with substitutions in the four loops, three of which contact the receptor in the structure of the arrestin-1-rhodopsin complex. Point mutations in the loop at the distal tip of the N-domain (Glu157Ala), in the C-loop (Phe255Ala), back loop (Lys313Ala), and one of the mutations in the finger loop (Gly65Pro) had mild variable effects on receptor binding. In contrast, the deletion of Gly65 at the beginning of the finger loop reduced the binding to all GPCRs tested, with the binding to dopamine D2 receptor being affected most dramatically. Thus, the presence of a glycine at the beginning of the finger loop appears to be critical for the arrestin-receptor interaction., Competing Interests: The authors have declared that no competing interests exist.

MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This review summarizes modern data on the structure and functions ofheteromersformed by D1 and D2 dopamine receptors focusing on their role in the mechanisms of drug dependence. This article discusses potential functional significance of heterodimeric D1-D2 dopamine receptorsdue to their localization in the brain as well as unique pharmacological propertiesversus constituent monomers. It is shown that heteromerization results in dramatic changes in activated signaling pathways compare to the corresponding monomers. These studies update our current knowledge of ligand-receptor interactions and provide better understanding of dopamine receptors pharmacology. Furthermore elucidation of significance of heterodimeric D1-D2 dopamine receptors as drug targets is important for the development of new effective drug addiction treatment.

The striatum plays important motor, associative and cognitive roles in brain functions. However, the rodent dorsolateral (the primate putamen) and dorsomedial (the primate caudate nucleus) striatum are not anatomically separated, making it difficult to distinguish their functions. By contrast, anatomical separation exists between the caudate nucleus and putamen in primates. Here, we successfully decreased dopamine D1 receptor (D1R) or D2R mRNA expression levels selectively in the marmoset caudate using shRNA knockdown techniques, as determined using positron emission tomography imaging with specific D1R and D2R ligands and postmortem in situ hybridization analysis. We then conducted a voxel-based correlation analysis between binding potential values of PET imaging and visual discrimination learning task performance in these genetically modified marmosets to find a critical role for the caudate D2R but no apparent role for the caudate D1R. This latter finding challenges the current understanding of the mechanisms underlying D1R activation in the caudate.

(-)-Stepholidine is an active ingredient of the Chinese herb Stephania and naturally occurring tetrahydroprotoberberine alkaloid with mixed dopamine receptor D1 agonistic and dopamine receptor D2 antagonistic activities. In this work, a series of novel hexahydrobenzo[4,5]azepino [2,1-a]isoquinolines were designed and synthesized as ring-expanded analogues of (-)-Stepholidine. Initial pharmacological assays demonstrated that a benzazepine replacement was associated with significant increase in selectivity and functional reversal at dopamine receptor D1 . Compound-(-)-15e (Ki  = 5.32 ± 0.01 nm) is more potent than (-)-Stepholidine (Ki  = 13 nm) and was identified as a selective dopamine receptor D1 antagonist (IC50  = 0.14 μm). Moreover, molecular modeling suggested that (-)-15e might exert its dopamine receptor D1 antagonistic activities through interacting with the transmembrane helix 7 of dopamine receptor D1 ., (© 2016 John Wiley & Sons A/S.)

A role for the dopamine D1-D2 receptor heteromer in the regulation of reward and addiction-related processes has been previously implicated. In the present study, we examined the effects of D1-D2 heteromer stimulation by the agonist SKF 83959 and its disruption by a selective TAT-D1 peptide on amphetamine-induced locomotor sensitization, a behavioral model widely used to study the neuroadaptations associated with psychostimulant addiction. D1-D2 heteromer activation by SKF 83959 did not alter the acute locomotor effects of amphetamine but significantly inhibited amphetamine-induced locomotor responding across the 5day treatment regimen. In addition, a single injection of SKF 83959 was sufficient to abolish the expression of locomotor sensitization induced by a priming injection of amphetamine after a 72-hour withdrawal. Conversely, inhibition of D1-D2 heteromer activity by the TAT-D1 peptide enhanced subchronic amphetamine-induced locomotion and the expression of amphetamine locomotor sensitization. Treatment solely with the TAT-D1 disrupting peptide during the initial 5day treatment phase was sufficient to induce a sensitized locomotor phenotype in response to the priming injection of amphetamine. Together these findings demonstrate that the dopamine D1-D2 receptor heteromer exerts a tonic inhibitory control on neurobiological processes involved in sensitization to amphetamine, indicating that the dopamine D1-D2 receptor heteromer may be a novel molecular substrate in addiction processes involving psychostimulants., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

A novel scaffold derived from l-SPD with a substituted thiophene group in the D ring were designed, synthesized, and evaluated for their binding affinities at dopamine (D1, D2 and D3) and serotonin (5-HT1A and 5-HT2A) receptors. Most of the tetracyclic compounds exhibited higher affinities for D2 and 5-HT1A receptors than l-SPD, while compound 23 e showed the highest Ki value of 7.54 nM at D2 receptor which was 14 times more potent than l-SPD. Additionally, compounds 23 d and 23 e were more potent than l-SPD at D3 receptor. According to the functional assays, 23 d and 23 e were demonstrated as full antagonists at D1 and D2 receptors and full agonists at 5-HT1A receptor. Since the combination of D2 antagonism and 5-HT1A agonism is considered effective in treating both the positive and negative symptoms of schizophrenia, these novel compounds are implicated as potential therapeutic agents., (Copyright © 2014. Published by Elsevier Ltd.)

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.

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 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.)

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.)

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.

A set of novel apomorphine derivatives were synthesized with diversely functionalized side chains in the proximity of position 2 of the aporphine skeleton. Amino and/or carboxylic functions were introduced to this region of the backbone to test their pharmacological effects. During the synthesis of 2-(S-3-mercaptopropionic acid)-derivative a heteroring-fused congener was also isolated. The structural elucidation confirmed that the formation of this product was in accordance with our previous observations on the reaction of thebaine (2) with thiosalycilic acid. All the novel apomorphine congeners 4a-g were neuropharmacologically characterized to discover their dopaminergic profiles. Two derivatives were identified as D(2) full agonists equipotent with apomorphine (1) having significantly increased D(2)/D(1) selectivity ratios., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR-NLS is retained at the cell surface, and following ligand removal, the GPCR-NLS translocated from the cell surface. The D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dopamine and its receptors play a critical role in diseases such as Parkinson's disease and schizophrenia. A problem with developing specific drugs for such diseases is that there are five subtypes of dopamine receptors that can be categorized as either D1 like or D2 like. Since the binding sites are quite similar, it is difficult to design the subtype specific agonists and antagonists required for therapy with minimal side effects. Thus, the aim of this study was to identify the molecular characteristics important to the selective binding of dopamine D1 like and D2 like receptors using quantitative structure activity relationships (QSARs). Datasets of 29 and 69 molecules capable of binding to cloned human D1 and D2 receptors were used to build QSAR models. The dissociation constants (Ki) for these molecules were taken from the literature. The optimized 3D structure of each molecule was encoded with 62 theoretical molecular descriptors. The QSAR, using hybrid neural network modeling, was built using categorical and continuous molecular descriptors as inputs, with dissociation constants (Ki) as outputs. Categorically assigned molecular descriptors improved performance in both models. Secondary amines and other nitrogen-containing moieties were shown to be important for the D1 like receptor selectivity, whereas molecular size, volume and tertiary and quaternary carbons were found to be of significant importance for the D2 like receptor selectivity.

It is often difficult to differentiate effectively between related G-protein coupled receptors and their subtypes when doing ligand-based drug design. GALAHAD uses a multi-objective scoring system to generate multiple alignments involving alternative trade-offs between the conflicting desires to minimize internal strain while maximizing pharmacophoric and steric (pharmacomorphic) concordance between ligands. The various overlays obtained can be associated with different subtypes by examination, even when the ligands available do not discriminate completely between receptors and when no specificity information has been used to bias the alignment process. This makes GALAHAD a potentially powerful tool for identifying discriminating models, as is illustrated here using a set of dopaminergic agonists that vary in their D1 vs. D2 receptor selectivity.

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically similar but biologically divergent target compounds served as molecular probes for radioligand displacement experiments, mutagenesis, and docking studies on homology models based on the recent crystal structure of the beta2-adrenergic receptor. Specific interactions with the highly conserved amino acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by nonconserved but frequently mediated by conserved amino acids.

In this report, we investigated whether the D5 dopamine receptor, given its structural and sequence homology with the D1 receptor, could interact with the D2 receptor to mediate a calcium signal similar to the G(q/11) protein-linked phospholipase C-mediated calcium signal resulting from the coactivation of D1 and D2 dopamine receptors within D1-D2 receptor heterooligomers. Fluorescent resonance energy transfer experiments demonstrated close colocalization of cell surface D5 and D2 receptors (<100 A), indicating hetero-oligomerization of D5 and D2 receptors in cells coexpressing both receptors. Coactivation of D5 and D2 receptors within the D5-D2 hetero-oligomers activated a calcium signal. However, unlike what is observed for D1 receptors, which activate extensive calcium mobilization only within a complex with the D2 receptors, a robust calcium signal was triggered by D5 receptors expressed alone. Hetero-oligomerization with the D2 receptor attenuated the ability of the D5 receptor to trigger a calcium signal. The D5 and D5-D2-associated calcium signals were G(q/11) protein-linked and phospholipase C-mediated but were also critically dependent on the influx of extracellular calcium through store-operated calcium channels, unlike the calcium release triggered by D1-D2 heterooligomers. Collectively, these results demonstrate that calcium signaling through D5-D2 receptor hetero-oligomers occurred through a distinct mechanism to achieve an increase in intracellular calcium levels.

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D(2) receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D(1) receptor (404EE405) on the receptors' localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D(1)-D(2) receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D(2)R, which may suggest that the indicated region of ic3 in D(2)R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D(1)-cyan fluorescent protein, D(2)-yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg-rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein-tagged D(2)R and cyan fluorescent protein-tagged D(1)R may result from differing localization of these proteins in the cell rather than from the possible role of the D(2)R basic domain in the mechanism of D(1)-D(2) receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D(1) receptor is engaged in the heterodimerization process.

The tight correlation between the clinical potency and the D2R blocking action of antipsychotic medications suggests that dopamine hyperactivity plays a significant role in psychosis. Clozapine, one of the most effective antipsychotic drugs, has been shown to display moderate affinity for various neurotransmitter receptors, including the dopamine D1 and D2 receptors; however, the exact mechanism of action of clozapine has not yet been fully elucidated. Here, we describe our working hypothesis pointing to the role of dopamine D1-D2 receptor hetero-dimerization as a mechanism of action of clozapine. It has been widely assumed that D1 and D2 receptors are segregated to separate neuronal populations; however, other data suggest that D1 and D2 receptors are co-expressed by a moderate to substantial proportion of striatal neurons, as well as in the medial prefrontal cortex. Our recent studies indicate that concomitant stimulation of both D1 and D2 dopamine receptors induces an increase in their hetero-dimerization. In order to confirm the working hypothesis that clozapine influences D1-D2 receptor oligomerization, we employed fluorescence resonance energy transfer (FRET) technology, using fluorescently tagged dopamine receptors and fluorescence lifetime microscopy of intact living cells. The effect of clozapine on D1R-D2R hetero-oligomerization was strongly dependent on the drug concentration; the lower concentration, which resulted in binding to the high affinity sites, decreased the transfer efficiency, while the higher concentration of clozapine enhanced transfer efficiency. Further investigation confirmed the idea that high affinity binding sites exist when the receptor is coupled with G protein, and also that clozapine attenuates the hetero-oligomerization of a high affinity pool of dopamine D1-D2 receptors. The results discussed in the present study, showing the effect of clozapine on D1-D2 receptor hetero-oligomerization, together with the data pointing to the importance of receptors forming hetero-oligomers as a novel level for pharmacological intervention help to increase the understanding of the molecular mechanism of action of antipsychotic drugs.

Dibenzo- and benzindolo-azecines represent a novel class of high-affinity dopamine receptor antagonists. To further characterize these drugs as potential neuroleptics, we selected a set of azecine derivatives and ring expanded homologues and we measured their antagonist activity at the 5-HT(2A) receptor in the porcine coronary artery. SARs found for the 5-HT(2A) receptor resemble those for the D1 but not the D2 receptor. The protein-ligand interactions were discussed with respect to the different binding pockets.

(-)-Stepholidine (SPD), an active ingredient of the Chinese herb Stephania, is the first compound found to have dual function as a dopamine receptor D1 agonist and D2 antagonist. Insights into dynamical behaviors of D1 and D2 receptors and their interaction modes with SPD are crucial in understanding the structural and functional characteristics of dopamine receptors. In this study a computational approach, integrating protein structure prediction, automated molecular docking, and molecular dynamics simulations were employed to investigate the dual action mechanism of SPD on the D1 and D2 receptors, with the eventual aim to develop new drugs for treating diseases affecting the central nervous system such as schizophrenia. The dynamics simulations revealed the surface features of the electrostatic potentials and the conformational "open-closed" process of the binding entrances of two dopamine receptors. Potential binding conformations of D1 and D2 receptors were obtained, and the D1-SPD and D2-SPD complexes were generated, which are in good agreement with most of experimental data. The D1-SPD structure shows that the K-167&#95;EL-2-E-302&#95;EL-3 (EL-2: extracellular loop 2; EL-3: extracellular loop 3) salt bridge plays an important role for both the conformational change of the extracellular domain and the binding of SPD. Based on our modeling and simulations, we proposed a mechanism of the dual action of SPD and a subsequent signal transduction model. Further mutagenesis and biophysical experiments are needed to test and improve our proposed dual action mechanism of SPD and signal transduction model.

When dopamine D1 and D2 receptors were coactivated in D1-D2 receptor hetero-oligomeric complexes, a novel phospholipase C-mediated calcium signal was generated. In this report, desensitization of this Gq/11-mediated calcium signal was demonstrated by pretreatment with dopamine or with the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-81297) or the D2-selective agonist quinpirole. Desensitization of the calcium signal mediated by D1-D2 receptor hetero-oligomers was initiated by agonist occupancy of either receptor subtype even though the signal was generated only by occupancy of both receptors. The efficacy, potency, and rate of calcium signal desensitization by agonist occupancy of the D1 receptor (t1/2, approximately 1 min) was far greater than by the D2 receptor (t1/2, approximately 10 min). Desensitization of the calcium signal was not mediated by depletion of calcium stores or internalization of the hetero-oligomer and was not decreased by inhibiting second messenger-activated kinases. The involvement of G protein-coupled receptor kinases 2 or 3, but not 5 or 6, in the desensitization of the calcium signal was shown, occurring through a phosphorylation independent mechanism. Inhibition of Gi protein function associated with D2 receptors increased D1 receptor-mediated desensitization of the calcium signal, suggesting that cross-talk between the signals mediated by the activation of different G proteins controlled the efficacy of calcium signal desensitization. Together, these results demonstrate the desensitization of a signal mediated only by hetero-oligomerization of two G protein-coupled receptors that was initiated by agonist occupancy of either receptor within the hetero-oligomer, albeit with differences in desensitization profiles observed.

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

We demonstrate a heteromeric D1-D2 dopamine receptor signaling complex in brain that is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. We provide evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase IIalpha in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity.

Evidence for hetero-oligomerization has recently been provided for various G protein-coupled receptors. In this paper, we have studied the possibility that dopamine D(1) and D(2) receptors physically interact with each other. Human dopamine D(1) and D(2) receptors were fluorescently tagged with derivatives of green fluorescence protein and transiently coexpressed in the membrane of human embryonic kidney 293 cells. Using qualitative fluorescence spectroscopy, as well as quantitative Förster resonance energy transfer (FRET) analysis, performed in a single cell by confocal microscopy and fluorescence lifetime microscopy, we show that dopamine D(1) and D(2) receptors can form hetero-oligomers in the plasma membrane. The degree of receptor protein-protein interaction is significantly enhanced by concomitant addition of D(1) and D(2) receptor subtype-specific agonists. Our investigations extend biochemical and electrophysiological studies and give insights into the regulation and synergistic mode of operation of dopamine receptors.

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.

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.

We provided evidence for the formation of a novel phospholipase C-mediated calcium signal arising from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity indicative of D1-D2 receptor heterooligomerization. The close proximity of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (+/-)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers.

Functional interactions between adenosine A2A and dopamine D2 receptors have been demonstrated both at the D2 agonist-binding and second messenger levels. The present studies use a [3H]dopamine-binding assay as a sensitive measure of A2A receptor-mediated modulation of D2 receptors. Co-incubation with an A2A receptor agonist increased the Kd value of high-affinity [3H]dopamine-binding sites of the D2 receptor without changing their Bmax values in a cotransfected cell line. This interaction was shown to be subtype specific, as the A2A receptor agonist did not modulate the affinity of the D1 receptor for [3H]dopamine. The domains of the D2 receptor important for the A2A/D2 receptor interaction were studied with chimeric dopamine D2/D1 receptors. The results showed that the A2A receptor agonist still strongly reduced the affinity of a D2/D1 chimera with the sixth transmembrane (TM) domain and third extracellular loop from the D1 receptor. However, the A2A receptor agonist was not able to modulate a D2/D1 chimeric receptor containing the fifth and sixth TM domains and the third intracellular and extracellular loops from the D1 receptor, indicating that the fifth TM domain and/or the third intracellular loop may be involved in the interaction between A2A and D2 receptors.

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.

This article describes D1 and D2 receptor subfamilies, their neuroanatomic localizations and the possibility of their neuronal co-localization and relation to the other dopamine autoreceptors and those coupled to G-proteins as well as the actions of antipsychotic drugs on D1 and D2 receptor subfamilies are also analyzed.

One of the key events in the actions of agonists and antagonists is their binding to receptors. Understanding this event is of interest in terms of understanding receptor function but it also has immense practical relevance for the design of drugs. If the ligand-binding process could be understood in detail, including the nature of the interactions made between ligand and receptor, then this could help in the design of more-selective drugs. The interaction of a ligand with its receptor is clearly of importance in determining the specificity of ligand action but ligand-receptor interaction also initiates the processes of signalling that are exhibited in the efficacy of ligand action. Here Philip Strange considers these events for catecholamine receptors, concentrating mostly on dopamine receptors; where necessary the discussion is widened to include other receptor systems.

Aim: To build up the structure models of dopamine receptors, then combined with the receptor models, to investigate the action mechanism of tetrahydroprotoberberines (THPB) on dopamine receptors at the molecular level., Methods: Using the three-dimensional structure of bacteriorhodopsin as a template, we have constructed dopamine D1 and D2 receptor models on computer. l-Stepholidine was selected as the leading compound of THPB and docked into D1 and D2 receptor active sites., Results: After manual adjustment and energy minimization, the ligand-receptor interaction models were achieved. Based on these models, the possible action mechanism of THPB on dopamine receptors was suggested that the protonated N atom of THPB form electrostatic interaction and hydrogen-bonding interaction with residue Asp in TM3 of the receptor, the two substituents in D ring of THPB form hydrogen-bonding interactions with two Ser residues in TM5 of the receptor, and the aryl groups form pi-pi interactions with some aryl residues of the receptor around ligand., Conclusion: Our ligand-receptor interaction models should be helpful for rational design of more potent drugs.

D1/D2 chimeras were constructed that had D1 dopamine receptor sequence at the amino-terminal end and D2 dopamine receptor sequence at the carboxyl-terminal end. The chimeras with the first four, five and six transmembrane domains of the D1 receptor (CH2, CH3, CH4, respectively) bound the D1 receptor antagonist [3H]SCH 23390 with high affinity. Reciprocal chimeras constructed with D2 receptor sequence at the amino-terminal end displayed no detectable specific binding of [3H]SCH 23390, [125I]epidepride, or [3H]spiperone. CH2, CH3, and CH4 had lower affinity than either D1 or D2 dopamine receptors for the nonselective antagonists and agonists and D2-selective antagonists tested. The chimeric receptors had affinities for three D1-selective ligands and the D2-selective agonist, quinpirole, that were intermediate between D1 and D2 receptor affinities for the drugs. The substantial loss or gain of affinity for three ligands upon replacement of D1 transmembrane VII with D2 sequence (CH4) suggests an important role for this region in the selectivity of these drugs. Stimulation of adenylyl cyclase activity by D1 agonists occurred in cells expressing CH3 and CH4, both of which included the D1 third cytoplasmic loop, but not in cells expressing CH1 or CH2, both with the D2 third cytoplasmic loop. However, only CH3 was able to mediate stimulation of adenylyl cyclase by quinpirole, implying that D2 receptor transmembrane domain VI was an important determinant of the selective efficacy of quinpirole. On the other hand, transmembrane domain VII was particularly important for the selective potency of quinpirole. Inhibition of beta-adrenergic receptor-stimulated adenylyl cyclase activity by dopamine was seen in cells expressing D2 receptors and CH1, but not CH2, CH3, or CH4. Thus, the third cytoplasmic loop of D1 dopamine receptors was crucial for the coupling of the receptors to Gs, but inhibition of adenylyl cyclase via Gi required structural features, such as the second cytoplasmic loop of the D2 receptor, in addition to the 3rd cytoplasmic loop.

In guanine nucleotide-binding regulatory protein- (G protein) coupled receptors, an amphipathic alpha-helix has been postulated to be the common structural determinant in the NH2- and COOH-terminal portions of the third intracellular loop representing the major interaction site with the G proteins. Here we assessed the ability of six peptides derived from these sites of the human dopamine D1-, D2-, and beta 1-adrenergic receptors to either activate G proteins directly or to uncouple the activity of their respective receptors in a native membrane environment. In addition, the cross-reactivity was analyzed. Nonspecific effects occurring at high concentrations were differentiated from G protein-specific effects. The peptide D2N derived from the NH2-terminal part of the third intracellular loop of the dopamine D2 receptor was the only one capable of specifically reversing the action of its receptor, the dopamine-mediated inhibition of the adenylyl cyclase. Furthermore, only D2N stimulated pertussis toxin-sensitive G proteins. However, D2N as the only peptide exhibiting specific effects did not exhibit the predicted amphipathic alpha-helix observed for mastoparan (Higashijima, T., Burnier, J., and Ross, E. M. (1990) J. Biol. Chem. 265, 14176-14186) as demonstrated by circular dichroism spectroscopy. In contrast, a peptide for which a certain degree of helicality was verified spectroscopically (D1C) was neither active in GTPase and adenylyl cyclase determinations, nor did it block the receptor-mediated cyclase activation. Hence, the amphipathic alpha-helix does not represent the main structural determinant for the receptor-G protein interaction site.