Allosterism in the adenosine A2A and cannabinoid CB2 heteromer.

BACKGROUND AND PURPOSE: Allosterism is a regulatory mechanism for GPCRs that can be attained by ligand-binding or protein-protein interactions with another GPCR. We have studied the influence of the dimer interface on the allosteric properties of the A2A receptor and CB2 receptor heteromer. EXPERIMENTAL APPROACH: We have evaluated cAMP production, phosphorylation of signal-regulated kinases (pERK1/2), label-free dynamic mass redistribution, ß-arrestin 2 recruitment and bimolecular fluorescence complementation assays in the absence and presence of synthetic peptides that disrupt the formation of the heteromer. Molecular dynamic simulations provided converging evidence that the heteromeric interface influences the allosteric properties of the A2AR-CB2R heteromer. KEY RESULTS: Apo A2AR blocks agonist-induced signalling of CB2R. The disruptive peptides, with the amino acid sequence of transmembrane (TM) 6 of A2AR or CB2R, facilitate CB2R activation, suggesting that A2AR allosterically prevents the outward movement of TM 6 of CB2R for G protein binding. Significantly, binding of the selective antagonist SCH 58261 to A2AR also facilitated agonist-induced activation of CB2R. CONCLUSIONS AND IMPLICATIONS: It is proposed that the A2AR-CB2R heteromer contains distinct dimerization interfaces that govern its functional properties. The molecular interface between protomers of the A2AR-CB2R heteromer interconverted from TM 6 for apo or agonist-bound A2AR, blocking CB2R activation, to mainly the TM 1/7 interface for antagonist-bound A2AR, facilitating the independent opening of intracellular cavities for G protein binding. These novel results shed light on a different type of allosteric mechanism and extend the repertoire of GPCR heteromer signalling.

17-ß-estradiol potentiates the neurotrophic and neuroprotective effects mediated by the dopamine D3/acetylcholine nicotinic receptor heteromer in dopaminergic neurons.

Dopaminergic neurons express a heteromer composed of the dopamine D3 receptor and the α4ß2 nicotinic acetylcholine receptor, the D3R-nAChR heteromer, activated by both nicotine and dopamine D2 and D3 receptors agonists, such as quinpirole, and crucial for dopaminergic neuron homeostasis. We now report that D3R-nAChR heteromer activity is potentiated by 17-ß-estradiol which acts as a positive allosteric modulator by binding a specific domain on the α4 subunit of the nicotinic receptor protomer. In mouse dopaminergic neurons, in fact, 17-ß-estradiol significantly increased the ability of nicotine and quinpirole in promoting neuron dendritic remodeling and in protecting neurons against the accumulation of α-synuclein induced by deprivation of glucose, with a mechanism that does not involve the classical estrogen receptors. The potentiation induced by 17-ß-estradiol required the D3R-nAChR heteromer since either nicotinic receptor or dopamine D3 receptor antagonists and interfering TAT-peptides, but not the estrogen receptor antagonist fulvestrant, specifically prevented 17-ß-estradiol effects. Evidence of estrogens neuroprotection, mainly mediated by genomic mechanisms, have been provided, which is in line with epidemiological data reporting that females are less likely to develop Parkinson's Disease than males. Therefore, potentiation of D3R-nAChR heteromer activity may represent a further mechanism by which 17-ß-estradiol reduces dopaminergic neuron vulnerability.

Cannabinoid and Orexigenic Systems Interplay as a New Focus of Research in Alzheimer's Disease.

Alzheimer's disease (AD) remains a significant health challenge, with an increasing prevalence globally. Recent research has aimed to deepen the understanding of the disease pathophysiology and to find potential therapeutic interventions. In this regard, G protein-coupled receptors (GPCRs) have emerged as novel potential therapeutic targets to palliate the progression of neurodegenerative diseases such as AD. Orexin and cannabinoid receptors are GPCRs capable of forming heteromeric complexes with a relevant role in the development of this disease. On the one hand, the hyperactivation of the orexins system has been associated with sleep-wake cycle disruption and Aß peptide accumulation. On the other hand, cannabinoid receptor overexpression takes place in a neuroinflammatory environment, favoring neuroprotective effects. Considering the high number of interactions between cannabinoid and orexin systems that have been described, regulation of this interplay emerges as a new focus of research. In fact, in microglial primary cultures of APPSw/Ind mice model of AD there is an important increase in CB2R-OX1R complex expression, while OX1R antagonism potentiates the neuroprotective effects of CB2R. Specifically, pretreatment with the OX1R antagonist has been shown to strongly potentiate CB2R signaling in the cAMP pathway. Furthermore, the blockade of OX1R can also abolish the detrimental effects of OX1R overactivation in AD. In this sense, CB2R-OX1R becomes a new potential therapeutic target to combat AD.

The Expression and Functionality of CB1R-NMDAR Complexes Are Decreased in A Parkinson's Disease Model.

One of the hallmarks of Parkinson's disease (PD) is the alteration in the expression and function of NMDA receptor (NMDAR) and cannabinoid receptor 1 (CB1R). The presence of CB1R-NMDAR complexes has been described in neuronal primary cultures. The activation of CB1R in CB1R-NMDAR complexes was suggested to counteract the detrimental NMDAR overactivation in an AD mice model. Thus, we aimed to explore the role of this receptor complex in PD. By using Bioluminescence Resonance Energy Transfer (BRET) assay, it was demonstrated that α-synuclein induces a reorganization of the CB1R-NMDAR complex in transfected HEK-293T cells. Moreover, α-synuclein treatment induced a decrease in the cAMP and MAP kinase (MAPK) signaling of both CB1R and NMDAR not only in transfected cells but also in neuronal primary cultures. Finally, the interaction between CB1R and NMDAR was studied by Proximity Ligation Assay (PLA) in neuronal primary cultures, where it was observed that the expression of CB1R-NMDAR complexes was decreased upon α-synuclein treatment. These results point to a role of CB1R-NMDAR complexes as a new therapeutic target in Parkinson's disease.

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

The adenosine A2A receptor in the basal ganglia: Expression, heteromerization, functional selectivity and signalling.

Adenosine is a neuroregulatory nucleoside that acts through four G protein-coupled receptors (GPCRs), A1, A2A, A2B and A3, which are widely expressed in cells of the nervous system. The A2A receptor (A2AR), the GPCR with the highest expression in the striatum, has a similar role to that of receptors for dopamine, one of the main neurotransmitters. Neuronal and glial A2ARs participate in the modulation of dopaminergic transmission and act in almost any action in which the basal ganglia is involved. This chapter revisits the expression of the A2AR in the basal ganglia in health and disease, and describes the diversity of signalling depending on whether the receptors are expressed as monomer or as heteromer. The A2AR can interact with other receptors as adenosine A1, dopamine D2, or cannabinoid CB1 to form heteromers with relevant functions in the basal ganglia. Heteromerization, with these and other GPCRs, provides diversity to A2AR-mediated signalling and to the modulation of neurotransmission. Thus, selective A2AR antagonists have neuroprotective potential acting directly on neurons, but also through modulation of glial cell activation, for example, by decreasing neuroinflammatory events that accompany neurodegenerative diseases. In fact, A2AR antagonists are safe and their potential in the therapy of Parkinson's disease has already led to the approval of one of them, istradefylline, in Japan and United States. The receptor also has a key role in reward circuits and, again, heteromers with dopamine receptors, but also with cannabinoid CB1 receptors, participate in the events triggered by drugs of abuse.

LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration.

BACKGROUND: G protein-coupled receptor heteromerization is believed to exert dynamic regulatory impact on signal transduction. CXC chemokine receptor 4 (CXCR4) and its ligand CXCL12, both of which are overexpressed in many cancers, play a pivotal role in metastasis. Likewise, lysophosphatidic acid receptor 1 (LPA1) is implicated in cancer cell proliferation and migration. In our preliminary study, we identified LPA1 as a prospective CXCR4 interactor. In the present study, we investigated in detail the formation of the CXCR4-LPA1 heteromer and characterized the unique molecular features and function of this heteromer. METHODS: We employed bimolecular fluorescence complementation, bioluminescence resonance energy transfer, and proximity ligation assays to demonstrate heteromerization between CXCR4 and LPA1. To elucidate the distinctive molecular characteristics and functional implications of the CXCR4-LPA1 heteromer, we performed various assays, including cAMP, BRET for G protein activation, ß-arrestin recruitment, ligand binding, and transwell migration assays. RESULTS: We observed that CXCR4 forms heteromers with LPA1 in recombinant HEK293A cells and the human breast cancer cell line MDA-MB-231. Coexpression of LPA1 with CXCR4 reduced CXCL12-mediated cAMP inhibition, ERK activation, Gαi/o activation, and ß-arrestin recruitment, while CXCL12 binding to CXCR4 remained unaffected. In contrast, CXCR4 had no impact on LPA1-mediated signaling. The addition of lysophosphatidic acid (LPA) further hindered CXCL12-induced Gαi/o recruitment to CXCR4. LPA or alkyl-OMPT inhibited CXCL12-induced migration in various cancer cells that endogenously express both CXCR4 and LPA1. Conversely, CXCL12-induced calcium signaling and migration were increased in LPAR1 knockout cells, and LPA1-selective antagonists enhanced CXCL12-induced Gαi/o signaling and cell migration in the parental MDA-MB-231 cells but not in LPA1-deficient cells. Ultimately, complete inhibition of cell migration toward CXCL12 and alkyl-OMPT was only achieved in the presence of both CXCR4 and LPA1 antagonists. CONCLUSIONS: The presence and impact of CXCR4-LPA1 heteromers on CXCL12-induced signaling and cell migration have been evidenced across various cell lines. This discovery provides crucial insights into a valuable regulatory mechanism of CXCR4 through heteromerization. Moreover, our findings propose a therapeutic potential in combined CXCR4 and LPA1 inhibitors for cancer and inflammatory diseases associated with these receptors, simultaneously raising concerns about the use of LPA1 antagonists alone for such conditions. Video Abstract.

Daily Δ9-Tetrahydrocannabinol and Withdrawal Increase Dopamine D1-D2 Receptor Heteromer to Mediate Anhedonia- and Anxiogenic-like Behavior Through a Dynorphin and Kappa Opioid Receptor Mechanism.

Background: Frequent cannabis use is associated with a higher risk of developing cannabis use disorder and other adverse consequences. However, rodent models studying the underlying mechanisms of the reinforcing and withdrawal effects of the primary constituent of cannabis, Δ9-tetrahydrocannabinol (THC), have been limited. Methods: This study investigated the effects of daily THC (1 mg/kg, intraperitoneal, 9 days) and spontaneous withdrawal (7 days) on hedonic and aversion-like behaviors in male rats. In parallel, underlying neuroadaptive changes in dopaminergic, opioidergic, and cannabinoid signaling in the nucleus accumbens were evaluated, along with a candidate peptide designed to reverse altered signaling. Results: Chronic THC administration induced anhedonic- and anxiogenic-like behaviors not attributable to altered locomotor activity. These effects persisted after drug cessation. In the nucleus accumbens, THC treatment and withdrawal catalyzed increased cannabinoid CB1 receptor activity without modifying receptor expression. Dopamine D1-D2 receptor heteromer expression rose steeply with THC, accompanied by increased calcium-linked signaling, activation of BDNF/TrkB (brain-derived neurotrophic factor/tropomyosin receptor kinase B) pathway, dynorphin expression, and kappa opioid receptor signaling. Disruption of the D1-D2 heteromer by an interfering peptide during withdrawal reversed the anxiogenic-like and anhedonic-like behaviors as well as the neurochemical changes. Conclusions: Chronic THC increases nucleus accumbens dopamine D1-D2 receptor heteromer expression and function, which results in increased dynorphin expression and kappa opioid receptor activation. These changes plausibly reduce dopamine release to trigger anxiogenic- and anhedonic-like behaviors after daily THC administration that persist for at least 7 days after drug cessation. These findings conceivably provide a therapeutic strategy to alleviate negative symptoms associated with cannabis use and withdrawal.

Neuroprotection afforded by targeting G protein-coupled receptors in heteromers and by heteromer-selective drugs.

G protein-coupled receptors (GPCRs) are the target of hundreds of approved drugs. Although these drugs were designed to target individual receptors, it is becoming increasingly apparent that GPCRs interact with each other to form heteromers. Approved drug targets are often part of a GPCR heteromer, and therefore new drugs can be developed with heteromers in mind. This review presents several strategies to selectively target GPCRs in heteromeric contexts, namely, taking advantage of i) heteromer-mediated biased agonism/signalling, ii) discovery of drugs with higher affinity for the receptor if it is part of a heteromer (heteromer selective drugs), iii) allosteric compounds directed against the interacting transmembrane domains and, eventually, iv) antagonists that block both GPCRs in a heteromer. Heteromers provide unique allosteric sites that should help designing a new type of drug that by definition would be a heteromer selective drug. The review also provides examples of rhodopsin-like class A receptors in heteromers that could be targeted to neuroprotect and/or delay the progression of diseases such as Parkinson's and Alzheimer's. GPCRs in heteromers (GriH) with the potential to address dyskinesias, a common complication of dopaminergic replacement therapy in parkinsonian patients, are also described.

Heterodimers Are an Integral Component of Chemokine Signaling Repertoire.

Chemokines are a family of signaling proteins that play a crucial role in cell-cell communication, cell migration, and cell trafficking, particularly leukocytes, under both normal and pathological conditions. The oligomerization state of chemokines influences their biological activity. The heterooligomerization occurs when multiple chemokines spatially and temporally co-localize, and it can significantly affect cellular responses. Recently, obligate heterodimers have emerged as tools to investigate the activities and molecular mechanisms of chemokine heterodimers, providing valuable insights into their functional roles. This review focuses on the latest progress in understanding the roles of chemokine heterodimers and their contribution to the functioning of the chemokine network.

The olfactory Olfr-78/51E2 receptor interacts with the adenosine A2A receptor. Effect of menthol and 1,8-cineole on A2A receptor-mediated signaling.

Heteromer formation is unknown for the olfactory family of G protein-coupled receptors (GPCRs). We here identified, in a heterologous system, heteromers formed by the adenosine A2A receptor (A2AR), which is a target for neuroprotection, and an olfactory receptor. A2AR interacts with the receptor family 51, subfamily E, member 2 (OR51E2), the human ortholog of the mouse Olfr-78, whose mRNA is differentially expressed in activated microglia treated with adenosine receptor ligands. Bioluminescence resonance energy transfer (BRET) assays were performed in HEK-293T cells expressing the human version of the receptors, OR51E2 and A2AR, fused, respectively, to Renilla luciferase (RLuc) and the yellow fluorescent protein (YFP). BRET data was consistent with a receptor-receptor interaction whose consequences at the functional level were measured by cAMP level determination in CHO cells. Results showed an olfactory receptor-mediated partial blockade of Gs coupling to the A2AR, i.e., the effect of the A2AR selective agonist on intracellular levels of cAMP was significantly reduced. Two odorants, menthol and 1,8-cineole, which failed to show Golf-mediated OR51E2 activation because they did not increase cytosolic cAMP levels, reduced the BRET readings in cells expressing A2AR-YFP and OR51E2-Rluc, most likely suggesting a conformational change of at least one receptor. These odorants led to an almost complete block of A2AR coupling to Gs.

The cannabinoid CB1 receptor interacts with the angiotensin AT2 receptor. Overexpression of AT2-CB1 receptor heteromers in the striatum of 6-hydroxydopamine hemilesioned rats.

It is of particular interest the potential of cannabinoid and angiotensin receptors as targets in the therapy of Parkinson's disease (PD). While endocannabinoids are neuromodulators that act through the CB1 and CB2 cannabinoid receptors, the renin angiotensin-system is relevant for regulation of the correct functioning of several brain circuits. Resonance energy transfer assays in a heterologous system showed that the CB1 receptor (CB1R) can directly interact with the angiotensin AT2 receptor (AT2R). Coactivation of the two receptors results in increased Gi-signaling. The AT2-CB1 receptor heteromer imprint consists of a blockade of AT2R-mediated signaling by rimonabant, a CB1R antagonist. Interestingly, the heteromer imprint, discovered in the heterologous system, was also found in primary striatal neurons thus demonstrating the expression of the heteromer in these cells. In situ proximity ligation assays confirmed the occurrence of AT2-CB1 receptor heteromers in striatal neurons. In addition, increased expression of the AT2-CB1 receptor heteromeric complexes was detected in the striatum of a rodent PD model consisting of rats hemilesioned using 6-hydroxydopamine. Expression of the heteromer was upregulated in the striatum of lesioned animals and, also, of lesioned animals that upon levodopa treatment became dyskinetic. In contrast, there was no upregulation in the striatum of lesioned rats that did not become dyskinetic upon chronic levodopa treatment. The results suggest that therapeutic developments focused on the CB1R should consider that this receptor can interact with the AT2R, which in the CNS is involved in mechanisms related to addictive behaviors and to neurodegenerative and neuroinflammatory diseases.

Preferential formation of human heteromeric SK2:SK3 channels limits homomeric SK channel assembly and function.

Three isoforms of small conductance, calcium-activated potassium (SK) channel subunits have been identified (SK1-3) that exhibit a broad and overlapping tissue distribution. SK channels have been implicated in several disease states including hypertension and atrial fibrillation, but therapeutic targeting of SK channels is hampered by a lack of subtype-selective inhibitors. This is further complicated by studies showing that SK1 and SK2 preferentially form heteromeric channels during co-expression, likely limiting the function of homomeric channels in vivo. Here, we utilized a simplified expression system to investigate functional current produced when human (h) SK2 and hSK3 subunits are co-expressed. When expressed alone, hSK3 subunits were more clearly expressed on the cell surface than hSK2 subunits. hSK3 surface expression was reduced by co-transfection with hSK2. Whole-cell recording showed homomeric hSK3 currents were larger than homomeric hSK2 currents or heteromeric hSK2:hSK3 currents. The smaller amplitude of hSK2:hSK3-mediated current when compared with homomeric hSK3-mediated current suggests hSK2 subunits regulate surface expression of heteromers. Co-expression of hSK2 and hSK3 subunits produced a current that arose from a single population of heteromeric channels as exhibited by an intermediate sensitivity to the inhibitors apamin and UCL1684. Co-expression of the apamin-sensitive hSK2 subunit and a mutant, apamin-insensitive hSK3 subunit [hSK3(H485N)], produced an apamin-sensitive current. Concentration-inhibition relationships were best fit by a monophasic Hill equation, confirming preferential formation of heteromers. These data show that co-expressed hSK2 and hSK3 preferentially form heteromeric channels and suggest that the hSK2 subunit acts as a chaperone, limiting membrane expression of hSK2:hSK3 heteromeric channels.

The ADORA1 mutation linked to early-onset Parkinson's disease alters adenosine A1-A2A receptor heteromer formation and function.

Adenosine modulates neurotransmission through inhibitory adenosine A1 receptors (A1Rs) and stimulatory A2A receptors (A2ARs). These G protein-coupled receptors are involved in motor function and related to neurodegenerative diseases such as Parkinson's disease (PD). An autosomal-recessive mutation (G2797.44S) within the transmembrane helix (TM) 7 of A1R (A1RG279S) has been associated with the development of early onset PD (EOPD). Here, we aimed at investigating the impact of this mutation on the structure and function of the A1R and the A1R-A2AR heteromer. Our results revealed that the G2797.44S mutation does not alter A1R expression, ligand binding, constitutive activity or coupling to transducer proteins (Gαi, Gαq, Gα12/13, Gαs, ß-arrestin2 and GRK2) in transfected HEK-293 T cells. However, A1RG279S weakened the ability of A1R to heteromerize with A2AR, as shown in a NanoBiT assay, which led to the disappearance of the heteromerization-dependent negative allosteric modulation that A1R imposes on the constitutive activity and agonist-induced activation of the A2AR. Molecular dynamic simulations allowed to propose an indirect mechanism by which the G2797.44S mutation in TM 7 of A1R weakens the TM 5/6 interface of the A1R-A2AR heteromer. Therefore, it is demonstrated that a PD linked ADORA1 mutation is associated with dysfunction of adenosine receptor heteromerization. We postulate that a hyperglutamatergic state secondary to increased constitutive activity and sensitivity to adenosine of A2AR not forming heteromers with A1R could represent a main pathogenetic mechanism of the EOPD associated with the G2797.44S ADORA1 mutation.

Functional rewiring of G protein-coupled receptor signaling in human labor.

Current strategies to manage preterm labor center around inhibition of uterine myometrial contractions, yet do not improve neonatal outcomes as they do not address activation of inflammation. Here, we identify that during human labor, activated oxytocin receptor (OTR) reprograms the prostaglandin E2 receptor, EP2, in the pregnant myometrium to suppress relaxatory/Gαs-cAMP signaling and promote pro-labor/inflammatory responses via altered coupling of EP2 from Gαq/11 to Gαi/o. The ability of EP2 to signal via Gαi/o is recapitulated with in vitro OT and only following OTR activation, suggesting direct EP2-OTR crosstalk. Super-resolution imaging with computational modeling reveals OT-dependent reorganization of EP2-OTR complexes to favor conformations for Gαi over Gαs activation. A selective EP2 ligand, PGN9856i, activates the relaxatory/Gαs-cAMP pathway but not the pro-labor/inflammatory responses in term-pregnant myometrium, even following OT. Our study reveals a mechanism, and provides a potential therapeutic solution, whereby EP2-OTR functional associations could be exploited to delay preterm labor.

GPCR heteromers: An overview of their classification, function and physiological relevance.

G protein-coupled receptors (GPCRs) are capable of interacting to form higher order structures such as homomers and heteromers. Heteromerisation in particular has implications for receptor function, with research showing receptors can attain unique expression, ligand binding, signalling and intracellular trafficking upon heteromerisation. As such, GPCR heteromers represent novel drug targets with extensive therapeutic potential. Changes to ligand affinity, efficacy and G protein coupling have all been described, with alterations to these pharmacological aspects now well accepted as common traits for heteromeric complexes. Changes in internalisation and trafficking kinetics, as well as ß-arrestin interactions are also becoming more apparent, however, few studies to date have explicitly looked at the implications these factors have upon the signalling profile of a heteromer. Development of ligands to target GPCR heteromers both experimentally and therapeutically has been mostly concentrated on bivalent ligands due to difficulties in identifying and developing heteromer-specific ligands. Improving our understanding of the pharmacology and physiology of GPCR heteromers will enable further development of heteromer-specific ligands with potential to provide therapeutics with increased efficacy and decreased side effects.

Unmasking allosteric-binding sites: novel targets for GPCR drug discovery.

INTRODUCTION: Unexpected non-apparent and hidden allosteric-binding sites are non-classical and non-apparent allosteric centers in 3-D X-ray protein structures until orthosteric or allosteric ligands bind to them. The orthosteric center of one protomer that modulates binding centers of the other protomers within an oligomer is also an unexpected allosteric site. Furthermore, another partner protein can also produce these effects, acting as an unexpected allosteric modulator. AREAS COVERED: This review summarizes both classical and non-classical allosterism. The authors focus on G protein-coupled receptor (GPCR) oligomers as a paradigm of allosteric molecules. Moreover, they show several examples of unexpected allosteric sites such as hidden allosteric sites in a protomer that appear after the interaction with other molecules and the allosterism exerted between orthosteric sites within GPCR oligomer, emphasizing on the allosteric modulations that can occur between binding sites. EXPERT OPINION: The study of these new non-classical allosteric sites will expand the diversity of allosteric control on the function of orthosteric sites within proteins, whether GPCRs or other receptors, enzymes, or transporters. Moreover, the design of new drugs targeting these hidden allosteric sites or already known orthosteric sites acting as allosteric sites in protein homo- or hetero-oligomers will increase the therapeutic potential of allosterism.

Dopamine D2 and Serotonin 5-HT1A Dimeric Receptor-Binding Monomeric Antibody scFv as a Potential Ligand for Carrying Drugs Targeting Selected Areas of the Brain.

Targeted therapy uses multiple ways of ensuring that the drug will be delivered to the desired site. One of these ways is an encapsulation of the drug and functionalization of the surface. Among the many molecules that can perform such a task, the present work focused on the antibodies of single-chain variable fragments (scFvs format). We studied scFv, which specifically recognizes the dopamine D2 and serotonin 5-HT1A receptor heteromers. The scFvD2-5-HT1A protein was analyzed biochemically and biologically, and the obtained results indicated that the antibody is properly folded and non-toxic and can be described as low-immunogenic. It is not only able to bind to the D2-5-HT1A receptor heteromer, but it also influences the cAMP signaling pathway and-when surfaced on nanogold particles-it can cross the blood-brain barrier in in vitro models. When administered to mice, it decreased locomotor activity, matching the effect induced by clozapine. Thus, we are strongly convinced that scFvD2-5-HT1A, which was a subject of the present investigation, is a promising targeting ligand with the potential for the functionalization of nanocarriers targeting selected areas of the brain.

Novel Pharmacology Following Heteromerization of the Angiotensin II Type 2 Receptor and the Bradykinin Type 2 Receptor.

The angiotensin type 2 (AT2) receptor and the bradykinin type 2 (B2) receptor are G protein-coupled receptors (GPCRs) that have major roles in the cardiovascular system. The two receptors are known to functionally interact at various levels, and there is some evidence that the observed crosstalk may occur as a result of heteromerization. We investigated evidence for heteromerization of the AT2 receptor and the B2 receptor in HEK293FT cells using various bioluminescence resonance energy transfer (BRET)-proximity based assays, including the Receptor Heteromer Investigation Technology (Receptor-HIT) and the NanoBRET ligand-binding assay. The Receptor-HIT assay showed that Gαq, GRK2 and ß-arrestin2 recruitment proximal to AT2 receptors only occurred upon B2 receptor coexpression and activation, all of which is indicative of AT2-B2 receptor heteromerization. Additionally, we also observed specific coupling of the B2 receptor with the Gαz protein, and this was found only in cells coexpressing both receptors and stimulated with bradykinin. The recruitment of Gαz, Gαq, GRK2 and ß-arrestin2 was inhibited by B2 receptor but not AT2 receptor antagonism, indicating the importance of B2 receptor activation within AT2-B2 heteromers. The close proximity between the AT2 receptor and B2 receptor at the cell surface was also demonstrated with the NanoBRET ligand-binding assay. Together, our data demonstrate functional interaction between the AT2 receptor and B2 receptor in HEK293FT cells, resulting in novel pharmacology for both receptors with regard to Gαq/GRK2/ß-arrestin2 recruitment (AT2 receptor) and Gαz protein coupling (B2 receptor). Our study has revealed a new mechanism for the enigmatic and poorly characterized AT2 receptor to be functionally active within cells, further illustrating the role of heteromerization in the diversity of GPCR pharmacology and signaling.