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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
CNS Neurol Disord Drug Targets. Author manuscript; available in PMC Mar 29, 2011.
Published in final edited form as:
PMCID: PMC3066024
NIHMSID: NIHMS280722

Heteromerization of G Protein-Coupled Receptors: Relevance to Neurological Disorders and Neurotherapeutics

Abstract

Because G protein-coupled receptors (GPCRs) are numerous, widely expressed and involved in major physiological responses, they represent a relevant therapeutic target for drug discovery, particularly regarding pharmacological treatments of neurological disorders. Among the biological phenomena regulating receptor function, GPCR heteromerization is an important emerging area of interest and investigation. There is increasing evidence showing that heteromerization contributes to the pharmacological heterogeneity of GPCRs by modulating receptor ontogeny, activation and recycling. Although in many cases the physiological relevance of receptor heteromerization has not been fully established, the unique pharmacological and functional properties of heteromers are likely to lead to new strategies in clinical medicine. This review describes the main GPCR heteromers and their implications for major neurological disorders such as Parkinson’s disease, schizophrenia and addiction. A better understanding of molecular mechanisms underlying drug interactions related to the targeting of receptor heteromers could provide more specific and efficient therapeutic agents for the treatment of brain diseases.

Keywords: Heteromerization, heteromer, GPCR, neurological disorder, drug discovery

INTRODUCTION

G protein-coupled receptors (GPCRs) are involved in most physiological responses, and alterations of their function are related to many pathological states such as hypertension, diabetes, pain, asthma, immunological and neurological disorders. 30% to 40% of current pharmaceutical drugs target GPCRs and the regulation of their function is essential for therapeutic strategies. GPCR heteromerization refers to the direct interaction between at least two different functional receptors forming a complex with specific biochemical and functional properties different from those of its component receptor units [1, 2]. Heteromerization is emerging as increasingly important in creating functional receptor diversity. Diverse studies using biochemical, pharmacological, functional or behavioural approaches indicate that GPCRs can exist as dimeric entities [313]. While nearly all domains of GPCRs have been identified as involved in interaction within specific heteromers, the majority of studies implicate the transmembrane helices and intracellular domains as the major interaction interfaces [1418]. The stochiometry of these complexes is not completely established. Future investigation will clarify the various structures and composition of these novel receptor complexes [2]. We focus here on current evidence for GPCR heteromers.

Prior to the recent evidence for receptor heteromers, experimental data relevant to functional communication between GPCRs following their ligand activation were attributed to crosstalk of distinct signaling pathways downstream of GPCRs. The universality of this classical interpretation of experimental results has been brought into question with the recent discovery that different GPCRs can interact directly. Thus some GPCR crosstalk mechanisms may occur directly at the level of the receptor complex. Consonant with this possibility, many studies have revealed functional consequences of GPCR heteromerization on each step of the life cycle of a receptor, from its targeting to the regulation of its pharmacological, signaling and internalization properties (Fig. 1).

Fig. (1)
Roles of GPCR heteromerization

Heterocomplex formation has been found to contribute to receptor maturation, folding and expression at the cell surface by modulating its targeting or internalization [19]. The chemokine receptor 1 (CXCR1)-chemokine receptor 2 (CXCR2) heteromer is an example of the role of receptor heteromerization in their targeting to the plasma membrane [20]. For β2-adrenergic receptor (β2AR)-delta opioid receptor (DOR) [21], mu opioid receptor (MOR)-somatostatin 2A receptor (SSTR2A) [22], α2A-adrenergic receptor (α2AR)-β1-adrenergic receptor (β1AR) [23] or α1A1B-adrenergic receptor [24] heteromers, activation of one receptor is sufficient to induce the internalization of both receptors. For DOR-kappa opioid receptor (KOR) [1], β2AR-KOR [21], β1AR-β2AR [25] or β2AR-β3-adrenergic receptor (β3AR) [26] heteromers, activation of one receptor prevents the internalization of the other one. Others studies show that GPCR heteromerization can modify receptor internalization by determining the interaction with β–arrestin [27].

Once at the cell surface, the crosstalk between GPCRs can lead to specific receptor pharmacological profiles [28]. Indeed, negative (SSTR2A-somatostatin SSTR3 [29], LH/CGR-TSHR [30] or chemokine CCR2-CCR5 [6]) and positive (β1AR-β2AR [31]) cooperative binding processes have been described for many heteromers [32, 33]. GPCR heteromerization has other important functional roles. Heteromers can regulate the receptor-activated signal transduction positively (D2R-somatostatin SSTR5 [34], angiotensin II type 1 receptor-bradykinin B2 receptor [35], α1B1D-adrenergic receptors [36] or α2C-adrenergic receptor-β2AR [37]) or negatively (SSTR2A-SSTR3 [29] or melatonin MT1 receptor-orphan GPR50 receptor [38]). In some cases, the heteromerization can change the selectivity of a GPCR for different types of G protein or β-arrestin, leading to the regulation of a new signaling pathway [39]. This occurs for DOR-MOR [40, 41], CCR2-CCR5 [15] and D1R-D2R [42, 43] heteromers.

GPCR heteromers introduce new properties leading to a large complexity of receptor-mediated regulatory responses. The combinatorial complexity introduced by this phenomenon of heterocomplex formation makes it more difficult to understand and to study the biological mechanisms of GPCRs. The existence of functional GPCR heteromers represents both a major challenge and an opportunity for neuroscience and drug discovery. The number of GPCRs for which evidence suggests a functional role of heteromerization is rapidly increasing. Given the large number of GPCR genes, their ability to form combinations raises the daunting possibility that there are tens or even hundreds of thousands of unique receptor heteromers in the brain and nervous system. Elucidating the function of each of these potential distinct receptor complexes represents a considerable challenge. On the other hand, this proliferation of functional complexes brings with it a potential for developing drugs targeted to specific heteromers that have greatly increased therapeutic specificity and reduced side-effects. Because GPCRs include many important therapeutic targets, a better understanding of GPCR heteromerization is essential to understand the pharmacology of potential drugs, especially in the treatment of neurological disorders [10, 11, 14, 44]. In this review, we examine salient examples of GPCR crosstalk mechanisms implicated in the pathophysiology of the central nervous system, with particular emphasis on functions that are more likely to result from their heteromeric association (Table 1). We also consider new therapeutic approaches targeting specifically these complexes.

Table 1
Examples of GPCR Heteromers with Implications in Neurological Disorders

ADENOSINE AND DOPAMINE RECEPTOR HETEROMERS

Adenosine (A1R-A2AR) and Dopamine (D1R-D2R; D1R-D3R; D2R-D3R) Receptor Heteromers

Adenosine and dopamine receptors are implicated in many neurological processes (motivation, pleasure, cognition, memory, learning, motor control) [45] and alterations of their signaling are involved in several neuropsychiatric disorders, including Parkinson's disease [46, 47], schizophrenia [48], drug addiction [49, 50] and Alzheimer’s disease [51]. Adenosine A1R and A2AR and dopamine D1R and D2R are major targets for neurologic drugs (antipsychotics or psychostimulants) and investigations of their interaction mechanisms are particularly relevant for the understanding of their functional crosstalk and the development of new therapeutic strategies.

The first evidence for adenosine and dopamine receptor dimerization has been provided for adenosine A1R, dopamine D1R and D2R homomers [5254]. The colocalization of these different receptor subtypes in the same areas led to the study of their possible heteromerization. Recently, A1R and A2AR heteromeric complexes have been identified in living cells and striatal nerve terminals by combining BRET (Bioluminescence Resonance Energy Transfer), FRET (Fluorescence Resonance Energy Transfer) and co-immunoprecipitation approaches [55]. Both D1R and D2R can also form heteromers and these complexes present specific signaling and internalization properties that are distinct from that of D1R and D2R homomers [42]. The D1R-D2R heteromer is associated to the coupling of Gq/11 protein and activates the phopholipase C cascade in the striatum, while D1R is specifically coupled to Gs protein and D2R to Gi/o protein [43]. Also D1R-D2R heteromers cointernalize after selective activation of either receptor [56]. Recently, D1R has been demonstrated to interact in living cells with another dopamine receptor subtype, D3R [57, 58]. Binding assays on striatal membrane preparations show modifications of receptor pharmacological properties, since D3R activation increases D1R agonist affinity. Moreover, behavioural effects mediated by D1R are enhanced when D3R is activated suggesting the presence of D1R-D3R heteromerization in striatum [57]. D2R-D3R heteromers detected in living cells and the receptor co-localization in striatopallidal gamma-aminobutyric acid (GABA) neurons are also potentially relevant to the pathophysiology and treatment of both Parkinson’s disease and schizophrenia [5961].

Due to the adenosine-dopamine antagonistic functional crosstalk in the central nervous system and the striatal co-localization of their different GPCR subtypes, the existence of functional complexes between them has been investigated. Indeed, adenosine and dopamine systems modulate activity of striatonigral and striatopallidal pathway neurons. The dopaminergic D2R neurons express both A1R and A2AR while the dopaminergic D1R neurons express only A1R. Based on co-immunoprecipitation, BRET and FRET experiments, several groups have provided evidence for specific A1R-D1R and A2AR-D2R heteromers both in living cells and striatum [6267].

Adenosine A1R-Dopamine D1R Heteromer

Co-immunoprecipitation experiments performed on fibroblast cells co-transfected with two receptors provide evidence for a specific A1R-D1R but not A1R-D2R heteromeric complex that is modulated by ligands. Pretreatment of cells with a selective A1R agonist increases the amount of A1R-D1R complex detected, whereas a D1R agonist has the opposite effect [67].

In brain, adenosine and dopamine antagonize each other’s pharmacological and biochemical effects; this may result in part from their activity at the A1R-D1R heteromer complex [45, 62]. Pharmacological experiments show that adenosine A1R agonists shift the dopamine D1R binding state from high-affinity to low-affinity. A1R expressed in presynaptic dopaminergic nerve terminals decreases dopamine release by inhibiting the D1R activation. A1R antagonists lead to a potentiation in the D1R-induced cyclic adenosine monophosphate (cAMP) response [68], an effect that could result from their regulation of G proteins having offsetting activities. D1R is predominantly coupled to Gs protein which stimulates adenylate cyclase and A1R to Gi/o proteins which have inhibitory effects. However, the heteromer introduces crosstalk effects that cannot be entirely attributed to the separate signaling effects of the two receptors. Coactivation of the A1R-D1R heteromer induces a decrease of the affinity of D1R for agonist and D1R-induced cAMP accumulation. A recent report demonstrates that adenosine A1R activation enhances dopamine D1R desensitization in human embryonic kidney 293 (HEK293) cells and this effect is blocked by A1R antagonists [69]. Another study shows that activation of A1R blocks D1R desensitization in COS-7 cells [70]. These differences also highlight the importance of the cellular context; HEK293 and COS7 cells do not express the same regulator proteins such as G protein-coupled receptor kinases (GRK) and β-arrestin protein that regulate D1R desensitization. Overall, these data suggest strongly that A1R-D1R heteromers alter receptor ligand binding, signaling and desensitization mechanisms [6973]. At the neuronal level, double immunofluorescence assays demonstrate a colocalization of adenosine A1R and dopamine D1R in soma and dendrites of cortical neurons [74, 75]. Many behavioral studies report functional antagonism of adenosine A1R-dopamine D1R. Selective adenosine agonists inhibit the motor activating effects induced by dopamine agonists while adenosine antagonists enhance the same effect [45]. Moreover, the last descriptions of pharmacological properties of the A1R-D1R heteromer provide a basis for the design of new pharmaceutical compounds to treat addiction. Interestingly, it was reported that cocaine, a potent stimulant of the central nervous system, targets the adenosine-dopamine heteromeric complex in rat nucleus accumbens by inhibiting the physical interaction between A1R and D1R [50].

Adenosine A2AR-Dopamine D2R Heteromer

In the early 1990s, evidence for adenosine A2AR-dopamine D2R heteromers in striatum was first described. The antagonistic receptor crosstalk found in the central nervous system leads to the consideration of A2AR antagonists as neuroprotective molecules and antiparkinsonian compounds [76, 77]. A2AR-D2R heteromer existence was subequently confirmed by co-immunoprecipitation [63, 78] and BRET / FRET methods [66, 79, 80] in living cells and striatum. Heteromerization between these receptors was described as constitutive since this phenomenon is A2AR and D2R agonist-independent. Moreover, it was found by using D1-D2 chimeric receptors that the fifth transmembrane domain and particularly an Arg-rich epitope of the N-terminus of the third intracellular loop of D2R may constitute interfaces that interact with a phosphorylated Ser-rich epitope of the C-terminal part of A2AR in the heteromer [18, 65].

The observation that activation of the A2AR leads to a decrease of D2R agonist binding site affinity suggests a close and functional association between A2AR and D2R [66, 76, 77]. However, which components of antagonist signaling between the two receptors occur at the level of the receptor heteromer or at the level of downstream signaling is not known. Some opposing effects of A2AR and D2R agonists may also take place downstream of the receptor since the receptors are coupled to G proteins than can have opposing effects, Gs protein and Gi/o protein, respectively. However, activation of the A2AR decreases coupling of the D2R to its Gi/o protein [81]. Furthermore, stimulation of D2R decreases the coupling of A2AR to its Gs protein [63, 82]. Thus the A2AR-D2R heteromer affects receptor binding, G protein coupling and may be also involved in the cross-desensitization mechanisms via agonist-induced coaggregation and cointernalization of both receptors [45, 63].

In the central nervous system, the striatonigral and the striatopallidal GABAergic neurons represent more than 90% of the striatal neuronal population. A1R-D1R interactions would be expected to modulate the function of striatonigral neurons. A2AR-D2R interactions would alter the function of striatopallidal neurons. Both populations play crucial roles in the pathophysiology of basal ganglia disorders [45]. Numerous studies have suggested that the A2AR-D2R heteromer in the central nervous system may provide a new therapeutic target for treating Parkinson's disease, schizophrenia and addiction [45, 81, 83, 84]. In animal models, adenosine agonists and antagonists are potent atypical neuroleptics and antiparkinsonian drugs, respectively [45, 8587]. Behavorial studies show that selective A2R agonists inhibit the motor activating effects induced by D2R agonists, while A2R antagonists enhance the same effects promoting motor inhibition [45]. This enhancement of dopamine D2R transmission by adenosine A2R antagonists help reverse the motor impairment observed in models of Parkinson’s disease (reduced D2R signaling). Thus A2R antagonists have antiparkinsonian activity [8891]. On the other hand, the striatopallidal GABAergic neurons, which contain A2AR-D2R heteromers, are also known to be targets for antipsychotic drugs, mainly D2R antagonists. An A2R agonist, CGS 21680, shows antipsychotic activity in both amphetamine and phencyclidine rodent models of schizophrenia [87, 92]. The antipsychotic activity of A2R agonists may be mediated through the inhibition of the D2R within the A2R-D2R heteromer [87, 92]. Moreover, it was found that an adenosine uptake inhibitor enhances the antipsychotic effects of a dopamine receptor antagonist, supporting the role of the dopaminergic transmission regulation in the treatment of schizophrenia [93]. Lastly, A2R agonists have also been suggested as cocaine addiction drugs by altering receptor signaling within striatal A2AR-D2R heteromers [84].

Adenosine A2AR-Dopamine D2R-Glutamate mGluR5 Heteromer

In addition to adenosine and dopamine transmission, glutamate transmission plays an important role in the function of striatal GABAergic efferent neurons originating in the nucleus accumbens. Adenosine A2R and dopamine D2R also colocalize with metabotropic glutamate mGlu5 receptor (mGluR5) in striatum. A2R and mGluR5 are co-expressed in nearly half of striatal glutamatergic nerve terminals. An association of A2R and mGluR5 has been suggested by co-immunoprecipitation studies [9496]. Pharmacological studies suggest an allosteric interaction in the D2R-mGluR5 heteromer; mGluR5 agonists decrease the affinity of the striatal D2R agonist binding sites [97, 98]. The A2R, D2R and mGluR5 heterocomplex has been speculated in striatum [83]. Consonant with this idea, A2R and mGluR5 agonists synergistically increase ventral pallidal extracellular level of GABA in the nucleus accumbens leading to a potential stability of the inhibitory dopaminergic D2R effects on the striato-pallidal GABA pathway [99]. In Parkinson’s disease, glutamate transmission is overactive due to the reduced inhibitory D2R effect. It has been reported that the treatment of Parkinsons leads to regulation of mGluR5 and A2R activation [100, 101]. Indeed, mGluR5 and A2R antagonists, which are known to be antiparkinsonian, could act synergistically by targeting the proposed A2R-D2R-mGluR5 heterocomplex [102]. It has been reported that mGluR5 antagonists induce their motor activator effects only when both A2R and D2R are co-expressed [101]. Furthermore, the intramembrane A2R-D2R interaction is positively regulated by mGluR5 activation [94, 97, 98, 103]. These results suggest that A2R and mGluR5 antagonists may have their antiparkinsonian effects in the glutamatergic synapses of the dorsal striato-pallidal GABA neurons by enhancing the dopamine D2R signaling through their A2R-D2R-mGluR5 heteromerization [102]. Therefore, a simultaneous combination between A2R and mGluR5 antagonists enhancing their efficacy in reversing Parkinsonian motor deficits may constitute a novel nondopaminergic therapy by avoiding the adverse chronic effects of dopaminergic drugs [100, 104, 105]. A role of the striatal A2R-mGluR5 interactions in addiction has also been proposed, which may partly explain the modulation of cocaine and methamphetamine dependence by mGluR5 agonists [103, 106]. Thus, the A2R-D2R-mGluR5 heteromer could have a role in controlling the ventral striato-pallidal GABA neurons involved in drug addiction and schizophrenia and provide an interesting potential target for future therapies.

Adenosine A2AR-Dopamine D2R-Cannabinoid CB1R Heteromer

Cannabinoid represents an important inhibitory neuromodulator acting in the central nervous system. The activation of cannabinoid CB1 receptors (CB1R) leads to motor depression. CB1R are co-expressed predominantly with D2R in the soma and dendrites of the ventral striato-pallidal GABA neurons and with A2R in corticostriatal glutamate terminals [107112]. Recently, co-immunoprecipitation and BRET experiments showed that CB1R and A2R interact together in living cells and striatum and that this interaction is functional since it mediates the cannabinoid motor effects. Biochemical experiments in the neuroblastoma cell line and behavioral tests in mice indicated that striatal CB1R activation-induced synaptic effects depend on A2R activation [113]. Indeed, A2R antagonists lead to an inhibition of CB1R agonist-induced motor depressant effects [114]. A reduction of alterations in the motor depression effects induced by CB1R agonists in A2R knockout mice was also described [115]. Therefore, the A2R-CB1R heteromer may mediate the motor depressant effects of CB1R agonists. Moreover, evidence for CB1R-D2R heteromers in cell lines and striatum has been provided by co-immunoprecipitation experiments, FRET experiments and pharmacological analysis indicating the reduction of the affinity of D2R agonist binding by CB1R agonists and the agonist-induced enhancement of CB1R-D2R heteromerization. Several studies have reported antagonistic interactions between CB1R and D2R activation, as well as the antiparkinsonian actions of CB1R antagonists [116118].

CB1R knockout mice show an increase in their baseline levels on anxiety [119]. Furthermore, CB1R activation mediates central effects and more particularly the addictive properties of cannabinoids such as tetrahydrocannabinol (THC), the main psychoactive ingredient of marihuana [120]. In view of the potential role of CB1R-D2R heteromer and A2R antagonists as potential therapeutic agents in drug dependence [121123], the antagonistic CB1R-D2R interactions may also involve the A2R [124126]. A2R may play a role in the behavioral inhibition exerted by the CB1R agonist on D2R agonist-induced locomotor hyperactivity. In support of this view, striatal A2R is known to directly interact with both D2R [66] and CB1R [114], which, as noted above, may suggest the formation of a A2R-CB1R-D2R multisubunit complex in brain. Furthermore, A2R-CB1R-D2R heteromers have been recently identified in living cells by using a method combining bimolecular fluorescence complementation and BRET techniques [127129].

In post-mortem brain of schizophrenics, pharmacological studies showed an alteration of A2R [130], D2R [131] and CB1R [132] expression. In animal models, antipsychotic treatment leads to a down-regulation of CB1R expression in nucleus accumbens [133] which could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release [134]. Taking these results together, the A2R-CB1R-D2R heteromer may also have a role in schizophrenia and represents a potential target for antipsychotic compounds.

SEROTONIN 5-HT2AR-GLUTAMATE mGluR2 HETEROMER

The function of serotonin in brain is strongly associated with specific physiological responses, ranging from modulation of neuronal activity and transmitter release to behavioural changes [135]. Glutamate serves as the principal neurotransmitter of the pyramidal cells, which are the sources of efferent and interconnecting pathways of the cerebral cortex and limbic systems. A physical and functional interaction between serotonin 5-HT2A receptor and metabotropic glutamate subtype 2 receptor (mGluR2) has been identified in cortical pyramidal neurons [136140]. This heteromer constitutes a new and uncommon example of a physical association between GPCRs of different classes since mostly heteromers which have been described involve GPCRs belonging to the same class. Fluorescent in situ hybridization (FISH) experiments showed the co-localization of 5-HT2AR and mGluR2 mRNA expression in layer V of mouse somatosensory cortex and mouse cortical primary cultures. Evidence for a receptor association has been provided by co-immunoprecipitation, BRET, FRET, binding assays in heterologous systems and brain cortex. The glutamate mGluR2-serotonin 5-HT2AR heteromer represents an example of GPCR association having specific consequences on the pharmacology, signaling and behavioural pharmacology of drugs acting at the receptor components [136]. Competition binding experiments showed that a mGluR2 agonist increases the affinity of hallucinogenic drugs for the 5-HT2AR-binding site, and a 5-HT2AR agonist decreases the affinity of agonists for the mGluR2 binding site (Fig. 2). Changes in high affinity binding caused by mGluR2-5-HT2AR crosstalk suggest that this complex may serve to integrate serotonin and glutamate receptor signaling and modulate G-protein coupling. Pharmacologically, similar hallucinogenic and non-hallucinogenic serotonin 5-HT2AR agonists elicit qualitatively different downstream signaling events as reflected in the pattern of gene transcription they induce [141]. More precisely, hallucinogenic 5-HT2AR agonists induce specific receptor conformational changes within the mGluR2-5-HT2AR heteromer leading to the recruitment of specific cortical 5-HT2AR mediated signaling pathways [142]. Hallucinogenic 5-HT2AR agonists induce through the heteromerization with mGluR2 the activation of both Gαq/11 and also Gαi/o proteins, while 5-HT2AR and mGluR2 alone are coupled to Gαq/11 and Gαi/o proteins, respectively (Fig. 2). Similar evidence for specification of G-protein subtype regulation was also observed for the endogeneous mGluR2-5-HT2AR complex with membranes from cortical primary cultures [136]. Interestingly, the mGluR2-5-HT2AR heteromer may constitute thus a new target for antipsychotic drugs in the treatment of schizophrenia since mGluR2 agonists inhibit via allosteric interactions within the heteromer the 5-HT2AR-mediated hallucinogen-specific Gαi/o signaling [143145].

Fig. (2)
Functional crosstalk between glutamate mGluR2 and serotonin 5-HT2AR resulting of their heteromerization

The differences in the capacity of the mGluR2 and mGluR3 to change the pharmacological properties of 5-HT2AR and their close sequence similarity were the basis to identify the specific mGluR2 domains responsible for heteromer formation. The study of a series of molecular chimeras of the mGluR2 and mGluR3 demonstrated that the segment containing transmembrane helices 4 and 5 of mGluR2 is necessary and sufficient for the heteromeric formation with 5-HT2AR [136]. These results provide structural evidence for the formation of a heteromer between members of different classes of GPCRs and identify the involvement of specific protein domains at the interface of a GPCR heteromer.

The expression of each component of the mGluR2-5-HT2AR heteromer was studied in the brain of patients with schizophrenia. The receptor densities in cortical membranes of untreated schizophrenic subjects were significantly altered, showing increased 5-HT2AR and decreased mGluR2 expression levels [136]. It is possible that this dysregulation of 5-HT2AR and/or mGluR2 expression may alter cortical integration of serotonin and glutamate signaling and contribute to the abnormalities of thought and behavior in schizophrenia. These results are consistent with the hypothesis that the mGluR2-5-HT2AR heteromer integrates serotonin and glutamate signaling to regulate the sensory gating functions of the cortex, a process that is disrupted in psychosis [137140]. This complex warrants further study of its possible role in the symptoms of schizophrenia and of its potential as a therapeutic target.

ADRENERGIC α2AR-OPIOID MOR HETEROMER

Another example of receptor association is the α2AR-MOR heteromer. α2AR and MOR are both members of GPCR class A that couple to the same Gi/o class of G proteins. These receptors affect the nociceptive system and are particularly involved in depression of neurotransmitter release in the spinal cord [146150]. Activation of MOR by agonists such as morphine results in strong antinociceptive effects [151, 152]. In vivo, evidence for a colocalization of α2AR and MOR was provided by immunocytochemical experiments in hippocampal neurons and in neurons of of the medial nucleus tractus solitarius, suggesting a crosstalk between the two systems [153, 154].

The first evidence supporting an association between the two receptors comes from studies using mice without functional α2AR. These mice show a decrease in the analgesic potency of spinally administered morphine compared with wild-type mice. These results are consonant with the effects depending on an interaction between α2AR and MOR [150]. A direct interaction between α2AR and MOR has been demonstrated using biophysical, biochemical and pharmacological studies in transfected cells and primary neurons [154156]. Treatment with either MOR or α2AR agonist increases the quantity of immunoprecipitable receptor complex. In addition, in transfect cells, expression of both receptors increases level of G protein activation and mitogen-activated protein kinase (MAPK) phosphorylation induced with either agonist. These results suggest that the α2AR-MOR complex increases the signaling and efficacy of specific MOR (morphine) or α2AR (clonidine) agonists [154]. The α2AR-MOR heteromer may play a role in the adrenergicopioid crosstalk seen in vivo in older studies, such as the potentiating effect of clonidine on morphine analgesia [157, 158]. While the link between the functional crosstalk of the two receptors and their physical interaction is controversial [155], recently, an approach based on FRET confirmed the existence of the α2AR-MOR heteromer and its effects on cell signaling [156]. Indeed, cross-conformational changes within the heteromer lead to a transinhibition of receptor activation [156]. Using MOR or α2AR agonists in electrophysiology experiments measuring voltage-gated Ca2+, a mutual cross-desensitization between MOR and α2AR-mediated current inhibition was demonstrated. This effect is closely associated with simultaneous internalization of MOR and α2AR. Furthermore, inhibition of p38 MAPK prevented the crossdesensitization as well as cointernalization of MOR and α2AR. Changes in receptor trafficking profiles suggested that p38 MAPK activity was required for initiating MOR internalization and maintaining possible MOR and α2AR association during their cointernalization. Moreover, it was demonstrated the p38 MAPK and β-arrestin 2 dependent cross-regulation between neuronal MOR and α2AR [159].

Biochemical assays in postmortem human brain also suggest that the α2AR-MOR heteromer might play a role in opioid addiction. Adaptive changes of α2AR density in brain have been demonstrated in animal models of opioid dependence [160, 161], as well as in postmortem human brain of opioid addicts [162]. It has also been suggested that, in cortical brain membranes from chronic abusers of opioids, the MOR-G protein coupling is unchanged, whereas the potency of the selective α2AR agonist UK14304 to stimulate [35S]GTPγS binding decreased [163]. Concurrently with results in vitro in tissue cultures, these findings in postmortem human brain of opioid addicts raise the possibility that α2AR down-regulation and desensitization may alter the signaling properties of the α2AR-MOR heteromer and therefore prevent the down-regulation of MOR and their functional uncoupling for G proteins in vivo. Further experiments are required in MOR and/or α2AR knock-out mouse models to test this hypothesis.

In summary, these results are consistent with the notion that the physical interaction between MOR and α2AR plays an important role in modulating their signaling. These effects may result from agonist-induced changes in receptor conformation and/or heteromer association.

OTHER GPCR HETEROMERS

Given the large number of GPCRs, it is likely that many other heteromers exist and contribute to normal brain function and disease pathogenesis. Evidence for the existence of various other GPCR heteromers has been reported. A functional interaction between adenosine A1R and metabotropic glutamate mGluR1α has been shown in transfected cells as well in cerebellar and primary cortical neurons and may constitute a possible therapeutic target for antipsychotic drugs [164]. In addition, the adenosine A1R could also interact with serotonin 5-HT2AR, since an A1R agonist abolishes the molecular and behavioural effects induced by a hallucinogenic 5-HT2AR agonist while a A1R antagonist produces opposite effects [165, 166]. Both A1R and mGluR2 couple to the Gi/o protein and their agonists have somewhat similar effects on the hallucinogen-induced 5-HT2AR activation.

Recently, opposing pharmacological and behavioral properties have been described for the dopamine D2R and histamine H3 receptor (H3R) in striatum. These results are consistent with an interaction between the two GPCRs, D2R-H3R heteromers have been identified by BRET in transfected cells [167]. It is conceivable that a complex can exist between A2AR-D2R-H3R in vivo since these three GPCR are co-expressed in striato-pallidal GABA neurons. In addition to adenosine A2AR antagonists and dopamine D2R agonists, histamine H3R antagonists may represent novel antiparkinsonian agents suitable for targeting the heteromer.

Other GPCR heteromers have been implicated in neuropsychiatric disorders. Vasopressin V1b receptor (V1bR) and the receptor of bird ortholog vasopressin (VT2R) heteromerize with corticotropin releasing hormone receptor type 1 (CRHR1). These two heteromers have recently been identified and correlated with altered receptor signaling [168170]. Due to the co-localization of these receptors in corticotrope pituitary cells and the respective role of each in the stress axis, these heteromeric complexes are interesting candidates for playing a role in mood disorder or depression.

GPCR HETEROMERS AND DRUG DISCOVERY

GPCR heteromeric complexes having unique biochemical and functional properties provide important new targets for drug discovery [44, 171173]. While the identification of heteromers has complicated the search for drugs by providing a combinatorial increase in the number of targets, this complexity brings with it a new possibility for increased therapeutic efficacy and reduced side effects. Receptor heteromerization represents a direct mechanism for crosstalk between two extracellular signals. The many emerging examples of modification of the pharmacological and signaling properties of a GPCR in presence of another one highlights the importance of identifying appropriate heteromeric complexes as drug targets. The potential roles of opioid receptor heteromers in analgesia and drug tolerance/dependence [174177] and of serotonin/glutamate heteromers in the effects of antipsychotics agents [143] suggest that heteromers will be increasingly important as drug targets for diseases of the brain and nervous system.

Presently, most ligands acting at heteromers are molecules that target preferentially one of the receptors components of the heteromer (Fig. 3). For example, SKF83959 which has antiparkinsonian effects, is believed to act at the D1R-D2R heteromer and induce Gq/11 protein signaling in the brain. SKF83959 binds preferentially to the D1R component of the complex [43, 178]. It is likely that a number of GPCR ligands having therapeutic potential (D2R, A2AR, 5-HT2AR, mGluR2, mGluR5, CB1R, and H3R ligands) exert some of their clinical effects in diseases ranging from Parkinson’s to schizophrenia from their activity at GPCR heteromers. Furthermore, some dopamine receptor ligands originally described as being selective for a single receptor may be heteromer-specific. An example is the antipsychotic l-stepholidine, which is pharmacologically a D1R agonist and a D2R antagonist, that may act specifically at D1R-D2R heteromers [179]. In the same manner, antiparkinsonian agents such as S32504, ropinirole and pramipexole also represent selective agonists for D2R-D3R heteromers [59]. Thus, while heteromer-specific compounds are more difficult to characterize, their use could reduce side effects in the treatment of neurological diseases.

Fig. (3)
GPCR heteromer-specific drug targets

A different approach is to develop bivalent ligands binding both individual components of the heteromer [180] (Fig. 3). These compounds contain two different GPCR ligands (two agonists, two antagonists or one of each) that are linked through amino acid spacers [181]. A recent study describes the development of new adenosine A2AR antagonist – dopamine D2R agonist bivalent ligands useful for the detection of A2AR-D2R heteromers [182]. In view of selective A2AR antagonists and D2R agonists as antiparkinsonian compounds, the adenosine / dopamine bivalent action could be used for the treatment of Parkinson’s disease by improving specificity and efficacy. In addition, GPCR ligands that act as allosteric modulators represent another potential therapeutic tool to target receptor heteromeric entities [8, 28, 183].

CONCLUSIONS

Experimental results on GPCR heteromers highlight the difficulty in identifying a causal link between functional crosstalk of two receptor systems and physical association of the receptor components in a complex. Determining factors regulating the receptor crosstalk mechanisms involved in neurological disorders is an increasingly important avenue for developing improved drugs. Before the “dimerization” era, GPCR crosstalk was attributed entirely to the integrative effects of distinct signaling pathways coupled to each receptor. As described above, GPCR dimerization is a likely mechanism for the interactive effects of drugs as well as of the responses to individual drugs [19, 184]. However, the role of heteromers in signaling crosstalk has only been demonsrated conclusively in a few instances and crosstalk resulting from the regulation of downstream signaling molecules will remain an important mechanism for interactive effects [185].

Until recently, most experiments demonstrating a direct GPCR interaction and its functional consequences have been performed in artificial cell systems using modified receptors. Nevertheless, some examples by using functional (knockout mice), biochemical (co-immunoprecipitation) and pharmacological (allosteric modulations) approaches strongly support the existence of functional heteromers in vivo [5, 6, 31, 35, 136, 164, 186191]. However, with the exception of positive cooperative binding [13, 192, 193], most results from such studies do not exclude mechanisms independent of direct receptor interaction. Techniques suitable for studying direct receptor-receptor interactions in native tissues require new reagents, including receptor / heteromer-specific antibodies, heteromer-specific ligands, ligands containing fluorophore labelling and approaches to study the functional effects of receptor complexes in specific subcellular compartments in vivo [194196]. One new approach is the development of a biophysical method to detect physical GPCR interactions in native tissues using time-resolved FRET and selective fluorescent ligands [197, 198]. The relationship of altered receptor heteromerization and symptoms of brain disease remains to be determined.

We have focused our attention on direct GPCR interactions with putative implications in neuropsychiatric disorders (Table 1). The allosteric interaction in the receptor heteromer namely the intermolecular interaction by which binding of a ligand to one of the receptor units in the heteromer changes the binding properties of another receptor unit constitutes the predominant biochemical effect [2, 199, 200]. Allosteric mechanisms within heteromers induce facilitating or inhibitory effects on ligand binding and also alter receptor signaling selectivity and trafficking [27, 43]. GPCR heteromerization leads to a diversity of pharmacological profiles and considerably increases the repertoire of GPCR functional responses. This diversity is increased if GPCRs exist as complexes comprising than two sub-units [2]. Indeed, recent studies report the existence of oligomers (or multimers) [128, 183, 201205]. The stochiometry of the receptor complexes and the functional consequences of complex formation are areas of intense current research.

The direct interactions between GPCRs can also depend on partner proteins at the extracellular, intramembrane or intracellular compartments [14, 199, 200]. Interactions between heteromers and various partners such as G proteins, β-arrestins, calmodulin, regulators of G protein signaling (RGS) or receptor activity modifying proteins (RAMP) have consequences in the regulation of signal transduction [206, 207]. Another challenge is determining the stoichiometry of interaction between heteromers and their signaling / adapter proteins. Studies in this area are likely to yield additional targets for the design of specific drugs for brain disease.

GPCR heteromerization provides promising insights into GPCR pathophysiology and the development of novel heteromer-targeted drugs. Recent studies have demonstrated incontrovertible existence for functional GPCR heteromers in a native context. The characterization of heteromerization represents a challenge and an opportunity for developing new drugs for brain disease with increased therapeutic efficacy and reduced side effects.

ABBREVIATIONS

GPCR
G protein-coupled receptor
A1R
Adenosine A1 receptor
A2AR
Adenosine A2A receptor
D1R
Dopamine D1 receptor
D2R
Dopamine D2 receptor
D3R
Dopamine D3 receptor
CB1R
Cannabinoid 1 receptor
mGluR5
Metabotropic glutamate receptor 5
mGluR2
Metabotropic glutamate receptor 2
5-HT2AR
Serotonin 2A receptor
α2AR
α2A-adrenergic receptor
β1AR
β1-adrenergic receptor
β2AR
β2-adrenergic receptor
MOR
Mu opioid receptor
DOR
Delta opioid receptor
SSTR2A
Somatostatin 2A receptor
H3R
Histamine H3 receptor
BRET
Bioluminescence resonance energy transfer
FRET
Fluorescence resonance energy transfer
GABA
Gamma-aminobutyric acid
MAPK
Mitogen-activated protein kinase

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