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ONC201 is a first-in-class imipridone compound that is in clinical trials for the treatment of high-grade gliomas and other advanced cancers. Recent studies identified that ONC201 antagonizes D2-like dopamine receptors at therapeutically relevant concentrations. In the current study, characterization of ONC201 using radioligand binding and multiple functional assays revealed that it was a full antagonist of the D2 and D3 receptors (D2R and D3R) with low micromolar potencies, similar to its potency for antiproliferative effects. Curve-shift experiments using D2R-mediated β -arrestin recruitment and cAMP assays revealed that ONC201 exhibited a mixed form of antagonism. An operational model of allostery was used to analyze these data, which suggested that the predominant modulatory effect of ONC201 was on dopamine efficacy with little to no effect on dopamine affinity. To investigate how ONC201 binds to the D2R, we employed scanning mutagenesis coupled with a D2R-mediated calcium efflux assay. Eight residues were identified as being important for ONC201's functional antagonism of the D2R. Mutation of these residues followed by assessing ONC201 antagonism in multiple signaling assays highlighted specific residues involved in ONC201 binding. Together with computational modeling and simulation studies, our results suggest that ONC201 interacts with the D2R in a bitopic manner where the imipridone core of the molecule protrudes into the orthosteric binding site, but does not compete with dopamine, whereas a secondary phenyl ring engages an allosteric binding pocket that may be associated with negative modulation of receptor activity. SIGNIFICANCE STATEMENT: ONC201 is a novel antagonist of the D2 dopamine receptor with demonstrated efficacy in the treatment of various cancers, especially high-grade glioma. This study demonstrates that ONC201 antagonizes the D2 receptor with novel bitopic and negative allosteric mechanisms of action, which may explain its high selectivity and some of its clinical anticancer properties that are distinct from other D2 receptor antagonists widely used for the treatment of schizophrenia and other neuropsychiatric disorders., (U.S. Government work not protected by U.S. copyright.)

We previously proposed that the dopamine D2 receptor-interacting protein S100B binds to a putative S100B-binding motif at residues R233-L240 toward the N terminus of the third intracellular loop. We used in vitro pull-down assays with FLAG-tagged fragments of the rat dopamine D2 receptor third intracellular loop (D2-IC3) and in vitro-synthesized S100B to evaluate this hypothesis. Our results indicate that the putative S100B-binding motif is neither necessary nor sufficient for strong binding of S100B to D2-IC3. Instead, two residues at the junction of the fifth membrane-spanning domain and the cytoplasmic extension of that α -helical domain, K211-I212, are required for robust, calcium-sensitive binding of S100B. This is also the approximate location of previously identified determinants for the binding of arrestin and calmodulin. A D2 receptor mutation converting I212 to phenylalanine has been described in patients with a hyperkinetic movement disorder. SIGNIFICANCE STATEMENT: S100B is a small calcium-binding protein that modulates signaling by the dopamine D2 receptor. New data suggest that the previous hypothesis about the involvement of an S100B-binding motif is incorrect, and that an important determinant of S100B binding includes a residue that is mutated in patients with a hyperkinetic movement disorder., (U.S. Government work not protected by U.S. copyright.)

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

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

In our previous studies, we demonstrated that the mutation of His393(6.55) to alanine results in an increased affinity of 1,4-disubstituted phenylpiperazines to the dopamine D(2L) receptor. This change most likely accounts for the reduced steric hindrance in this part of the binding pocket. In this work, we investigated the role of the steric hindrance imposed by the residue His393(6.55) for the receptor activation modulated by 1,4-disubstituted aromatic piperidines/piperazines. Site-directed mutagenesis and ligand modifications were used to probe the structural basis of ligand efficacy. The operational model of agonism was used to quantify the ligand bias between the ability of compounds to inhibit cAMP accumulation and stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Whereas substantial ligand-biased signaling was observed for the D(2L) wild-type receptor, an overall increase in agonism was observed for the D(2L) H393(6.55)A mutant without noteworthy functional selectivity. Targeted chemical modification of the phenylpiperazine moiety at the site of its interaction with the residue His393(6.55) led to the functionally selective ligand {3-[4-(2,3-dihydro-benzofuran-7-yl)-piperazin-1-yl]-propyl}-pyrazol[1,5-a]pyridine-3-carboxamide (FAUC350) that has distinct signaling profiles toward adenylyl cyclase and ERK1/2. FAUC350 behaves as an antagonist in the inhibition of cAMP accumulation and as a partial agonist in the stimulation of ERK1/2 phosphorylation (efficacy = 55%). Overall, the residue His393(6.55) and proximate molecular substructures of receptor ligands were identified to be crucial for multidimensional ligand efficacy.

The quaternary structure of G protein-coupled receptors (GPCRs) can influence their trafficking and ability to transduce signals. GPCR oligomers are generally portrayed as long-lived entities, although the stability of these complexes has not been studied. Here we show that D2 dopamine receptor protomers interact transiently at a specific oligomer interface. Selective immobilization of cyan fluorescent protein-D2 receptors (C-D2Rs) in the plasma membrane failed to completely immobilize coexpressed D2-venus receptors (D2R-Vs), suggesting that the two did not form stable oligomers with each other. Oxidative cross-linking stabilized C-D2R-D2R-V oligomers such that immobilization of C-D2R also immobilized D2R-V. This stabilization required the presence in both C-D2R and D2R-V of a cysteine residue in transmembrane domain 4 (TM4), a region identified as a putative oligomer interface in these and other class A GPCRs. These results suggest that the interaction of D2 receptor protomers at TM4 is transient unless stabilized and that the quaternary structure of these receptors may thus be subject to physiological or pharmacological regulation.

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.

Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

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.

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.

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.

Adenosine A(2A) and dopamine D(2) receptors have been shown previously to form heteromeric complexes and interact at the level of agonist binding, G protein coupling, and trafficking. Because dopamine D(2) and D(3) receptors show a high degree of sequence homology, A(2A) and D(3) receptors may also interact in a similar manner. The present studies with confocal microscopy showed that A(2A)-yellow fluorescent protein (YFP) and D(3)-green fluorescent protein 2 (GFP2) receptors colocalize in the plasma membrane. Furthermore, fluorescence resonance energy transfer (FRET) analysis demonstrated that A(2A)-YFP and D(3)-GFP2 receptors give a positive FRET efficiency and are thereby likely to exist as heteromeric A(2A)/D(3) receptor complexes. Saturation experiments with [(3)H]dopamine demonstrated that the A(2A) receptor agonist 4-[2-[[6-amino-9(N-ethyl-beta-d-ribofuranuronaminoamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS-21680) reduced the affinity of the high-affinity agonist binding state of the D(3) receptor for [(3)H]dopamine. The A(2A) and D(2A) receptors seem to interact also at the level of G protein coupling, because the adenosine A(2A) receptor agonist CGS-21680 fully counteracted the D(3) receptor-mediated inhibition of a forskolin-mediated increase in cAMP levels. Taken together, when coexpressed in the same neuron, A(2A) and D(3) receptors seem to form A(2A)/D(3) heteromeric receptor complexes in which A(2A) receptors antagonistically modulate both the affinity and the signaling of the D(3) receptors. D(3) receptor is one of the therapeutic targets for treatment of schizophrenia, and therefore, the A(2A)/D(3) receptor interactions could provide an alternative antischizophrenic treatment.

We previously demonstrated that, in the D4 dopamine receptor, the aromatic microdomain that spans the interface of the second and third transmembrane segments influences the high-affinity interactions with the D4-selective ligand L750,667 [3-[[4-(4-iodophenyl) piperazin-1-yl]methyl]-1H-pyrrolo[2,3-b]pyridine] and the D2-selective ligands methylspiperone, aripiprazole, and its congener OPC4392 [7-[3-(4-(2,3-dimethylphenyl) piperazinyl) propoxy] 2-(1H)-quinolinone] (Schetz et al., 2000). Here we tested a variety of 1,4-disubstituted aromatic piperidines/piperazines (1,4-DAPs) with different subtype selectivities and functional properties against a panel of D4 receptor mutations in the aromatic microdomain to ascertain whether these ligands recognize this common site. Mutant D4 receptors were constructed by substituting the nonconserved amino acid(s) from the corresponding locations in the D2 receptor. The D4-L2.60W, D4-F2.61V, and D4-LM3.28-3.29FV substitutions result in alterations of the relative position of members of the aromatic microdomain. From these results and molecular models of the ligand-receptor complexes, we conclude that 9 of the 11 D4-selective 1,4-DAPs, including L750,667, have a common pattern of ligand-receptor recognition that depends upon favorable interactions with the phenylalanine at position 2.61 (D4-F2.61V, 20-96-fold decrease). Like methylspiperone, aripiprazole, and OPC4392, the two D4-selective 1,4-DAPs that are insensitive to the D4-F2.61V mutation are sensitive to aromatics at position 2.60 (D4-L2.60W, 7-20-fold increase), and they all have longer spacer arms that permit their tethered aromatics to adopt alternative orientations in the binding-site crevice. All 11 of the D4-selective 1,4-DAPs were sensitive to the D4-LM3.28-3.29FV mutation (13-494-fold decrease) but not the moderately D2-selective methylspiperone. The inferences suggest that subtype selectivity involves two different modes of interaction with the microdomain for the D4-selective 1,4-DAPs and a third mode for D2-selective 1,4-DAPs.

A homology model of the dopamine D2 receptor was constructed based on the crystal structure of rhodopsin. A putative sodium-binding pocket identified in an earlier model (PDB ) was revised. It is now defined by Asn-419 backbone oxygen at the apex of a pyramid and Asp-80, Ser-121, Asn-419, and Ser-420 at each vertex of the planar base. Asn-423 stabilizes this pocket through hydrogen bonds to two of these residues. Highly conserved Asn-52 is positioned near the sodium pocket, where it hydrogen-bonds with Asp-80 and the backbone carbonyl of Ser-420. Mutation of three of these residues, Asn-52 in helix 1, Ser-121 in helix 3, and Ser-420 in helix 7, profoundly altered the properties of the receptor. Mutants in which Asn-52 was replaced with Ala or Leu or Ser-121 was replaced with Leu exhibited no detectable binding of radioligands, although receptor immunoreactivity in the membrane was similar to that in cells expressing the wild-type D2L receptor. A mutant in which Asn-52 was replaced with Gln, preserving hydrogen-bonding capability, was similar to D2L in affinity for ligands and ability to inhibit cAMP accumulation. Mutants in which either Ser-121 or Ser-420 was replaced with Ala or Asn had decreased affinity for agonists (Ser-121), but increased affinity for the antagonists haloperidol and clozapine. Interestingly, the affinity of these Ser-121 and Ser-420 mutants for substituted benzamide antagonists showed little or no dependence on sodium, consistent with our hypothesis that Ser-121 and Ser-420 contribute to the formation of a sodium-binding pocket.

The availability of a high-resolution structure of rhodopsin now allows us to reconsider research attempts to understand structure-function relationships in other G protein-coupled receptors (GPCRs). A comparison of the rhodopsin structure with the results of previous sequence analysis and molecular modeling that incorporated experimental results demonstrates a high degree of success for these methods in predicting the helix ends and protein-protein interface of GPCRs. Moreover, the amino acid residues inferred to form the surface of the binding-site crevice based on our application of the substituted-cysteine accessibility method in the dopamine D(2) receptor are in remarkable agreement with the rhodopsin structure, with the notable exception of some residues in the fourth transmembrane segment. Based on our analysis of the data reviewed, we propose that the overall structures of rhodopsin and of amine receptors are very similar, although we also identified localized regions where the structure of these receptors may diverge. We further propose that several of the highly unusual structural features of rhodopsin are also present in amine GPCRs, despite the absence of amino acids that might have thought to have been critical to the adoption of these features. Thus, different amino acids or alternate microdomains can support similar deviations from regular alpha-helical structure, thereby resulting in similar tertiary structure. Such structural mimicry is a mechanism by which a common ancestor could diverge sufficiently to develop the selectivity necessary to interact with diverse signals, while still maintaining a similar overall fold. Through this process, the core function of signaling activation through a conformational change in the transmembrane segments that alters the conformation of the cytoplasmic surface and subsequent interaction with G proteins is presumably shared by the entire Class A family of receptors, despite their selectivity for a diverse group of ligands.

Numerous mutant G protein-coupled receptors with diminished or no function have been described that are naturally occurring or that are the product of gene manipulation. It has largely been assumed that receptor mutants do not affect the function of the wild-type receptor; however, the occurrence of G protein-coupled receptor dimerization suggests the possibility that an intermolecular interaction between mutant and wild-type receptors can occur. We have shown previously that the D2 dopamine receptor (D2DR) exists as dimers in cell lines and brain tissue. In this study, we demonstrated that mutant D2DR can modulate the function of the wild-type D2DR. While attempting to elucidate the structure of the D2DR dimer, we demonstrated that nonfunctional D2DR substitution and truncation mutants antagonized wild-type D2DR function. Furthermore, from analyses of this interaction between the receptor mutants and the D2DR, using photoaffinity labeling, we provide evidence that the D2DR is oligomeric in the cell.

The molecular determinants that govern selective ligand binding to the rat D(4) dopamine receptor were investigated by substituting D(2) dopamine receptor sequences into a D(4) dopamine receptor background. The resulting mutant D(4) dopamine receptors were then screened with a panel of 10 selective and nonselective ligands, which included two allosteric modulators as sensitive measures of protein conformational changes. Mutation of a phenylalanine at position 88 in the second transmembrane-spanning domain (TMS2) of the D(4) receptor to the corresponding valine in the D(2) receptor D(4)-F88V resulted in an approximately 100-fold decrease in the affinity of the highly D(4)-selective drug 3-([4-(4-iodophenyl) piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-750,667) without substantially affecting the binding of the other ligands. Mutations at the extracellular side of D(4)-TMS3 produced moderate decreases in L-750,667 binding affinities with concomitant increases in binding affinity for the D(2)/D(3)-selective antagonist (-)-raclopride. However, the binding affinities of these same D(4)-TMS3 mutants for the allosteric modulator isomethylbutylamiloride also were an anomalous 6- to 20-fold higher than either wild-type receptor. In the combined D(4)-F88V/TMS3 mutants, L-750,667 binding affinity was further decreased, but this decrease was not additive. More importantly, the combined D(4)-F88V/TMS3 mutants had (-)-raclopride and isomethylbutylamiloride binding properties that reverted back to those of the wild-type D(4)-receptor. In contrast to the D(4)-F88V mutant, the adjacent D(4)-L87W mutant had an increased affinity for ligands with extended structures, but had essentially no effect on ligands with compact structures. These findings demonstrate that two residues near the extracellular side of D(4)-TMS2 are critical molecular determinants for the selective binding of L-750,667 and ligands with extended structures.

To investigate the roles of individual transmembrane segments (TM) of the human D3 dopamine receptor in its ligand-receptor interactions, we generated chimeric receptors in which its TMs were replaced, one at a time, partially or entirely, by the corresponding TM of the homologous human D1 receptor. Ligand binding properties of the chimeras, as expressed heterologously in Sf9 cells using recombinant baculoviruses, indicate that the critical binding regions for D3-selective (over D1) ligands reside at narrow regions (6 to 8 residues) near the extracellular surface for TMI, II, IV and VI, while TMV seems to be minimally involved in the ligand selectivity. For TMIII and TMVII, the critical regions seem to be deeper, involving at least the 10 residues near the extracellular surface for TMIII, and the entire TM segment for TMVII. This is based on our current observations that the chimeras with the D3 sequence in the critical regions, although the rest of the TM is of D1 origin (except TMVII), showed the binding properties indistinguishable from those of the wild-type receptor. The chimeras with the D1 sequence in the regions, on the other hand, showed ligand binding characteristics wildly variable depending on substituted TMs: Most marked decreases in ligand affinities were observed with the chimeras of TMIII and VII, and intermediate changes with those of TMIV and VI. Replacements of TMV produced no appreciable effects on the affinities of 14 test ligands (except for one). The chimeras of TMI and II with the D1 sequence in the critical regions showed no appreciable specific binding for several radioactive D3-selective ligands, possibly reflecting their critical roles in assembly and folding of the receptor. These critical regions of the D3 receptor were highly homologous to those of the D2 receptor, except for several nonconservatively substituted residues, which could be exploited to develop ligands selective for the D3 over D2 dopamine receptor or vice versa.