L-DOPA Receptor GPR143 Functionally Couples with Adrenergic α1B Receptor at the Second Transmembrane Interface.

Adrenergic receptors (ADRs) are widely distributed in the peripheral and central nervous systems. We previously reported that L-3,4-dihydroxyphenylalanine (L-DOPA), the precursor of dopamine, sensitizes adrenergic α1 receptor (ADRA1) through a G protein-coupled receptor GPR143. Chimeric analysis, in which the transmembrane (TM) domains of GPR143 were replaced with those of GPR37, revealed that the second TM region was essential for the potentiation of phenylephrine-induced extracellular signal-regulated kinase (ERK) phosphorylation by GPR143. In HEK293T cells expressing ADRA1B, phenylephrine-induced ERK phosphorylation was augmented by the co-expression of GPR143, compared to the mock vector. Immunoprecipitation analysis revealed that a synthetic transactivator of the transcription peptide fused with TM2 of GPR143 (TAT-TM2) disrupts the interaction between GPR143 and ADRA1B. This TAT-TM2 peptide suppressed the augmentation of phenylephrine-induced ERK phosphorylation by GPR143 in HEK293T cells co-expressing ADRA1B and GPR143. These results indicate that the interaction between GPR143 and ADRA1B is required for the potentiation of ADRA1B-mediated signaling by GPR143. The TM2 region of GPR143 is a crucial dimeric interface for the functional coupling between ADRA1B and GPR143.

Pharmacological evidence for transactivation within melatonin MT2 and serotonin 5-HT2C receptor heteromers in mouse brain.

Association of G protein-coupled receptors into heterodimeric complexes has been reported for over 50 receptor pairs in vitro but functional in vivo validation remains a challenge. Our recent in vitro studies defined the functional fingerprint of heteromers composed of Gi -coupled melatonin MT2 receptors and Gq -coupled serotonin 5-HT2C receptors, in which melatonin transactivates phospholipase C (PLC) through 5-HT2C . Here, we identified this functional fingerprint in the mouse brain. Gq protein activation was probed by [35 S]GTPγS incorporation followed by Gq immunoprecipitation, and PLC activation by determining the inositol phosphate levels in brain lysates of animals previously treated with melatonin. Melatonin concentration-dependently activated Gq proteins and PLC in the hypothalamus and cerebellum but not in cortex. These effects were inhibited by the 5-HT2C receptor-specific inverse agonist SB-243213, and were absent in MT2 and 5-HT2C knockout mice, fully recapitulating previous in vitro data and indicating the involvement of MT2 /5-HT2C heteromers. The antidepressant agomelatine had a similar effect than melatonin when applied alone but blocked the melatonin-promoted Gq activation due to its 5-HT2C antagonistic component. Collectively, we provide strong functional evidence for the existence of MT2 /5-HT2C heteromeric complexes in mouse brain. These heteromers might participate in the in vivo effects of agomelatine.

Signaling profiles in HEK 293T cells co-expressing GLP-1 and GIP receptors.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are regarded as 'incretins' working closely to regulate glucose homeostasis. Unimolecular dual and triple agonists of GLP-1R and GIPR have shown remarkable clinical benefits in treating type 2 diabetes. However, their pharmacological characterization is usually carried out in a single receptor-expressing system. In the present study we constructed a co-expression system of both GLP-1R and GIPR to study the signaling profiles elicited by mono, dual and triple agonists. We show that when the two receptors were co-expressed in HEK 293T cells with comparable receptor ratio to pancreatic cancer cells, GIP predominately induced cAMP accumulation while GLP-1 was biased towards ß-arrestin 2 recruitment. The presence of GIPR negatively impacted GLP-1R-mediated cAMP and ß-arrestin 2 responses. While sharing some common modulating features, dual agonists (peptide 19 and LY3298176) and a triple agonist displayed differentiated signaling profiles as well as negative impact on the heteromerization that may help interpret their superior clinical efficacies.

Physiological roles of heteromerization: focus on the two-pore domain potassium channels.

Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.

TRESK and TREK-2 two-pore-domain potassium channel subunits form functional heterodimers in primary somatosensory neurons.

Two-pore-domain potassium channels (K2P) are the major determinants of the background potassium conductance. They play a crucial role in setting the resting membrane potential and regulating cellular excitability. These channels form homodimers; however, a few examples of heterodimerization have also been reported. The K2P channel subunits TRESK and TREK-2 provide the predominant background potassium current in the primary sensory neurons of the dorsal root and trigeminal ganglia. A recent study has shown that a TRESK mutation causes migraine because it leads to the formation of a dominant negative truncated TRESK fragment. Surprisingly, this fragment can also interact with TREK-2. In this study, we determined the biophysical and pharmacological properties of the TRESK/TREK-2 heterodimer using a covalently linked TRESK/TREK-2 construct to ensure the assembly of the different subunits. The tandem channel has an intermediate single-channel conductance compared with the TRESK and TREK-2 homodimers. Similar conductance values were recorded when TRESK and TREK-2 were coexpressed, demonstrating that the two subunits can spontaneously form functional heterodimers. The TRESK component confers calcineurin-dependent regulation to the heterodimer and gives rise to a pharmacological profile similar to the TRESK homodimer, whereas the presence of the TREK-2 subunit renders the channel sensitive to the selective TREK-2 activator T2A3. In trigeminal primary sensory neurons, we detected single-channel activity with biophysical and pharmacological properties similar to the TRESK/TREK-2 tandem, indicating that WT TRESK and TREK-2 subunits coassemble to form functional heterodimeric channels also in native cells.

Regulation of the thrombin/protease-activated receptor 1 axis by chemokine (CXC motif) receptor 4.

The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce ß-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced ß-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (CXC motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption.

Cross-talk between dopamine D1 and corticotropin releasing factor type 2 receptors leads to occlusion of their ERK1/2 signaling.

One manner in which G protein-coupled receptors potentiate, increase, and change their functionality is through the formation of heteromers in a specific cellular context. Previously, we have shown that dopamine D1 receptor (D1R) and the corticotropin releasing factor receptor type-2α (CRF2α) heteromerize in HEK293T cells, enabling D1R to mobilize intracellular calcium in response to D1R agonists. In this study, we further investigated the pharmacological properties of the CRF2α-D1R heteromer and the consequences of the heteromerization in their signaling and subcellular localization when both receptors are co-expressed in HEK293T cells. Using immunoprecipitation assays, we observed that the addition of 10 µM dopamine in the incubation medium significantly decreased the amount of CRF2α on the cell surface of cells expressing both receptors. The presence of agonists of both receptors increased the interaction between CRF2α and D1R as assessed by co-immunoprecipitation. However, the presence of agonists of both receptors resulted in a lesser efficient activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase. Using a synaptosomal preparation of rat prefrontal cortex devoid of post-synaptic elements, we found that CRF2α and D1R co-localize in synaptic terminals of the rat medial prefrontal cortex and that the simultaneous activation of both receptors also occluded phosphorylation of extracellular signal-regulated kinase. These results strengthen the idea that the heteromer CRF2a-D1R is an entity functionally different from each receptor that composes it and suggests that its formation is enhanced by CRF and dopamine co-transmission, as occurs in stress and addiction.

In vivo actions of SCTR/AT1aR heteromer in controlling Vp expression and release via cFos/cAMP/CREB pathway in magnocellular neurons of PVN.

With an increasing body of evidence regarding GPCR oligomerization and its clinical implications over the last decade, the modulation and dynamics of GPCR homo- and hetero-oligomers has more recently become an area of intense research focus. Previously, our lab showed in vitro heteromer formation between angiotensin II receptor type 1 subtype a (AT1aR) and secretin receptor (SCTR), which is involved in in vivo control of hyperosmolality-induced water drinking behavior. Because the secretin (SCT)/SCTR axis is crucial to the central actions of angiotensin II (ANGII) and both SCT and ANGII are capable of triggering vasopressin (Vp) release from hypothalamus, we investigated here the in vivo role of SCTR-AT1aR heteromer in regulating Vp release in hypothalamus using transmembrane peptides as tools. We showed that SCTR-AT1aR heteromer mediates stimulatory actions of both SCT and ANGII in hypothalamic Vp expression and release as well as neuronal activities via the immediate early gene cFos. The results from this study not only are consistent with our hypothesis that SCT and ANGII interact at the receptor level to mediate their water homeostatic activities but also provide evidence for in vivo functions of cross-class GPCR heteromers.-Mak, S. O. K., Zhang, L., Chow, B. K. C. In vivo actions of SCTR/AT1aR heteromer in controlling Vp expression and release via cFos/cAMP/CREB pathway in magnocellular neurons of PVN.

TRPC1 as a negative regulator for TRPC4 and TRPC5 channels.

Transient receptor potential canonical (TRPC) channels are calcium permeable, non-selective cation channels with wide tissue-specific distribution. Among 7 TRPC channels, TRPC 1/4/5 and TRPC3/6/7 are subdivided based on amino acid sequence homology. TRPC4 and TRPC5 channels exhibit cationic current with homotetrameric form, but they also form heterotetrameric channel such as TRPC1/4 or TRPC1/5 once TRPC1 is incorporated. The expression of TRPC1 is ubiquitous whereas the expressions of TRPC4 and TRPC5 are rather focused in nervous system. With the help of conditional knock-out of TPRC1, 4 and/or 5 genes, TRPC channels made of these constituents are reported to be involved in various pathophysiological functions such as seizure, anxiety-like behaviour, fear, Huntington's disease, Parkinson's disease and many others. In heterologous expression system, many issues such as activation mechanism, stoichiometry and relative cation permeabilites of homomeric or heteromeric channels have been addressed. In this review, we discussed the role of TRPC1 channel per se in plasma membrane, role of TRPC1 in heterotetrameric conformation (TRPC1/4 or TRPC1/5) and relationship between TRPC1/4/5 channels, calcium influx and voltage-gated calcium channels.

Nicotine prevents alpha-synuclein accumulation in mouse and human iPSC-derived dopaminergic neurons through activation of the dopamine D3- acetylcholine nicotinic receptor heteromer.

We recently found that in mouse dopaminergic neurons, the heteromer formed by the dopamine D3 receptor (D3R) and the ß2 subunit of acetylcholine nicotinic receptor (nAChR) exerts neurotrophic effects when activated by nicotine, leading to neurons with enlarged cell bodies and increased dendrite arborization. Beside this action, we now show that nicotine, by activating the D3R-nAChR heteromer, protects dopaminergic neurons against neuronal injury. In primary cultures of mouse dopaminergic neurons, in fact, the ability of nicotine to inhibit both the pathological accumulation of alpha-synuclein induced by glucose deprivation and the consequent morphological defects were strongly prevented by disrupting the D3R-nAChR heteromer with specific interfering TAT-peptides; the relevance of the phosphoinositide 3-kinase (PI3K) intracellular signaling in mediating nicotine prevention of alpha-synuclein aggregation has been also demonstrated. Moreover, the ability of nicotine in restoring the ubiquitin-proteasome system has been found as a mechanism contributing to the neuroprotective properties of nicotine. By using the proximity ligation assay, we have shown that the D3R-nAChR heteromer is also expressed in human dopaminergic neurons derived from induced pluripotent stem cells. In this human cell model, nicotine exerts neuroprotective effects specifically acting through the D3R-nAChR complex thus indicating that this heteromer is a relevant molecular effector involved in the protection of human dopaminergic neurons.

Mixing and matching TREK/TRAAK subunits generate heterodimeric K2P channels with unique properties.

The tandem of pore domain in a weak inwardly rectifying K(+) channel (Twik)-related acid-arachidonic activated K(+) channel (TRAAK) and Twik-related K(+) channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K2P) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs.

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

Plant and Mammal Aquaporins: Same but Different.

Aquaporins (AQPs) constitute an ancient and diverse protein family present in all living organisms, indicating a common ancient ancestor. However, during evolution, these organisms appear and evolve differently, leading to different cell organizations and physiological processes. Amongst the eukaryotes, an important distinction between plants and animals is evident, the most conspicuous difference being that plants are sessile organisms facing ever-changing environmental conditions. In addition, plants are mostly autotrophic, being able to synthesize carbohydrates molecules from the carbon dioxide in the air during the process of photosynthesis, using sunlight as an energy source. It is therefore interesting to analyze how, in these different contexts specific to both kingdoms of life, AQP function and regulation evolved. This review aims at highlighting similarities and differences between plant and mammal AQPs. Emphasis is given to the comparison of isoform numbers, their substrate selectivity, the regulation of the subcellular localization, and the channel activity.

Formation of Functional Heterodimers by TREK-1 and TREK-2 Two-pore Domain Potassium Channel Subunits.

Two-pore domain (K2P) potassium channels are the major molecular correlates of the background (leak) K(+) current in a wide variety of cell types. They generally play a key role in setting the resting membrane potential and regulate the response of excitable cells to various stimuli. K2P channels usually function as homodimers, and only a few examples of heteromerization have been previously reported. Expression of the TREK (TWIK-related K(+) channel) subfamily members of K2P channels often overlaps in neurons and in other excitable cells. Here, we demonstrate that heterologous coexpression of TREK-1 and TREK-2 subunits results in the formation of functional heterodimers. Taking advantage of a tandem construct (in which the two different subunits were linked together to enforce heterodimerization), we characterized the biophysical and pharmacological properties of the TREK-1/TREK-2 current. The heteromer was inhibited by extracellular acidification and by spadin similarly to TREK-1, and its ruthenium red sensitivity was intermediate between TREK-1 and TREK-2 homodimers. The heterodimer has also been distinguished from the homodimers by its unique single channel conductance. Assembly of the two different subunits was confirmed by coimmunoprecipitation of epitope-tagged TREK-1 and TREK-2 subunits, coexpressed in Xenopus oocytes. Formation of TREK-1/TREK-2 channels was also demonstrated in native dorsal root ganglion neurons indicating that heterodimerization may provide greater diversity of leak K(+) conductances also in native tissues.

Loop B serine of a plasma membrane aquaporin type PIP2 but not PIP1 plays a key role in pH sensing.

In the plant kingdom, the plasma membrane intrinsic aquaporins (PIPs) constitute a highly conserved group of water channels with the capacity of rapidly adjusting the water permeability (Pf) of a cell by a gating response. Most evidence regarding this mechanism was obtained by different biophysical approaches including the crystallization of a Spinaca olaracea PIP2 aquaporin (SoPIP2;1) in an open and close conformation. A close state seems to prevail under certain stimuli such as cytosolic pH decrease, intracellular Ca2+ concentration increase and dephosphorylation of specific serines. In this work we decided to address whether the state of phosphorylation of a loop B serine - highly conserved in all PIPs - combined with cytosolic acidification can jointly affect the gating response. To achieve this goal we generated loop B serine mutants of two PIP types of Fragaria×ananassa (FaPIP2;1S121A and FaPIP1;1S131A) in order to simulate a dephosphorylated state and characterize their behavior in terms of Pf and pH sensitivities. The response was tested for different co-expressions of PIPs (homo and heterotetramers combining wild-type and mutant PIPs) in Xenopus oocytes. Our results show that loop B serine phosphorylation status affects pH gating of FaPIP2;1 but not of FaPIP1;1 by changing its sensitivity to more alkaline pHs. Therefore, we propose that a counterpoint of different regulatory mechanisms - heterotetramerization, serine phosphorylation status and pH sensitivity - affect aquaporin gating thus ruling the Pf of a membrane that expresses PIPs when fast responses are mandatory.

Convergence of melatonin and serotonin (5-HT) signaling at MT2/5-HT2C receptor heteromers.

Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2C receptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT2 and 5-HT2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT2/5-HT2C heteromers amplified the 5-HT-mediated Gq/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT2C protomer of MT2/5-HT2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows "biased signaling." These observations demonstrate the existence of functionally unique MT2/5-HT2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT2/5-HT2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.

Dopamine D2 and serotonin 5-HT1A receptor interaction in the context of the effects of antipsychotics - in vitro studies.

The serotonin 5-HT1A receptor (5-HT1 A R) and dopamine D2 receptor (D2 R) have been implicated as important sites of action in antipsychotics. Several lines of evidence indicate the key role of G protein-coupled receptors (GPCRs) heteromers in pathophysiology of schizophrenia and highlight these complexes as novel drug targets. Because heterodimers can form only on those cells co-expressing constituent receptors, they present a target of high pharmacological specificity in the context of biochemical effects induced by antipsychotic drugs. In studies conducted in the HEK 293 cell line, we demonstrated that 5-HT1 A R and D2 R are able to form constitutive heterodimers, and antipsychotic drugs (clozapine, olanzapine, aripiprazole, and lurasidone) enhanced this process, with clozapine being most effective. Various functional tests (cAMP and IP1 as well as ERK activation) indicated that the drugs had different effects on signal transduction by the heteromer. Interestingly, co-incubation of heterodimer-expressing HEK 293 cells with clozapine and the 5-HT1 A R agonist 8-OH DPAT potentiated post-synaptic effects, especially with respect to ERK activation. Our results indicate that the D2 -5-HT1A complex possesses biochemical, pharmacological, and functional properties distinct from those of mono- and homomers. This result has implications for the development of improved pharmacotherapy for schizophrenia or other disorders (activating the heteromer might be cognitive enhancing, since it is expressed in frontal cortex) through the specific targeting of heterodimers. We reported the constitutive formation of D2 -5-HT1A heteromers, which possess biochemical, pharmacological, and functional properties distinct from those of mono- and homomers, as revealed by antipsychotics action. We also showed that these two receptors are co-expressed in mouse cortical neurons; therefore their potential to heterodimerize may comprise an essential target for the development of novel strategies for schizophrenia treatment.

Evidence for the heterotetrameric structure of the adenosine A2A-dopamine D2 receptor complex.

Heteromers of G-protein-coupled receptors (GPCRs) have emerged as potential novel targets for drug development. Accumulating evidence indicates that GPCRs can form homodimers and heteromers, with homodimers being the predominant species and oligomeric receptors being formed as multiples of dimers. Recently, heterotetrameric structures have been proposed for dopamine D1receptor (D1R)-dopamine D3receptor (D3R) and adenosine A2Areceptor (A2AR)-dopamine D2receptor (D2R) heteromers. The structural model proposed for these complexes is a heteromer constituted by two receptor homodimers. The existence of GPCR homodimers and heteromers provides a structural basis for inter-protomer allosteric mechanisms that might account for a multiplicity of unique pharmacological properties. In this review, we focus on the A2AR-D2R heterotetramer as an example of an oligomeric structure that is key in the modulation of striatal neuronal function. We also review the interfaces involved in this and other recently reported heteromers of GPCRs. Furthermore, we discuss several published studies showing theex vivoexpression of A2AR-D2R heteromers. The ability of A2AR agonists to decrease the affinity of D2R agonists has been reported and, on the basis of this interaction, A2AR antagonists have been proposed as potential drugs for the treatment of Parkinson's disease. The heterotetrameric structure of the A2AR-D2R complex offers a novel model that can provide new clues about how to adjust the drug dosage to the expected levels of endogenous adenosine.

Heteromerization of G2A and OGR1 enhances proton sensitivity and proton-induced calcium signals.

Proton-sensing G-protein-coupled receptors (GPCRs; OGR1, GPR4, G2A, TDAG8), with full activation at pH 6.4 ∼ 6.8, are important to pH homeostasis, immune responses and acid-induced pain. Although G2A mediates the G13-Rho pathway in response to acid, whether G2A activates Gs, Gi or Gq proteins remains debated. In this study, we examined the response of this fluorescence protein-tagged OGR1 family to acid stimulation in HEK293T cells. G2A did not generate detectable intracellular calcium or cAMP signals or show apparent receptor redistribution with moderate acid (pH ≥ 6.0) stimulation but reduced cAMP accumulation under strong acid stimulation (pH ≤ 5.5). Surprisingly, coexpression of OGR1- and G2A-enhanced proton sensitivity and proton-induced calcium signals. This alteration is attributed to oligomerization of OGR1 and G2A. The oligomeric potential locates receptors at a specific site, which leads to enhanced proton-induced calcium signals through channels.

Heteromeric TRPC3 with TRPC1 formed via its ankyrin repeats regulates the resting cytosolic Ca2+ levels in skeletal muscle.

The main tasks of skeletal muscle are muscle contraction and relaxation, which are mediated by changes in cytosolic Ca(2+) levels. Canonical-type transient receptor potential 3 (TRPC3) contains an ankyrin repeat (AR) region at the N-terminus (38-188 amino acids) and forms extracellular Ca(2+)-entry channels by homo or heteromerization with other TRP subtypes in various cells including skeletal myotubes. However, previous research has not determined which region(s) of TRPC3 is responsible for the heteromerization, whether the AR region participates in the heteromerizations, or what is the role of heteromeric TRPC3s in skeletal muscle. In the present study, the heteromerization of TRPC3 with TRPC1 was first examined by GST pull-down assays of TRPC3 portions with TRPC1. The portion containing the AR region of TRPC3 was bound to the TRPC1, but the binding was inhibited by the very end sub-region of the TRPC3 (1-37 amino acids). In-silico studies have suggested that the very end sub-region possibly induces a structural change in the AR region. Second, the very end sub-region of TRPC3 was expressed in mouse primary skeletal myotubes, resulting in a dominant-negative inhibition of heteromeric TRPC3/1 formation. In addition, the skeletal myotubes expressing the very end sub-region showed a decrease in resting cytosolic Ca(2+) levels. These results suggest that the AR region of TRPC3 could mediate the heteromeric TRPC3/1 formation, and the heteromeric TRPC3/1 could participate in regulating the resting cytosolic Ca(2+) levels in skeletal muscle.