Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2’-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

N-Methyl-D-aspartate (NMDA) and cannabinoid CB2 receptors form functional complexes in cells of the central nervous system: insights into the therapeutic potential of neuronal and microglial NMDA receptors

Background The cannabinoid CB2 receptor (CB2R), which is a target to afford neuroprotection, and N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, which are key in mediating excitatory neurotransmission, are expressed in both neurons and glia. As NMDA receptors are the target of current medication in Alzheimer’s disease patients and with the aim of finding neuromodulators of their actions that could provide benefits in dementia, we hypothesized that cannabinoids could modulate NMDA function. Methods Immunocytochemistry was used to analyze the colocalization between CB2 and NMDA receptors; bioluminescence resonance energy transfer was used to detect CB2-NMDA receptor complexes. Calcium and cAMP determination, mitogen-activated protein kinase (MAPK) pathway activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify CB2-NMDA heteromer expression in mouse primary cultures and in the brain of APPSw/Ind transgenic mice, an Alzheimer’s disease model expressing the Indiana and Swedish mutated version of the human amyloid precursor protein (APP). Results In a heterologous system, we identified CB2-NMDA complexes with a particular heteromer print consisting of impairment by cannabinoids of NMDA receptor function. The print was detected in activated primary microglia treated with lipopolysaccharide and interferon-γ. CB2R activation blunted NMDA receptor-mediated signaling in primary hippocampal neurons from APPSw/Ind mice. Furthermore, imaging studies showed that in brain slices and in primary cells (microglia or neurons) from APPSw/Ind mice, there was a marked overexpression of macromolecular CB2-NMDA receptor complexes thus becoming a tool to modulate excessive glutamate input by cannabinoids. Conclusions The results indicate a negative cross-talk in CB2-NMDA complexes signaling. The expression of the CB2-NMDA receptor heteromers increases in both microglia and neurons from the APPSw/Ind transgenic mice, compared with levels in samples from age-matched control mice.

Profiling novel pharmacology of receptor complexes using Receptor-HIT

Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. This review will discuss various applications of Receptor-HIT, including its use with different classes of receptors (e.g. G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and others), its use to monitor receptor interactions both intracellularly and extracellularly, and also its use with genome-edited endogenous proteins.

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

Functional Fine-Tuning of Metabolic Pathways by the Endocannabinoid System—Implications for Health and Disease

The endocannabinoid system (ECS) employs a huge network of molecules (receptors, ligands, and enzymatic machinery molecules) whose interactions with other cellular networks have still not been fully elucidated. Endogenous cannabinoids are molecules with the primary function of control of multiple metabolic pathways. Maintenance of tissue and cellular homeostasis by functional fine-tuning of essential metabolic pathways is one of the key characteristics of the ECS. It is implicated in a variety of physiological and pathological states and an attractive pharmacological target yet to reach its full potential. This review will focus on the involvement of ECS in glucose and lipid metabolism, food intake regulation, immune homeostasis, respiratory health, inflammation, cancer and other physiological and pathological states will be substantiated using freely available data from open-access databases, experimental data and literature review. Future directions should envision capturing its diversity and exploiting pharmacological options beyond the classical ECS suspects (exogenous cannabinoids and cannabinoid receptor monomers) as signaling through cannabinoid receptor heteromers offers new possibilities for different biochemical outcomes in the cell.

Investigation of Receptor Heteromers Using NanoBRET Ligand Binding

Receptor heteromerization is the formation of a complex involving at least two different receptors with pharmacology that is distinct from that exhibited by its constituent receptor units. Detection of these complexes and monitoring their pharmacology is crucial for understanding how receptors function. The Receptor-Heteromer Investigation Technology (Receptor-HIT) utilizes ligand-dependent modulation of interactions between receptors and specific biomolecules for the detection and profiling of heteromer complexes. Previously, the interacting biomolecules used in Receptor-HIT assays have been intracellular proteins, however in this study we have for the first time used bioluminescence resonance energy transfer (BRET) with fluorescently-labeled ligands to investigate heteromerization of receptors on the cell surface. Using the Receptor-HIT ligand binding assay with NanoBRET, we have successfully investigated heteromers between the angiotensin II type 1 (AT1) receptor and the β2 adrenergic receptor (AT1-β2AR heteromer), as well as between the AT1 and angiotensin II type 2 receptor (AT1-AT2 heteromer).

Volatile allosteric antagonists of mosquito odorant receptors inhibit human-host attraction

Odorant-dependent behaviors in insects are triggered by the binding of odorant ligands to the variable subunits of heteromeric olfactory receptors. Previous studies have shown, however, that specific odor binding to ORco, the common subunit of odorant receptor heteromers, may allosterically alter olfactory receptor function and profoundly affect subsequent behavioral responses. Using an insect cell-based screening platform, we identify and characterize several antagonists of the odorant receptor co-receptor of the African malaria vector Anopheles gambiae (AgamORco) in a small collection of natural volatile organic compounds (VOCs). Because some of the identified antagonists were previously shown to strongly repel Anopheles and Culex mosquitoes, we examined the bioactivities of the identified antagonists against Aedes, the third major genus of the Culicidae family. The tested antagonists inhibited the function of Ae. aegypti ORco ex vivo and repelled adult Asian tiger mosquitoes (Ae. albopictus). Binary mixtures of specific antagonists elicited higher repellency than single antagonists, and binding competition assays suggested that this enhanced repellence is due to antagonist interaction with distinct ORco sites. Our results also suggest that the enhanced mosquito repellency by antagonist mixtures is due to additive rather than synergistic effects of the specific antagonist combinations on ORco function. Taken together, these findings provide novel insights concerning the molecular aspects of odorant receptor function. Moreover, our results demonstrate that a simple screening assay may be used for the identification of allosteric modifiers of olfactory-driven behaviors capable of providing enhanced personal protection against multiple mosquito-borne infectious diseases.