AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus <Emphasis Type="Italic">Branchiostoma floridae</Emphasis>: evolutionary aspects of the catecholaminergic system during development

Catecholamine receptors mediate wide-ranging functions in vertebrates and invertebrates but are largely unknown in invertebrate chordates such as amphioxus. Catecholaminergic cells have been described in amphioxus adults, but few data are known about the transmembrane signal transduction pathways and the expression pattern of related receptors during development. In Branchiostoma floridae, we cloned a full-length cDNA (AmphiD1/β) that corresponds to the dopamine D1/β receptor previously cloned from a related species of amphioxus, Branchiostoma lanceolatum, but no expression studies have been performed for such receptor in amphioxus. In B. floridae, AmphiD1/β encodes a polypeptide with typical G-protein-coupled receptor features, characterized by highest sequence similarity with D1 dopamine and β-adrenergic receptors. The expression of AmphiD1/β mRNA in different regions of the cerebral vesicle corresponds to that of D1-like receptors in vertebrate homologous structures. Furthermore, in situ experiments show that during development, the expression in the nervous system is restricted to cells located anteriorly. A further expression was found in larvae at the level of the endostyle, but it has no counterpart in the predominant expression domains of vertebrate dopamine and/or adrenergic receptor genes. At the same time, we compared the dopaminergic system, consisting of AmphiTH-expressing cells, with the AmphiD1/β expression. In conclusion, the identification of the AmphiD1/β receptor provides further basis for understanding the evolutionary history of the dopaminergic system at the transition from invertebrates and vertebrates.     

Characterisation of AmphiAmR11, an Amphioxus (Branchiostoma floridae) D2-Dopamine-Like G Protein-Coupled Receptor

The evolution of the biogenic amine signalling system in vertebrates is unclear. However, insights can be obtained from studying the structures and signalling properties of biogenic amine receptors from the protochordate, amphioxus, which is an invertebrate species that exists at the base of the chordate lineage. Here we describe the signalling properties of AmphiAmR11, an amphioxus (Branchiostoma floridae) G protein-coupled receptor which has structural similarities to vertebrate α₂-adrenergic receptors but which functionally acts as a D₂ dopamine-like receptor when expressed in Chinese hamster ovary -K1 cells. AmphiAmR11 inhibits forskolin-stimulated cyclic AMP levels with tyramine, phenylethylamine and dopamine being the most potent agonists. AmphiAmR11 also increases mitogen-activated protein kinase activity and calcium mobilisation, and in both pathways, dopamine was found to be more potent than tyramine. Thus, differences in the relative effectiveness of various agonists in the different second messenger assay systems suggest that the receptor displays agonist-specific coupling (biased agonism) whereby different agonists stabilize different conformations of the receptor which lead to the enhancement of one signalling pathway over another. The present study provides insights into the evolution of α₂-adrenergic receptor signalling and support the hypothesis that α₂-adrenergic receptors evolved from D₂-dopamine receptors. The AmphiAmR11 receptor may represent a transition state between D₂-dopamine receptors and α₂-adrenergic receptors.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

Identification and characterization of a novel amphioxus dopamine D1-like receptor

Dopamine receptors function to control many aspects of motor control and other forms of behaviour in both vertebrates and invertebrates. They can be divided into two main groups (D₁ and D₂) based on sequence similarity, ligand affinity and effector coupling. However, little is known about the pharmacology and functionality of dopamine receptors in the deuterostomian invertebrates, such as the cephalochordate amphioxus ( Branchiostoma floridae) which has recently been placed as the most basal of all the chordates. A bioinformatic study shows that amphioxus has at least three dopamine D₁-like receptor sequences. One of these receptors, AmphiD₁/β, was found to have high levels of sequence similarity to both vertebrate D₁ receptors and to β-adrenergic receptors. Here, we report on the cloning of AmphiD₁/β from an adult amphioxus cDNA library, and its pharmacological characterization subsequent to its expression in both mammalian cell lines and Xenopus oocytes. It was found that AmphiD₁/β has a similar pharmacology to vertebrate D₁ receptors, including responding to benzodiazepine ligands. The pharmacology of the receptor exhibits 'agonist-specific coupling' depending upon the second messenger pathway to which it is linked. Moreover, no pharmacological characteristics were observed to suggest that AmphiD₁/β may be an amphioxus orthologue of vertebrate β-adrenergic receptors.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum

Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species.The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca²⁺ signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 – amino- 6,7 – dihydroxy – 1,2,3,4 – tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.     

Amphioxus connectin exhibits merged structure as invertebrate connectin in I-band region and vertebrate connectin in A-band region

Connectin is an elastic protein found in vertebrate striated muscle and in some invertebrates as connectin-like proteins. In this study, we determined the structure of the amphioxus connectin gene and analyzed its sequence based on its genomic information. Amphioxus is not a vertebrate but, phylogenetically, the lowest chordate. Analysis of gene structure revealed that the amphioxus gene is approximately 430 kb in length and consists of regions with exons of repeatedly aligned immunoglobulin (Ig) domains and regions with exons of fibronectin type 3 and Ig domain repeats. With regard to this sequence, although the region corresponding to the I-band is homologous to that of invertebrate connectin-like proteins and has an Ig-PEVK region similar to that of the Neanthes sp. 4000K protein, the region corresponding to the A-band has a super-repeat structure of Ig and fibronectin type 3 domains and a kinase domain near the C-terminus, which is similar to the structure of vertebrate connectin. These findings revealed that amphioxus connectin has the domain structure of invertebrate connectin-like proteins at its N-terminus and that of vertebrate connectin at its C-terminus. Thus, amphioxus connectin has a novel structure among known connectin-like proteins. This finding suggests that the formation and maintenance of the sarcomeric structure of amphioxus striated muscle are similar to those of vertebrates; however, its elasticity is different from that of vertebrates, being more similar to that of invertebrates.     

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermate): biochemical and functional characterization of MPR 46 protein

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr^(−/−) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.     

昆虫多巴胺及其受体的研究进展

多巴胺（dopamine, DA）是脊椎动物和无脊椎动物体内一种重要的生物胺, 其参与调控了昆虫的多种生理反应和行为过程, 如学习与记忆、 认知、 性取向、 抉择、 运动以及型变等。多巴胺主要通过结合特异性的G蛋白偶联受体, 即多巴胺受体（dopamine receptors, DARs）来发挥生理作用。本文综述了多巴胺在昆虫中的调控、 分布及所参与的生理功能, 如多巴胺调控昆虫的交配、 发育、 嗅觉以及运动行为等, 特别对DARs的信号转导、 生理功能以及药理学等方面进行了详细评述。昆虫的DARs大致可分为两大类： D1-like DARs和D2-like DARs。D1-like DARs包含有2种亚型, 分别为DOP1和DOP2。DOP1仅能偶联胞内cAMP的上升, 而DOP2不仅可以起胞内cAMP的上升, 还可偶联胞内Ca²⁺的释放。 D2-like DARs仅包含有1种亚型DOP3, 其被激活后引起胞内cAMP的降低。DA通过激活不同的DARs可偶联不同的第二信使系统, 所产生的下游细胞反应则与昆虫的各种行为相关, 而对昆虫DARs的药理学研究将有助于我们开发特异性的杀虫剂用于害虫防治。     

Molecular and pharmacological characterization of two D(1)-like dopamine receptors in the Lyme disease vector, Ixodes scapularis

Advancements in tick neurobiology may impact the development of acaricides to control those species that transmit human and animal diseases. Here, we report the first cloning and pharmacological characterization of two neurotransmitter binding G protein-coupled receptors in the Lyme disease (blacklegged) tick, Ixodes scapularis. The genes IscaGPRdop1 and IscaGPRdop2 were identified in the I. scapularis genome assembly and predicted as orthologs of previously characterized D₁-like dopamine receptors in the fruit fly Drosophila melanogaster and honeybee Apis mellifera. Heterologous expression in HEK 293 cells demonstrated that each receptor functioned as a D₁-like dopamine receptor because significant increases in levels of intracellular cyclic adenosine monophosphate (cAMP) were detected following dopamine treatment. Importantly, the receptors were distinct in their pharmacological properties regarding concentration-dependent response to dopamine, constitutive activity, and response to other biogenic amines. Exposure to a variety of dopamine receptor agonists and antagonists further demonstrated a D₁-like pharmacology of these dopamine receptors and highlighted their differential activities in vitro.     

D1- and D2-like receptors differentially mediate the effects of dopaminergic transmission on cost–benefit evaluation and motivation in monkeys

It has been widely accepted that dopamine (DA) plays a major role in motivation, yet the specific contribution of DA signaling at D₁-like receptor (D₁R) and D₂-like receptor (D₂R) to cost–benefit trade-off remains unclear. Here, by combining pharmacological manipulation of DA receptors (DARs) and positron emission tomography (PET) imaging, we assessed the relationship between the degree of D₁R/D₂R blockade and changes in benefit- and cost-based motivation for goal-directed behavior of macaque monkeys. We found that the degree of blockade of either D₁R or D₂R was associated with a reduction of the positive impact of reward amount and increasing delay discounting. Workload discounting was selectively increased by D₂R antagonism. In addition, blocking both D₁R and D₂R had a synergistic effect on delay discounting but an antagonist effect on workload discounting. These results provide fundamental insight into the distinct mechanisms of DA action in the regulation of the benefit- and cost-based motivation, which have important implications for motivational alterations in both neurological and psychiatric disorders.

Using quantitatively controlled pharmacological manipulations, this study teases apart the role of D1- and D2-like dopamine receptors in motivation and goal-directed behavior in monkeys, revealing complementary roles of two dopamine receptor subtypes in the computation of the cost/benefit trade-off to guide action.     

Identification and tissue-specific expression of a promethin-like homolog in amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.     

Dopamine Reduction of GABA Currents in Striatal Medium-sized Spiny Neurons is Mediated Principally by the D<Subscript>1</Subscript> Receptor Subtype

Dopamine modulates voltage- and ligand-gated currents in striatal medium-sized neurons (MSNs) through the activation of D1- and D2-like family receptors. GABA_A receptor-mediated currents are reduced by D1 receptor agonists, but the relative contribution of D₁ or D₅ receptors in this attenuation has been elusive due to the lack of selective pharmacological agents. Here we examined GABA_A receptor-mediated currents and the effects of D1 agonists on MSNs from wildtype and D₁ or D₅ receptor knockout (KO) mice. Immunohistochemical and single-cell RT-PCR studies demonstrated a lack of compensatory effects after genetic deletion of D₁ or D₅ receptors. However, the expression of GABA_A receptor α1 subunits was reduced in D₅ KO mice. At the functional level, whole-cell patch clamp recordings in dissociated MSNs showed that GABA peak current amplitudes were smaller in cells from D₅ KO mice indicating that lack of this receptor subtype directly affected GABA_A-mediated currents. In striatal slices, addition of a D1 agonist reduced GABA currents significantly more in D₅ KO compared to D₁ KO mice. We conclude that D₁ receptors are the main D1-like receptor subtype involved in the modulation of GABA currents and that D₅ receptors contribute to the normal expression of these currents in the striatum. Special issue dedicated to Anthony Campagnoni.     

Coupling of D1 and D5 dopamine receptors to multiple G proteins

Dopamine receptors are a subclass of the super family of G protein-coupled receptors, that transduce their effects by coupling to specific G proteins. Within the dopamine receptor family, the adenylyl cyclase stimulatory receptors include the D₁ and D₅ subtypes. The D₁ and D₅ dopamine receptors are genetically distinct, sharing >80% sequence homology within the highly conserved seven transmembrane spanning domains, but displaying only 50% overall homology at the amino acid level. When expressed in transfected GH₄C₁ rat pituitary cells, both D₁ and D₅ receptors stimulate adenylyl cyclase and have identical affinities toward dopaminergic agonists and antagonists. In order to analyze specific signaling pathways mediated by activation of either D₁ or D₅ receptors, we have identified the G proteins that are coupled to these receptors. Through functional analyses and competition binding studies, and from immunoprecipitation techniques, using antisera against the various α subunits of G proteins, we have established that both D₁ and D₅ receptors couple to G_sα. In addition, D₁ receptors are also coupled to G_oα. Since G_oα has been implicated in the regulation of Ca²⁺, K⁺, and Na⁺ channels, this finding would suggest that D₁ receptors can mediate the functional activity of these ion channels. There is also evidence to indicate that D₅ receptors couple to G_zα, a novel G protein abundantly expressed in neurons. Thus, despite similar pharmacological properties, such differential coupling of D₁ and D₅ receptors to G proteins other than G_sα, indicates that dopamine can transduce varied signaling responses upon the simultaneous stimulation of both these receptors.     

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.     

Systemic Blockade of Dopamine D2-Like Receptors Increases High-Voltage Spindles in the Globus Pallidus and Motor Cortex of Freely Moving Rats

High-voltage spindles (HVSs) have been reported to appear spontaneously and widely in the cortical–basal ganglia networks of rats. Our previous study showed that dopamine depletion can significantly increase the power and coherence of HVSs in the globus pallidus (GP) and motor cortex of freely moving rats. However, it is unclear whether dopamine regulates HVS activity by acting on dopamine D₁-like receptors or D₂-like receptors. We employed local-field potential and electrocorticogram methods to simultaneously record the oscillatory activities in the GP and primary motor cortex (M1) in freely moving rats following systemic administration of dopamine receptor antagonists or saline. The results showed that the dopamine D₂-like receptor antagonists, raclopride and haloperidol, significantly increased the number and duration of HVSs, and the relative power associated with HVS activity in the GP and M1 cortex. Coherence values for HVS activity between the GP and M1 cortex area were also significantly increased by dopamine D₂-like receptor antagonists. On the contrary, the selective dopamine D₁-like receptor antagonist, {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390, had no significant effect on the number, duration, or relative power of HVSs, or HVS-related coherence between M1 and GP. In conclusion, dopamine D₂-like receptors, but not D₁-like receptors, were involved in HVS regulation. This supports the important role of dopamine D₂-like receptors in the regulation of HVSs. An siRNA knock-down experiment on the striatum confirmed our conclusion.     

The Caenorhabditis elegans D2-like dopamine receptor DOP-2 physically interacts with GPA-14, a Gαi subunit

Dopaminergic inputs are sensed on the cell surface by the seven-transmembrane dopamine receptors that belong to a superfamily of G-protein-coupled receptors (GPCRs). Dopamine receptors are classified as D1-like or D2-like receptors based on their homology and pharmacological profiles. In addition to well established G-protein coupled mechanism of dopamine receptors in mammalian system they can also interact with other signaling pathways. In C. elegans four dopamine receptors (dop-1, dop-2, dop-3 and dop-4) have been reported and they have been implicated in a wide array of behavioral and physiological processes. We performed this study to assign the signaling pathway for DOP-2, a D2-like dopamine receptor using a split-ubiquitin based yeast two-hybrid screening of a C. elegans cDNA library with a novel dop-2 variant (DOP-2XL) as bait. Our yeast two-hybrid screening resulted in identification of gpa-14, as one of the positively interacting partners. gpa-14 is a G?? coding sequence and shows expression overlap with dop-2 in C. elegans ADE deirid neurons. In-vitro pull down assays demonstrated physical coupling between dopamine receptor DOP-2XL and GPA-14. Further, we sought to determine the DOP-2 region necessary for GPA-14 coupling. We generated truncated DOP-2XL constructs and performed pair-wise yeast two-hybrid assay with GPA-14 followed by in-vitro interaction studies and here we report that the third intracellular loop is the key domain responsible for DOP-2 and GPA-14 coupling. Our results show that the extra-long C. elegans D2-like receptor is coupled to gpa-14 that has no mammalian homolog but shows close similarity to inhibitory G-proteins. Supplementing earlier investigations, our results demonstrate the importance of an invertebrate D2-like receptor's third intracellular loop in its G-protein interaction.     

Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D₁-like family of dopamine receptors and inputs of aversion coming from neurons containing the D₂-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D₁ reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D₂ receptors function, we present evidence of σ₁ receptor molecular and functional interaction with dopamine D₂ receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D₂ receptors (the long isoform of the D₂ receptor) can complex with σ₁ receptors, a result that is specific to D₂ receptors, as D₃ and D₄ receptors did not form heteromers. We demonstrate that the σ₁-D₂ receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ₁ -D₂ receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ₁ knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D₂ receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D₁ and D₂ receptor containing neurons in the brain.