Dopamine D3 receptor ligands—Recent advances in the control of subtype selectivity and intrinsic activity

Various pharmacological studies have implicated the dopamine D₃ receptor as an interesting therapeutic target in the treatment of different neurological disorders. Because of these putative therapeutic applications, D₃ receptor ligands with diverse intrinsic activities have been an active field of research in recent years. Separation of purely D₃-mediated drug effects from effects produced by interactions with similar biogenic amine receptors allows to verify the therapeutic impact of D₃ receptors and to reduce possible side-effects caused by “promiscuous” receptor interactions. The requirement to gain control of receptor selectivity and in particular subtype selectivity has been a challenging task in rational drug discovery for quite a few years. In this review, recently developed structural classes of D₃ ligands are discussed, which cover a broad spectrum of intrinsic activities and show interesting selectivities.     

Aromatic ring functionalization of benzolactam derivatives: new potent dopamine D3 receptor ligands

Since the discovery of the dopamine D₃ receptor, an intensive effort has been directed toward the development of potent and selective ligands in order to elucidate the function and potential therapeutic advantages of targeting D₃ receptors. As a part of our efforts, a novel series of substituted benzolactams derivatives was synthesized mostly through palladium-catalyzed reactions. Their affinities on D₁-D₄ receptors were evaluated and the data led us to highly potent D₃ ligands, some of them highly selective for D₃ receptor, compared to the related dopamine receptor subtypes. Functional D₃ activity assays of the most relevant compounds have been carried out revealing antagonist as well as partial agonist activity.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

KLHL12 Promotes Non-Lysine Ubiquitination of the Dopamine Receptors D4.2 and D4.4, but Not of the ADHD-Associated D4.7 Variant

Dopamine D₄ Receptor Polymorphism

The dopamine D₄ receptor has an important polymorphism in its third intracellular loop that is intensively studied and has been associated with several abnormal conditions, among others, attention deficit hyperactivity disorder.

KLHL12 Promotes Ubiquitination of the Dopamine D₄ Receptor on Non-Lysine Residues

In previous studies we have shown that KLHL12, a BTB-Kelch protein, specifically interacts with the polymorphic repeats of the dopamine D₄ receptor and enhances its ubiquitination, which, however, has no influence on receptor degradation. In this study we provide evidence that KLHL12 promotes ubiquitination of the dopamine D₄ receptor on non-lysine residues. By using lysine-deficient receptor mutants and chemical approaches we concluded that ubiquitination on cysteine, serine and/or threonine is possible.

Differential Ubiquitination of the Dopamine D₄ Receptor Polymorphic Variants

Additionally, we show that the dopamine D_4.7 receptor variant, which is associated with a predisposition to develop attention deficient hyperactivity disorder, is differentially ubiquitinated compared to the other common receptor variants D_4.2 and D_4.4. Together, our study suggests that GPCR ubiquitination is a complex and variable process.     

Differential voltage-sensitivity of D2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D_2L receptor and we now extend our studies to include the other members of the D₂-like receptor subfamily; the D_2S, D₃, and D₄ dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D_2S, D₃, or D₄ receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration-response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D_2S and D₄ upon depolarization. In contrast, the concentration-response relationships for D₃-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.     

Intestinal 1,25-dihydroxyvitamin D3 binding protein: Specificity of binding

The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D₃ and 1,25-dihydroxyvitamin D₂ are equally competitive and are the most active. Of the structural features of the compounds, the 1α-hydroxyl is most important followed by the 25-hydroxyl and the 3β-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D₃ was much more effective than vitamin D₃ but less effective than 25-hydroxyvitamin D₃. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D₃ binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D₃ and the cytosol receptor.     

Mapping of D1 dopamine receptor mRNA by non-radioactive in situ hybridization

 In order to improve the identification and characterization of dopaminoceptive neurons, the rat brain was mapped for D₁ dopamine receptor mRNA by non-radioactive in situ hybridization (ISH) with a 45mer digoxigenin-labeled oligonucleotide probe. The specificity of the results was controlled with the help of a 396-bp D₁ receptor riboprobe. Labeled hybrids were visualized with an alkaline phosphatase-coupled anti-digoxigenin antibody. The high resolution obtained permitted individual labeled cells to be identified and to distinction between cell bodies and processes. D₁ mRNA was largely confined to neurons. With the exception of ependymal cells, glial cells were not distinctly labeled. Subcellularly, D₁ mRNA was localized to perikarya but not to dendrites or axons. D₁ receptor-expressing neurons were present in all of the known terminal fields of mesencephalic or diencephalic dopaminergic neurons. However, D₁ message was also detected in brain areas which are not known to contain D₁ ligand binding sites or in which the presence or the cellular source of this receptor subtype had previously not been unequivocally established, such as the hippocampus or cerebellar cortex. Moreover, labeled neurons were present in regions not known to receive dopaminergic projections, such as the thalamic and some brainstem nuclei. We conclude that this ISH technique provides a considerable gain in sensitivity and resolution with regard to neurotransmitter receptor mapping. Accepted: 25 August 1997     

Distribution of 1,25-Dihydroxyvitamin D3 Receptor Immunoreactivity in the Rat Olfactory System

1.The rat olfactory system contains numerous target sites for 1,25-dihydroxyvitamin D₃, as determined by receptor protein (VDR) immunocytochemistry and in situ hybidization.2.Nuclear and cytoplasmic VDR immunoreactivity as well as the corresponding hybridization signal was observed in neurons in the olfactory epithelium, the olfactory bulb, and throughout the limbic system in locations also known to be glucocorticoid targets.3.The widespread distribution of VDR indicates the distinct functional importance of 1,25-dihydroxyvitamin D₃ for olfactory perception.     

Dopamine Receptor Subtype Agonists and Feeding Behavior

Stimulation or blockade of various dopamine receptor subtypes is associated with reduced feeding. For example, D₂ receptor agonists suppress feeding in food-deprived and free-feeding rats, and in rats given access to a highly palatable diet Similarly, reduced food intake is associated with the actions of diverse D₁ receptor agonists, and these compounds can interact synergistically with D₂ receptor agonists to potentiate reductions in feeding. Using micro-structural analysis to compare D₁ and D₂ agonist effects, specific differences emerge in their modes of action. D₁ agonists reduce the duration of feeding, primarily by decreasing the frequency of feeding bouts, whereas D₂ agonists reduce the local rate of eating. However, since D₁ agonists uniquely reduce feeding in the absence of other behavioral impairments and are less disruptive of the pattern of feeding behavior, it has been suggested that D₁ agonists are more likely than D₂ agonists to act on central mechanisms regulating food intake. Moreover, only D₁ agonists are effective in suppressing sucrose sham-feeding, suggesting that D₁ receptor stimulation may promote satiety. Nevertheless, many questions remain. For example, antagonist studies have implicated 5-HT receptor stimulation in the anorectic effects of D₁ agonists, suggesting that further pharmacological and behavioral analyses of receptor-subtype agonist effects are required. Above all, recent developments in the classification of dopamine receptor subtypes reveal the need for new studies examining the involvement of D₃, D₄ and D₅ receptors in feeding.     

Dopamine-induced functional activation of Gαq mediated by dopamine D1-like receptor in rat cerebral cortical membranes

Although multiple roles of dopamine through D₁-like (D₁ and D₅) and D₂-like (D₂, D₃, and D₄) receptors are initiated primarily through stimulation or inhibition of adenylyl cyclase via G_s/olf or G_i/o, respectively, there have been many reports indicating diverse signaling mechanisms that involve alternative G protein coupling. In this study, dopamine-induced Gα_q activation in rat brain membranes was investigated. Agonist-induced Gα_q activation was assessed by increase in guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding to Gα_q determined by [³⁵S]GTPγS binding/immunoprecipitation assay in rat brain membranes. Dopamine-stimulated Gα_q functionality was highest in cortex as compared to hippocampus or striatum. In cerebral cortical membranes, this effect was mimicked by benzazepine derivatives with agonist properties at dopamine D₁-like receptors, that is, SKF83959, SKF83822, R(+)-SKF81297, R(+)-SKF38393, and SKF82958, but not by the compounds with dopamine D₂-like receptor agonist properties except for aripiprazole. Against expectation, stimulatory effects were also induced by SKF83566, R(+)-SCH23390, and pergolide. The pharmacological profiling by using a series of antagonists indicated that dopamine-induced response was mediated through dopamine D₁-like receptor, which was distinct from the receptor involved in 5-HT-induced response (5-HT_2A receptor). Conversely, the responses induced by SKF83566, R(+)-SCH23390, and pergolide were most likely mediated by 5-HT_2A receptor, but not by dopamine D₁-like receptor. Caution should be paid when interpreting the experimental data, especially in behavioral pharmacological research, in which SKF83566 or R(+)-SCH23390 is used as a standard selective dopamine D₁-like receptor antagonist. Also, possible clinical implications of the agonistic effects of pergolide on 5-HT_2A receptor has been mentioned.     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

Anxiolytic effect of the GPR103 receptor agonist peptide P550 (homolog of neuropeptide 26RFa) in mice. Involvement of neurotransmitters

The GPR103 receptor is a G protein-coupled receptor, which plays a role in several physiological functions. However, the role of the GPR103 receptor in anxiety has not been clarified. The first aim of our study was to elucidate the involvement of the GPR103 receptor in anxious behavior. Mice were treated with peptide P550, which is the mouse homolog of neuropeptide 26RFa and has similar activity for the GPR103 receptor as neuropeptide 26RFa. The anxious behavior was investigated using an elevated plus-maze paradigm. The second aim of our study was to investigate the underlying neurotransmissions. Accordingly, mice were pretreated with a nonselective muscarinic acetylcholine receptor antagonist, atropine, a γ-aminobutyric acid subunit A (GABA_A) receptor antagonist, bicuculline, a non-selective 5-HT₂ serotonergic receptor antagonist, cyproheptadine, a mixed 5-HT₁/5-HT₂ serotonergic receptor antagonist, methysergide, a D₂, D₃, D₄ dopamine receptor antagonist, haloperidol, a nonselective α-adrenergic receptor antagonist, phenoxybenzamine and a nonselective β-adrenergic receptor antagonist, propranolol. Our results demonstrated that peptide P550 reduces anxious behavior in elevated plus maze test in mice. Our study shows also that GABA_A-ergic, α- and β-adrenergic transmissions are all involved in this action, whereas 5-HT₁ and 5-HT₂ serotonergic, muscarinic cholinergic and D₂, D₃, D₄ dopaminergic mechanisms may not be implicated.     

Amplification of the action of subthreshold doses of aldosterone by 19-hydroxyandrost-4-ene-3, 17-dione.

Various 1α-hydroxylated side chain analogs of vitamin D₃ have been studied for their ability to compete with 1α,25-dihydroxy[³H]vitamin D₃ for binding to the chick intestinal receptor. Of the analogs examined, 1α,24R-dihydroxyvitamin D₃ was found to be nearly equivalent to 1α,25-dihydroxyvitamin D₃ in its ability to compete for receptor binding. However, this near equivalence was not shared by its stereoisomer, 1α,24S-dihydroxyvitamin D₃, which was only 10% as effective a competitor. It is proposed that the ability of a 24R-hydroxyl group to mimic the 25-hydroxyl group is not due to a lack of side chain specificity on the part of the receptor, but is instead due to the similar orientation of the 25-hydroxyl and the 24R-hydroxyl such that they can be accommodated equivalently by the receptor.     

K(v) 7 (KCNQ) channel openers normalize central 2-deoxyglucose uptake in a mouse model of mania and increase prefrontal cortical and hippocampal serine-9 phosphorylation levels of GSK3β

In the CNS, an antagonistic interaction has been shown between adenosine A_2A and dopamine D₂ receptors (A_2ARs and D₂Rs) that may be relevant both in normal and pathological conditions (i.e., Parkinson's disease). Thus, the molecular determinants mediating this receptor–receptor interaction have recently been explored, as the fine tuning of this target (namely the A_2AR/D₂R oligomer) could possibly improve the treatment of certain CNS diseases. Here, we used a fluorescence resonance energy transfer‐based approach to examine the allosteric modulation of the D₂R within the A_2AR/D₂R oligomer and the dependence of this receptor–receptor interaction on two regions rich in positive charges on intracellular loop 3 of the D₂R. Interestingly, we observed a negative allosteric effect of the D₂R agonist quinpirole on A_2AR ligand binding and activation. However, these allosteric effects were abolished upon mutation of specific arginine residues (217–222 and 267–269) on intracellular loop 3 of the D₂R, thus demonstrating a major role of these positively charged residues in mediating the observed receptor–receptor interaction. Overall, these results provide structural insights to better understand the functioning of the A_2AR/D₂R oligomer in living cells.     

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D3-specific receptor

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃ has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D₃ to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D₃ binding activity. However, when KCl is present at a concentration of 300 mm during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D₃ binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D₃ binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 mm dithiothreitol. Likewise, 5.0 mm monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D₃ binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D₃ binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.     

Intestinal cytosol binders of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3

The binding of 25-hydroxy-[26,27-³H]vitamin D₃ and 1,25-dihydroxy-[26,27-³H]vitamin D₃ to the cytosol of intestinal mucosa of chicks and rats has been studied by sucrose gradient analysis. The cytosol from chick mucosa showed variable binding of 1,25-dihydroxyvitamin D₃ to a 3.0S macromolecule which has high affinity and low capacity for this metabolite. However, when the mucosa was washed extensively before homogenization, a 3.7S macromolecule was consistently observed which showed considerable specificity and affinity for 1,25-dihydroxyvitamin D₃. Although 3.7S binders for 1,25-dihydroxyvitamin D₃ could also be located in other organs, competition experiments with excess nonradioactive 1,25-dihydroxyvitamin D₃ suggested that they were not identical to the 3.7S macromolecule from intestinal mucosal cytosol. As the 3.7S macromolecule was allowed to stand at 4 °C with bound 1,25-dihydroxy-[³H]vitamin D₃, the 1,25-dihydroxy-[³H]vitamin D₃ became increasingly resistant to displacement by non-radioactive 1,25-dihydroxyvitamin D₃. The 1,25-dihydroxy-[³H]vitamin D₃ remained unchanged and easily extractable with lipid solvents through this change, making unlikely the establishment of a covalent bond. Unlike the chick, mucosa from rats yielded cytosol in which no specific binding of 1,25-dihydroxy-[³H]vitamin D₃ was detected. Instead, a 5-6S macromolecule which binds both 1,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃ was found. This protein which was also found in chick mucosa shows preferential binding for 25-hydroxyvitamin D₃. It could be removed by washing the mucosa with buffer prior to homogenization which suggests that it may not be a cytosolic protein. Although the 3.7S protein from chick mucosa has properties consistent with its possible role as a receptor, the 5-6S macromolecule does not appear to have “receptor”-like properties.     

An hydroxylapatite batch assay for the quantitation of 1alpha,25-dihydroxyvitamin D3-receptor complexes.

A versatile hydroxylapatite batch assay for 1α,25-dihydroxyvitamin D₃-receptor complex from chick intestinal mucosa has been developed. The assay has been characterized with respect to time and temperature of incubations, protein concentration, amount of hydroxylapatite required to bind receptor-steroid complexes, pH, and effects of KCl and phosphate. Triton X-100 (0,5%, $\text{v}\text{v}$) was found to be essential for the removal of nonspecifically bound ligand. The hydroxylapatite was shown to bind the 1α,25-dihydroxy-vitamin D₃ receptor as demonstrated by the specificity and high affinity for 1α,25-dihydroxy-vitamin D₃ and the sedimentation properties of the phosphate-extracted hydroxylapatite-bound complex on sucrose density gradients. Binding appears to be nearly quantitative. The efficient separation of bound from free ligand utilizing this assay makes it possible to examine a number of aspects of the binding of this steroid hormone to its cytoplasmic receptor that has not previously been possible.     

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors

D₂ and D₃ dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D₂ receptor couples only to inhibitory G proteins, D₃ receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D₂ and D₃ receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D₂ receptor couple to Gi protein in a pattern characteristic of D₂ receptor. On the other hand chimeras containing a third cytoplasmic loop of D₃ receptor have coupling characteristics like those of D₃ receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D₂ and D₃ to G proteins.