Heteromeric Receptor Complexes Research Articles

Cholesterol modulates type I/II TGF-β receptor complexes and alters the balance between Smad and Akt signaling in hepatocytes

Cholesterol mediates membrane compartmentalization, affecting signaling via differential distribution of receptors and signaling mediators. While excessive cholesterol and aberrant transforming growth factor-β (TGF-β) signaling characterize multiple liver diseases, their linkage to canonical vs. non-canonical TGF-β signaling remained unclear. Here, we subjected murine hepatocytes to cholesterol depletion (CD) or enrichment (CE), followed by biophysical studies on TGF-β receptor heterocomplex formation, and output to Smad2/3 vs. Akt pathways. Prior to ligand addition, raft-dependent preformed heteromeric receptor complexes were observed. Smad2/3 phosphorylation persisted following CD or CE. CD enhanced phospho-Akt (pAkt) formation by TGF-β or epidermal growth factor (EGF) at 5 min, while reducing it at later time points. Conversely, pAkt formation by TGF-β or EGF was inhibited by CE, suggesting a direct effect on the Akt pathway. The modulation of the balance between TGF-β signaling to Smad2/3 vs. pAkt (by TGF-β or EGF) has potential implications for hepatic diseases and malignancies.

Abstract 2936: INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1

Abstract Transforming growth factor-β (TGFβ) signaling is common in many solid tumors and is initiated by binding of the high affinity canonical ligands TGFβ1, 2, οr 3 to TGFβR2, which forms a heteromeric receptor complex with TGFβR1 (Derynck et al, Nature Review Clinical Oncology 2020). Activation of the pathway results in potent suppression of immune cell-mediated anti-tumor immunity and has been reported to predict poor response to PD-(L)1 targeted therapy in patients (Mariathasan et al, Nature 2018; Kieffer et al, Cancer Discovery 2020). However, TGFβ drug development has been hampered by the occurrence of adverse events. INCA33890 is a dual PD-1 and TGFβR2 binding bispecific Biclonics® antibody, developed to antagonize the TGFβ signaling pathway specifically in cells co-expressing PD-1 and TGFBR2. Additionally, it potently antagonizes the PD-1 axis independently of TGFβR2 co-expression. The cell-selective action of INCA33890 was designed to mitigate risks of the known adverse effects associated with TGFβ-pathway inhibition in tissues requiring active TGFβ signaling. ΙΝCΑ33890 mediates its specificity through a PD-1 binding arm with a >10-fold higher affinity relative to the TGFβR2 binding arm. Consistent with this profile, in isogenic Jurkat cells expressing TGFβR2 ± PD-1, INCA33890 potently inhibits TGFβ1-induced pSMAD activation in a PD-1-correlated manner. Additionally, in two independent PD-1 reporter assays, INCA33890 inhibited SHP recruitment and enhanced NFAT activation with a potency within an order of magnitude to that of pembrolizumab. In mixed lymphocyte reaction assays with exhausted primary human T-cells, INCA33890 was found to induce a similar level of anti-tumor cytokine production as the combination of pembrolizumab and an anti-TGFβR2 antagonist mAb. Treatment of primary ovarian ascites with INCA33890 ex vivo induced IFNγ production in all donors tested, while pembrolizumab had no activity. Similarly, in human CD34+ cell-engrafted NSG mice, INCA33890 significantly inhibited the growth of human MDA-MB-231 and A375 subcutaneous xenograft tumors, whereas pembrolizumab or an anti-TGFβR2 antibody had little or no monotherapy activity. INCA33890 had a balanced pharmacokinetic and potency profile and was well tolerated in NHPs at exposures required for pharmacodynamic and tumor growth inhibiting activity in rodents. Encouragingly, there was no evidence of adverse effects in NHPs due to TGFβ-pathway blockade. Collectively, these results provide compelling data for an effective and specific approach to simultaneously antagonizing TGFβ and PD-1 signaling in tumors. Clinical development of INCA33890 in checkpoint inhibitor-resistant and other cancers has been initiated. Citation Format: Liang-Chuan S. Wang, Rinse Klooster, Ashwini Kulkarni, Amaya Garcia de Vinuesa, Maxim Soloviev, Linda JA Hendriks, Lu Huo, Michael Weber, Arpita Mondal, Yonghong Zhao, Shane Harvey, Xin He, Hong Chang, April Horsey, Alla Volgina, Yue Zhang, Veethika Pandey, Yan-Ou Yang, Jonathan Rios-Doria, Evgeniy Eruslanov, Daniel J. Powell, Steven M. Albelda, John de Kruif, Horacio Nastri, Cecile Geuijen, Patrick A. Mayes. INCA33890, a novel PD-1×TGFꞵR2 bispecific antibody conditionally antagonizes TGFꞵ signaling in primary immune cells co-expressing PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2936.

Use of NanoBiT and NanoBRET to characterise interleukin-23 receptor dimer formation in living cells.

Interleukin-23 (IL-23) and its receptor are important drug targets for the treatment of auto-inflammatory diseases. IL-23 binds to a receptor complex composed of two single transmembrane spanning proteins IL23R and IL12Rβ1. In this study, we aimed to gain further understanding of how ligand binding induces signalling of IL-23 receptor complexes using the proximity-based techniques of NanoLuc Binary Technology (NanoBiT) and Bioluminescence Resonance Energy Transfer (BRET). To monitor the formation of IL-23 receptor complexes, we developed a split luciferase (NanoBiT) assay whereby heteromerisation of receptor subunits can be measured through luminescence. The affinity of NanoBiT complemented complexes for IL-23 was measured using NanoBRET, and cytokine-induced signal transduction was measured using a phospho-STAT3 AlphaLISA assay. NanoBiT measurements demonstrated that IL-23 receptor complexes formed to an equal degree in the presence and absence of ligand. NanoBRET measurements confirmed that these complexes bound IL-23 with a picomolar binding affinity. Measurement of STAT3 phosphorylation demonstrated that pre-formed IL-23 receptor complexes induced signalling following ligand binding. It was also demonstrated that synthetic ligand-independent signalling could be induced by high affinity (HiBit) but not low affinity (SmBit) NanoBiT crosslinking of the receptor N-terminal domains. These results indicate that receptor complexes form prior to ligand binding and are not sufficient to induce signalling alone. Our findings indicate that IL-23 induces a conformational change in heteromeric receptor complexes, to enable signal transduction. These observations have direct implications for drug discovery efforts to target the IL-23 receptor.

Heteromeric interactions of ripening-related ethylene receptors in tomato fruit.

Ripening of climacteric fruits is initiated when the gaseous plant hormone ethylene is perceived by the cell. Ethylene binding to membrane-associated ethylene receptors (ETRs) triggers a series of biochemical events through multiple components, resulting in the induction of numerous ripening-related genes. In tomato (Solanum lycopersicum L.), there are seven members of the ETR family, which each contribute to the regulation of fruit ripening. However, the relative contribution of each individual receptor to ethylene signaling remains unknown. Here, we demonstrated the formation of heteromeric receptor complexes across the two ETR subfamilies in tomato fruit. Immunoprecipitation of subfamily II SlETR4 resulted in co-purification of subfamily I (SlETR1, SlETR2, and SlETR3), but not subfamily II members (SlETR5, SlETR6, and SlETR7). Such biased interactions were verified in yeast two-hybrid assays, and in transgenic Arabidopsis plants, in which heterologous SlETR4 interacts with subfamily I ETRs. Our analysis also revealed that the receptor complexes engage the Raf-like protein kinases SlCTR1 and SlCTR3, which are potential regulators of signaling. Here, we suggest that tomato receptor members form heteromeric complexes to fine-tune signal output to the downstream pathway, which is similar to that of the Arabidopsis system but appears to be partially diverged.

α1B/D-adrenoceptors regulate chemokine receptor–mediated leukocyte migration via formation of heteromeric receptor complexes

It is known that catecholamines regulate innate immune functions. The underlying mechanisms, however, are not well understood. Here we show that at least 20 members of the human chemokine receptor (CR) family heteromerize with one or more members of the α1-adrenergic receptor (AR) family in recombinant systems and that such heteromeric complexes are detectable in human monocytes and the monocytic leukemia cell line THP-1. Ligand binding to α1-ARs inhibited migration toward agonists of the CR heteromerization partners of α1B/D-ARs with high potency and 50 to 77% efficacy but did not affect migration induced by a noninteracting CR. Incomplete siRNA knockdown of α1B/D-ARs in THP-1 cells partially inhibited migration toward agonists of their CR heteromerization partners. Complete α1B-AR knockout via CRISPR-Cas9 gene editing in THP-1 cells (THP-1_ADRA1BKO) resulted in 82% reduction of α1D-AR expression and did not affect CR expression. Migration of THP-1_ADRA1BKO cells toward agonists of CR heteromerization partners of α1B/D-ARs was reduced by 82 to 95%. Our findings indicate that CR:α1B/D-AR heteromers are essential for normal function of CR heteromerization partners, provide a mechanism underlying neuroendocrine control of leukocyte trafficking, and offer opportunities to modulate leukocyte and/or cancer cell trafficking in disease processes.

Expression of the ionotropic glutamate receptors on neuronostatin neurons in the periventricular nucleus of the hypothalamus.

Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when given intracerebroventricularly. Although the effect mechanisms of neuronostatin have been shown in different studies, there are no reports in the literature describing the mechanisms controlling neuronostatin neurons. In this study, we aimed to determine the presence of the ionotropic glutamate receptor subunits (iGluRs) in neuronostatin neurons in the periventricular nucleus of the hypothalamus. The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunohistochemistry. Immunohistochemistry was performed on 40 μm thick coronal brain sections with antibodies against AMPA (GluA1-4), kainate (GluK1/2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. The results showed that the neuronostatin neurons expressed most of the NMDA and non-NMDA receptor subunits. The neuronostatin neurons in the anterior hypothalamic periventricular nucleus were particularly immunopositive for GluA1, GluA4, GluK1/2/3, GluK5 and GluN1 antibodies. No expression was observed for GluA2, GluA3 and GluN2A antibodies. For the first time in the literature, our study demonstrated that the neuronostatin neurons express glutamate receptor subunits which may form homomeric or heteromeric functional receptor complexes. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission on neuronostatin neurons in the anterior hypothalamic periventricular nucleus.

Human macrophage C-type lectin forms a heteromeric receptor complex with Mincle but not Dectin-2.

MCL, Mincle and Dectin-2 are C-type lectin receptors expressed by subsets of myeloid cells, and their genes cluster together in the APLEC/Dectin-2 gene complex. We have previously shown that MCL and Mincle form a heterodimer in the rat, and others have shown that MCL and Dectin-2 form a heterodimer in the mouse. In the rat, Dectin-2 is a pseudogene, but here, we examine the association of the three receptors in human. In co-transfection experiments analyzed with flow cytometry and immunoprecipitation, we here show that human MCL and Mincle form a disulphide-linked heterodimer that associates with the signalling adaptor molecule FcεRIγ, in accordance with our previous findings in the rat. In contrast to previous findings in the rat, data in this paper indicate a direct association of MCL with FcεRIγ, as previously shown for mouse MCL. We were unable to demonstrate the formation of a heterodimer between human MCL and Dectin-2. Thus, despite similarities, there may be important differences in the conformation of these receptors between rat, mouse and human, and this may have functional consequences.

Probing the binding of interleukin-23 to individual receptor components and the IL-23 heteromeric receptor complex in living cells using NanoBRET

SummaryInterleukin-23 (IL-23) is a pro-inflammatory cytokine involved in the host defense against pathogens but is also implicated in the development of several autoimmune disorders. The IL-23 receptor has become a key target for drug discovery, but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL-23 for its individual receptor components (IL23R and IL12Rβ1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase-tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL-23 has a greater than 7-fold higher affinity for IL12Rβ1 than IL23R. However, in the presence of both receptor subunits, IL-23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL-23 induces a potent change in the position of the N-terminal domains of the two receptor subunits, consistent with a conformational change in the heteromeric receptor structure.

Probing the Binding of Interleukin-23 to Individual Receptor Components and the IL23 Heteromeric Receptor Complex in Living Cells Using NanoBRET

Interleukin-23 (IL23) is a pro-inflammatory cytokine involved in the host defence against pathogens, but also implicated in the development of several autoimmune disorders. The IL23 receptor has become a key target for drug discovery but the exact mechanism of the receptor ligand interaction remains poorly understood. In this study the affinities of IL23 for its individual receptor components (IL23R and IL12Rβ1) and the heteromeric complex formed between them have been measured in living cells using NanoLuciferase tagged full-length proteins. Here, we demonstrate that TAMRA-tagged IL23 has a greater than seven fold higher affinity for IL12Rβ1 than IL23R. However, in the presence of both receptor subunits IL23 affinity is increased more than three orders of magnitude to 27 pM. Furthermore, we show that IL23 induces a potent change in the position of the N -terminal domains of the two receptor subunits consistent with a conformational change in the heteromeric receptor structure.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

A P2RX7 single nucleotide polymorphism haplotype promotes exon 7 and 8 skipping and disrupts receptor function.

P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.

Tezepelumab: a novel biological therapy for the treatment of severe uncontrolled asthma

ABSTRACTIntroduction: Thymic stromal lymphopoietin (TSLP) is overexpressed in the airways of severe asthmatics and is an upstream cytokine that orchestrates inflammatory responses in asthma. TSLP exerts its effects by binding to a high affinity heteromeric receptor complex composed of TSLPR and IL-7Rα. An association of polymorphisms in TSLP with airway hyperresponsiveness, IgE, eosinophilia and asthma has been documented. TSLP has been implicated in asthma pathophysiology. Tezepelumab is a first-in-class human monoclonal antibody that binds to TSLP, thus inhibiting its interaction with TSLP receptor complex. Tezepelumab given as an add-on-therapy to patients with severe uncontrolled asthma has shown safety, tolerability and efficacy. Several trials are evaluating the long-term safety and the efficacy of tezepelumab in adults and adolescents with severe uncontrolled asthma.Areas covered: We provide an overview of the monoclonal antibody therapeutics market for severe uncontrolled asthma, examine the underlying pathophysiology that drives TSLP and discuss the use of tezepelumab for the treatment of severe uncontrolled asthma,Expert opinion: TSLP is a promising target for T2-high and perhaps some patients with T2-low asthma. The results of preliminary clinical trials are encouraging. Several unanswered questions concerning basic pathophysiological aspects of TSLP variants, the long-term safety and efficacy of tezepelumab with different phenotypes/endotypes of asthma should be addressed.

Identification of Modifiers of Odor‐Triggered Mosquito Behaviors Acting through Binding to the ORco Subunit of Odorant Receptor Heteromers

Insect olfactory receptors (ORs) are heteromeric ion channels consisting of a common subunit, ORco, and one of many variable subunits, ORx, to which odorants acting as receptor agonists or antagonists, bind in an ORx‐specific manner. In cells expressing only ORco, ORco homomers form functional channels that are activated by specific ORco agonists. We have previously shown that mosquito repellents that block odor‐specific responses of multiple ORs of Anopheles gambiae mosquitoes, act as ORco antagonists capable of suppressing in vitro its activation by the ORco agonists. In parallel studies, we have also shown that the in vitro responses of heteromeric receptors induced by ORx‐specific agonists are enhanced by one to two orders of magnitude in the presence of an ORco‐specific agonist. The latter suggested the induction of conformational rearrangements in ORx‐bound heteromeric receptor complexes caused by the binding of the ORco agonist and resulting in enhanced ion influx into the receptor‐expressing cells.To expand on these findings, we have designed a lepidopteran insect cell‐based ORco expression platform that allows the fast screening of collections of chemical compounds for the presence of mosquito ORco agonists and antagonists in a single screening assay. Because mosquitoes are able to detect the presence of olfaction‐relevant molecules in their natural environments, we screened a small collection of volatile organic compounds (VOCs) of natural, plant, insect and bacterial, origin for the presence of relevant bioactive molecules. The screening effort resulted in the identification of several hits acting as ORco‐specific antagonists but no ligands behaving as ORco‐specific agonists. Examination of the bioactivity of three, randomly selected antagonist hits by in vivo repellence assays against Aedes albopictus mosquitoes, which vector a number of infectious agents, established that two of them displayed significant repellent activity with one of them showing anosmia‐causing activity equal to or slightly better than that of an equivalent dose of DEET. These results provide additional proof for the concept that volatile ORco antagonists may modify olfaction‐based mosquito behaviors in a predicted fashion. They also suggest that the newly developed screening platform may yield additional possibilities for enhanced personal protection against indoor and outdoor transmission of malaria and other mosquito‐borne infectious diseases. Screening of additional VOCs aimed at the identification of new ORco agonists and antagonists is in progress.Support or Funding InformationThis work has been supported by the ENAROMaTIC consortium project (grant No FP7‐222927 of the EC) and was carried out in the facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cell‐Based Screening Platforms for Identification of Modifiers of Odor‐Triggered Mosquito Behaviors Acting through Binding to the ORco Subunit of Odorant Receptor Heteromers

Insect olfactory receptors (ORs) are heteromeric ion channels consisting of a common subunit, ORco, and one of many variable subunits, ORx, to which odorants, receptor agonists or antagonists, bind in an ORx‐specific manner. In cells expressing the ORco subunit alone, however, ORco may also form a functional channel that is activated by ORco‐specific agonists. We have previously shown that mosquito repellents that block odor‐specific responses of multiple ORs of Anopheles gambiae mosquitoes, act as ORco antagonists that are capable of suppressing its activation by ORco‐specific agonists. We have also shown that the in vitro responses of heteromeric receptors that are induced by ORx‐specific agonists are enhanced by one to two orders of magnitude in the presence of either of two ORco‐specific agonists, VUAA1 and OrcoRAM2. The latter finding suggests the induction of allosteric changes in ORx‐bound heteromeric receptor complexes, which are caused by the binding of the ORco agonist and result in enhanced ion influx into the receptor‐expressing cells. To expand on these findings, we have designed a lepidopteran insect cell‐based expression platform that allows the fast screening of collections of pure compounds and compound mixtures for the presence of A. gambiae ORco agonists and antagonists in a single screening assay. Because mosquitoes should be able to detect the presence of olfaction‐relevant bioactive molecules in their natural environments, we screened for the presence of relevant compounds in (i) collections of essential oils obtained from aromatic plants and fractions thereof, which contain volatile constituents, and (ii) collections of pure volatiles of plant or bacterial origin. The initial screening effort resulted in the identification of a small number of hits acting as ORco‐specific agonists or antagonists from both types of examined collections. These hits are currently examined further to establish their functional relevance and effectiveness against mosquito populations in vivo. Should this testing prove the validity of the hypothesis that newly‐identified volatile ORco ligands may modify olfaction‐based mosquito behaviors in a predicted fashion, additional capacities will be generated for enhanced personal protection against indoor and outdoor transmission of malaria and other mosquito‐borne infectious diseases. We also note that equivalent screening platforms could be designed for identification of behavior modifiers of the same type for other arthropod species of interest.Support or Funding InformationThis work has been supported by the ENAROMaTIC consortium project (grant No FP7‐222927 of the EC) and was carried out in the facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Asymmetrical ligand-induced cross-regulation of chemokine (C-X-C motif) receptor 4 by \u03b11-adrenergic receptors at the heteromeric receptor complex

Recently, we reported that chemokine (C-X-C motif) receptor (CXCR)4 and atypical chemokine receptor 3 regulate α1-adrenergic receptors (α1-AR) through the formation of hetero-oligomeric complexes. Whether α1-ARs also regulate chemokine receptor function within such heteromeric receptor complexes is unknown. We observed that activation of α1b-AR within the α1b-AR:CXCR4 heteromeric complex leads to cross-recruitment of β-arrestin2 to CXCR4, which could not be inhibited with AMD3100. Activation of CXCR4 did not cross-recruit β-arrestin2 to α1b-AR. A peptide analogue of transmembrane domain 2 of CXCR4 interfered with α1b-AR:CXCR4 heteromerization and inhibited α1b-AR-mediated β-arrestin2 cross-recruitment. Phenylephrine (PE) induced internalization of CXCR4 in HEK293 cells co-expressing CXCR4 and α1b-AR and of endogenous CXCR4 in human vascular smooth muscle cells (hVSMC). The latter was detectable despite blockade of CXCR4 with the neutralizing antibody 12G5. hVSMC migrated towards CXCL12 and PE, but not towards a combination of CXCL12 and PE. PE inhibited CXCL12-induced chemotaxis of hVSMC (IC50: 77 ± 30 nM). Phentolamine cross-inhibited CXCL12-induced chemotaxis of hVSMC, whereas AMD3100 did not cross-inhibit PE-induced chemotaxis. These data provide evidence for asymmetrical cross-regulation of CXCR4 by α1-adrenergic receptors within the heteromeric receptor complex. Our findings provide mechanistic insights into the function of α1-AR:CXCR4 heteromers and suggest alternative approaches to modulate CXCR4 in disease conditions.

Dopamine receptors: homomeric and heteromeric complexes in L-DOPA-induced dyskinesia.

The current standard treatment for Parkinson disease focuses on restoring striatal dopamine levels using L-3,4-dihydroxyphenylalanine (L-DOPA). However, disease progression and chronic treatment are associated with motor side effects such as L-DOPA-induced dyskinesia (LID). Dopamine receptor function is strongly associated with the mechanisms underlying LID. In fact, increased D1R signaling is associated with this motor side effect. Compelling evidence demonstrates that dopamine receptors in the striatum can form heteromeric complexes, and heteromerization can lead to changes in the functional and pharmacological properties of receptors compared to their monomeric subtypes. Currently, the most promising strategy for therapeutic intervention in dyskinesia originates from investigations of the D1R-D3R heteromers. Interestingly, there is a correlation between the expression of D1R-D3R heteromers and the development of LID. Moreover, D3R stimulation can potentiate the D1R signaling pathway. The aim of this review is to summarize current knowledge of the distinct roles of heteromeric dopaminergic receptor complexes in LID.

Identification and functional characterization of arginine vasopressin receptor 1A : atypical chemokine receptor 3 heteromers in vascular smooth muscle.

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α1-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP3) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP3 production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.

Interaction network of ABA receptors in grey poplar.

The plant hormone abscisic acid (ABA) is a key player in responses to abiotic stress. ABA regulates a plant's water status and mediates drought tolerance by controlling stomatal gas exchange, water conductance and differential gene expression. ABA is recognized and bound by the Regulatory Component of ABA Receptors (RCARs)/PYR1/PYL (Pyrabactin Resistance 1/PYR1-like). Ligand binding stabilizes the interaction of RCARs with type 2C protein phosphatases (PP2C), which are ABA co-receptors. While the core pathway of ABA signalling has been elucidated, the large number of different ABA receptors and co-receptors within a plant species generates a complexity of heteromeric receptor complexes that has not functionally been resolved in any plant species to date. In this study, we characterized ABA receptors and co-receptors of grey poplar (Populus x canescens [Ait.] Sm.) and their capacity to regulate ABA responses. We observed a high number of regulatory combinations of holo-receptor complexes, but also some preferential and selective RCAR-PP2C interactions. Poplar and Arabidopsis ABA receptor components revealed a strong structural and functional conservation. Heterologous receptor complexes of poplar and Arabidopsis components showed functionality invitro and regulated ABA-responsive gene expression in cells of both species. ABA-responsive promoters of Arabidopsis were also active in poplar, which was explored to generate poplar reporter lines expressing green fluorescent protein in response to ABA. The study presents a detailed analysis of receptor complexes of a tree species and shows high conservation of ABA receptor components between an annual and a perennial plant.

α1-Adrenergic Receptors Function Within Hetero-Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C-X-C motif) Receptor 4 in Vascular Smooth Muscle Cells.

BackgroundRecently, we provided evidence that α1‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed that CXCR4 controls α1‐ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk between ACKR3 and α1‐ARs is unknown.Methods and ResultsWe show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α1A/B/D‐AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α1B/D‐AR:ACKR3, CXCR4:ACKR3, and α1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α1A/B/D‐AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α1B/D‐AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α1A/B‐AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α1b‐AR in β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries.Conclusionsα1‐ARs form hetero‐oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α1B/D‐AR function, and activation of ACKR3 negatively regulates α1‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α1‐ARs function within a network of hetero‐oligomeric receptor complexes.

Distracting the hungry: mosquito anosmia‐inducing and odor perception‐enhancing compounds of natural origin targeting ORco function for control of transmission of malaria and other mosquito‐borne infectious diseases

ORco, the ubiquitous co‐receptor of insect olfactory receptors, is an essential constituent of the insect olfactory machinery. Mutations affecting ORco function and RNAi‐mediated reduction in ORco abundance impact negatively on the organisms' capacity to detect the normal odor repertoire. On the other hand, compounds acting as ORco agonists may impact in a positive fashion on the function of olfactory receptors. In the current study, we have employed lepidopteran (Bombyx mori and Trichoplusia ni) cell‐based screening platforms expressing ORco and olfactory receptor variable subunits (ORx) of Anopheles gambiae in conjunction with specific ligand‐dependent functional assays to assess the effects of specific constituents of essential oils on receptor functionality. We show that natural compounds, previously selected through screening work employing A. gambiae odorant binding proteins (OBPs) with female expression bias, and displaying spatial repellent properties against Anopheles and Culex mosquitoes similar to or stronger than those of DEET, are, in fact, ORco antagonists. These antagonists cause chemical ablation of the function of all tested heteromeric olfactory receptor complexes containing ORco, and may induce anosmia‐like disorientation effects on mosquitoes. On the other hand, we show that members of a known class of synthetic ORco agonists induce allosteric changes on receptor heteromers resulting in significant ORx ligand‐specific enhancement in odor perception in vitro. Because these ORco agonists are not volatile and thus cannot be detected by mosquitoes at a distance, we are using the odorant receptor expression platforms to screen essential oils obtained from Mediterranean aromatic plants for the presence of natural volatiles acting as specific ORco agonists with functional properties similar to those of the synthetic agonists. In parallel, we are expanding the screens for the isolation of additional natural ORco antagonists. Due to their volatile nature, the natural anosmia‐inducing compounds would be expected to act at a distance and thus be suitable for use as spatial disorienting agents. The same principle should be applicable to the natural olfactory enhancers, which may be used as additives to human odor blend‐containing mosquito traps for more effective trapping. The combined deployment of anosmia‐inducing and olfactory capacity‐enhancing compounds targeting ORco function may provide a most effective approach for protection against indoor and outdoor transmission of malaria and other mosquito‐borne infectious diseases.Support or Funding InformationThis work has been supported by the ENAROMaTIC consortium project (grant No FP7‐222927 of the EC) and was carried out in facilities of the OPENSCREEN‐GR National (Greece) Research Infrastructure.