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Most neurodegenerative disorders, including the two most common, Alzheimer’s disease (AD) and Parkinson’s disease (AD), course with activation of microglia, the resident innate immune cells of the central nervous system. A3 adenosine receptor (A3R) agonists have been proposed to be neuroprotective by regulating the phenotype of activated microglia. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with 2-Cl-IB-MECA, a selective A3R agonist. The results showed that the number of negatively regulated genes in the presence of 2-Cl-IB-MECA was greater than the number of positively regulated genes. Gene ontology enrichment analysis showed regulation of genes participating in several cell processes, including those involved in immune-related events. Analysis of known and predicted protein-protein interactions showed that Smad3 and Sp1 are transcription factors whose genes are regulated by A3R activation. Under the conditions of cell activation and agonist treatment regimen, 2-Cl-IB-MECA did not lead to any tendency to favor the expression of genes related to neuroprotective microglia (M2).

Following approval of antagonists in Parkinson's disease therapy, the AThis review focuses on the therapeutic potential of targeting AThere is a pending issue, namely, how to demonstrate the neuroprotective potential of A

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB2 and serotonin 5HT1A receptors included. These two receptors may interact to form heteromers (CB2–5HT1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB2–5HT1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB2–5HT1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB2 and 5HT1A receptor–receptor interaction. Both cannabinoids regulated the effects of CB2 and 5HT1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB2–5HT1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB2–5HT1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB2–5HT1A-Hets and by reducing the aberrant expression of the receptor–receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A1, A2A, A2B, and A3, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A1 and A2A, A2A and A2B, and A2A and A3 receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.

Adenosine (Ado) receptors have been instrumental in the detection of heteromers and other higher-order receptor structures, mainly via interactions with other cell surface G-protein-coupled receptors. Apart from the first report of the A

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.

We have here assessed, using Δ9 -tetrahydrocannabinol (Δ9 -THC) for comparison, the effect of Δ9 -tetrahydrocannabinolic acid (Δ9 -THCA) and of Δ9 -tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9 -THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9 -THCA and Δ9 -THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9 -THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9 -THCV and Δ9 -THCA and Δ9 -THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2′ -chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation

We have here assessed, using Δ

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB2R is overexpressed in activated microglia, showing a neuroprotective effect. These two receptors may interact, forming CB2-OX1-Hets and becoming a new target to combat Alzheimer’s disease. Aims: Demonstrate the potential role of CB2-OX1-Hets expression and function in microglia from animal models of Alzheimer’s disease. Receptor heteromer expression was detected by immunocytochemistry, bioluminescence resonance energy transfer (BRET) and proximity ligation assay (PLA) in transfected HEK-293T cells and microglia primary cultures. Quantitation of signal transduction events in a heterologous system and in microglia cells was performed using the AlphaScreen® SureFire® kit, western blot, the GCaMP6 calcium sensor and the Lance Ultra cAMP kit (PerkinElmer). The formation of CB2-OX1 receptor complexes in transfected HEK-293T cells has been demonstrated. The tetrameric complex is constituted by one CB2R homodimer, one OX1R homodimer and two G proteins, a Gi and a Gq. The use of TAT interfering peptides showed that the CB2-OX1 receptor complex interface is TM4-TM5. At the functional level it has been observed that the OX1R antagonist, SB334867, potentiates the action induced by CB2R agonist JWH133. This effect is observed in transfected HEK-293T cells and microglia, and it is stronger in the Alzheimer’s disease (AD) animal model APPSw/Ind where the expression of the complex assessed by the proximity ligation assay indicates an increase in the number of complexes compared to resting microglia. The CB2-OX1 receptor complex is overexpressed in microglia from AD animal models where OX1R antagonists potentiate the neuroprotective actions of CB2R activation. Taken together, these results point to OX1R antagonists as drugs with therapeutic potential to combat AD. Data access statement: Raw data will be provided by the corresponding author upon reasonable requirement.

The composition of the aqueous humor of patients with type 2 diabetes is relevant to understanding the underlying causes of eye-related comorbidities. Information on the composition of aqueous humor in healthy subjects is limited due to the lack of adequate controls. To carry out a metabolomics study, 31 samples of aqueous humor from healthy subjects without ocular pathology, submitted to refractive surgery and seven samples from patients with type 2 diabetes without signs of ocular pathology related to diabetes were used. The level of 25 molecules was significantly (p < 0.001) altered in the aqueous humor of the patient group. The concentration of a single molecule, N-acetylornithine, makes it possible to discriminate between control and diabetes (sensitivity and specificity equal to 1). In addition, receptor operating characteristic curve and principal component analysis for the above-mentioned six molecules yielded significantly (p < 0.001) altered in the aqueous humor of the patient group. In addition, receptor operating characteristic curve and principal component analysis for six compounds yielded cut-off values and remarkable sensitivity, specificity, and segregation ability. The altered level of N-acetylornithine may be due to an increased amount of acetate in diabetes. It is of interest to further investigate whether this alteration is related to the pathogenesis of the disease. The increase in the amino form of pyruvate, alanine, in diabetes is also relevant because it could be a means of reducing the formation of lactate from pyruvate.

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called “hunger” hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB2 receptors at the central nervous system (CNS) level. In a heterologous system we identified CB2-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB2 receptor/Gi-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB2 receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB2-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called “hunger” hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB2 receptors at the central nervous system (CNS) level. In a heterologous system we identified CB2-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB2 receptor/Gi-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB2 receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB2-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.

Neuroprotective M2-skewed microglia appear as promising to alter the course of neurodegenerative diseases and G protein-coupled receptors (GPCRs) are potential targets to achieve such microglial polarization. A common feature of adenosine A2A (A2A R) and cannabinoid CB2 (CB2 R) GPCRs in microglia is that their expression is upregulated in Alzheimer's disease (AD). On the one hand, CB2 R seems a target for neuroprotection, delaying neurodegenerative processes like those associated to AD or Parkinson's diseases. A2A R antagonists reduce amyloid burden and improve cognitive performance and memory in AD animal models. We here show a close interrelationship between these two receptors in microglia; they are able to physically interact and affect the signaling of each other, likely due to conformational changes within the A2A -CB2 receptor heteromer (A2A -CB2 Het). Particularly relevant is the upregulation of A2A -CB2 Het expression in samples from the APPSw ,Ind AD transgenic mice model. The most relevant finding, confirmed in both heterologous cells and in primary cultures of microglia, was that blockade of A2A receptors results in increased CB2 R-mediated signaling. This heteromer-specific feature suggests that A2A R antagonists would potentiate, via microglia, the neuroprotective action of endocannabinoids with implications for AD therapy.

Stress is one of the factors underlying drug seeking behavior that often goes in parallel with loss of appetite. We here demonstrate that orexin 1 receptors (OX1R) may form complexes with the corticotropin releasing factor CRF2 receptor. Two specific features of the heteromer were a cross-antagonism and a blockade by CRF2 of OX1R signaling. In cells expressing one of the receptors, agonist-mediated signal transduction mechanisms were potentiated by amphetamine. Sigma 1 (σ1) and 2 (σ2) receptors are targets of drugs of abuse and, despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is not known. We here show that σ1 receptors interact with CRF2 receptors and that σ2 receptors interact with OX1R. Moreover, we show that amphetamine effect on CRF2 receptors was mediated by σ1R whereas the effect on OX1 receptors was mediated by σ2R. Amphetamine did potentiate the negative cross-talk occurring within the CRF2-OX1 receptor heteromer context, likely by a macromolecular complex involving the two sigma receptors and the two GPCRs. Finally, in vivo microdialysis experiments showed that amphetamine potentiated orexin A-induced dopamine and glutamate release in the ventral tegmental area (VTA). Remarkably, the in vivo orexin A effects were blocked by a selective CRF2R antagonist. These results show that amphetamine impacts on the OX1R-, CRF2R- and OX1R/CRF2R-mediated signaling and that cross-antagonism is instrumental for in vivo detection of GPCR heteromers. This article is part of the Special Issue entitled ‘Receptor heteromers and their allosteric receptor-receptor interactions’.

Neuronal survival depends on the glia, that is, on the astroglial and microglial support. Neurons die and microglia are activated not only in neurodegenerative diseases but also in physiological aging. Activated microglia, once considered harmful, express two main phenotypes: the pro-inflammatory or M1, and the neuroprotective or M2. When neuroinflammation, i.e., microglial activation occurs, it is important to achieve a good M1/M2 balance, i.e., at some point M1 microglia must be skewed into M2 cells to impede chronic inflammation and to afford neuronal survival. G protein-coupled receptors in general and adenosine receptors in particular are potential targets for increasing the number of M2 cells. This article describes the mechanisms underlying microglial activation and analyzes whether these cells exposed to a first damaging event may be ready to be preconditioned to better react to exposure to more damaging events. Adenosine receptors are relevant due to their participation in preconditioning. They can also be overexpressed in activated microglial cells. The potential of adenosine receptors and complexes formed by adenosine receptors and cannabinoids as therapeutic targets to provide microglia-mediated neuroprotection is here discussed. Keywords: neurodegeneration; aging; Parkinson's disease; Alzheimer's disease; neuroprotection; neuronal survival; cannabinoids; receptor heteromers

Cocaine not only increases brain dopamine levels but also activates the sigma1 receptor (σ1 R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D1 (D1 R), ghrelin (GHS-R1a ), and σ1 receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca2+ , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ1 R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ1 R, D1 R, and GHS-R1a is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ1 R, as confirmed using samples from σ1 R-/- mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ1 R, D1 R, and GHS-R1a complex constitutes a functional unit that is altered upon cocaine binding to the σ1 R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca2+ and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A

The aim of this paper was to check the possible interaction of two of the four purinergic P1 receptors, the A2A and the A3. Discovery of the A2A–A3 receptor complex was achieved by means of immunocytochemistry and of bioluminescence resonance energy transfer. The functional properties and heteromer print identification were addressed by combining binding and signaling assays. The physiological role of the novel heteromer is to provide a differential signaling depending on the pre-coupling to signal transduction components and/or on the concentration of the endogenous agonist. The main feature was that the heteromeric context led to a marked decrease of the signaling originating at A3 receptors. Interestingly from a therapeutic point of view, A2A receptor antagonists overrode the blockade, thus allowing A3 receptor-mediated signaling. The A2A–A3 receptor heteromer print was detected in primary cortical neurons. These and previous results suggest that all four adenosine receptors may interact with each other. Therefore, each adenosine receptor could form heteromers with distinct properties, expanding the signaling outputs derived from the binding of adenosine to its cognate receptors.

Recent approved medicines whose active principles are ΔCannabinoid CBDetermination of the affinity of phytocannabinoids for cannabinoid receptors and functional assessment of effects promoted by these compounds when interacting with cannabinoid receptors.A heterologous system expressing the human versions of CBAffinity of cannabinoids depend on the ligand of reference and may be different in membranes and in living cells. All tested phytocannabinoids have agonist-like behavior but behaved as inverse-agonists in the presence of selective receptor agonists. CBGV displayed enhanced potency in many of the functional outputs. However, the most interesting result was a biased signaling that correlated with differential affinity, i.e. the overall results suggest that the binding mode of each ligand leads to specific receptor conformations underlying biased signaling outputs.Results here reported and the recent elucidation of the three-dimensional structure of CB

(1) Background. N-methyl d-aspartate (NMDA) ionotropic glutamate receptor (NMDAR), which is one of the main targets to combat Alzheimer’s disease (AD), is expressed in both neurons and glial cells. The aim of this paper was to assess whether the adenosine A2A receptor (A2AR), which is a target in neurodegeneration, may affect NMDAR functionality. (2) Methods. Immuno-histo/cytochemical, biophysical, biochemical and signaling assays were performed in a heterologous cell expression system and in primary cultures of neurons and microglia (resting and activated) from control and the APPSw,Ind transgenic mice. (3) Results. On the one hand, NMDA and A2A receptors were able to physically interact forming complexes, mainly in microglia. Furthermore, the amount of complexes was markedly enhanced in activated microglia. On the other hand, the interaction resulted in a novel functional entity that displayed a cross-antagonism, that could be useful to prevent the exacerbation of NMDAR function by using A2AR antagonists. Interestingly, the amount of complexes was markedly higher in the hippocampal cells from the APPSw,Ind than from the control mice. In neurons, the number of complexes was lesser, probably due to NMDAR not interacting with the A2AR. However, the activation of the A2AR receptors resulted in higher NMDAR functionality in neurons, probably by indirect mechanisms. (4) Conclusions. A2AR antagonists such as istradefylline, which is already approved for Parkinson’s disease (Nouriast® in Japan and Nourianz® in the US), have potential to afford neuroprotection in AD in a synergistic-like fashion. i.e., via both neurons and microglia.

(1) Background. N-methyl d-aspartate (NMDA) ionotropic glutamate receptor (NMDAR), which is one of the main targets to combat Alzheimer&rsquo, s disease (AD), is expressed in both neurons and glial cells. The aim of this paper was to assess whether the adenosine A2A receptor (A2AR), which is a target in neurodegeneration, may affect NMDAR functionality. (2) Methods. Immuno-histo/cytochemical, biophysical, biochemical and signaling assays were performed in a heterologous cell expression system and in primary cultures of neurons and microglia (resting and activated) from control and the APPSw,Ind transgenic mice. (3) Results. On the one hand, NMDA and A2A receptors were able to physically interact forming complexes, mainly in microglia. Furthermore, the amount of complexes was markedly enhanced in activated microglia. On the other hand, the interaction resulted in a novel functional entity that displayed a cross-antagonism, that could be useful to prevent the exacerbation of NMDAR function by using A2AR antagonists. Interestingly, the amount of complexes was markedly higher in the hippocampal cells from the APPSw,Ind than from the control mice. In neurons, the number of complexes was lesser, probably due to NMDAR not interacting with the A2AR. However, the activation of the A2AR receptors resulted in higher NMDAR functionality in neurons, probably by indirect mechanisms. (4) Conclusions. A2AR antagonists such as istradefylline, which is already approved for Parkinson&rsquo, s disease (Nouriast&reg, in Japan and Nourianz&reg, in the US), have potential to afford neuroprotection in AD in a synergistic-like fashion. i.e., via both neurons and microglia.

Angiotensin-converting enzyme 2 (ACE2) is a membrane peptidase and a component of the renin-angiotensin system (RAS) that has been found in cells of all organs, including the lungs. While ACE2 has been identified as the receptor for severe acute respiratory syndrome (SARS) coronaviruses, the mechanism underlying cell entry remains unknown. Human immunodeficiency virus infects target cells via CXC chemokine receptor 4 (CXCR4)-mediated endocytosis. Furthermore, CXCR4 interacts with dipeptidyl peptidase-4 (CD26/DPPIV), an enzyme that cleaves CXCL12/SDF-1, which is the chemokine that activates this receptor. By analogy, we hypothesized that ACE2 might also be capable of interactions with RAS-associated G-protein coupled receptors. Using resonance energy transfer and cAMP and mitogen-activated protein kinase signaling assays, we found that human ACE2 interacts with RAS-related receptors, namely the angiotensin II type 1 receptor (AT1R), the angiotensin II type 2 receptor (AT2R), and the MAS1 oncogene receptor (MasR). Although these interactions led to various alterations of signal transduction, but, more importantly, ligand binding to AT1R resulted in the downregulation of ACE2 cell surface expression, while ligand binding to AT2R, but not to MasR, resulted in upregulation of ACE2 cell surface expression. Proximity ligation assays performed in situ revealed macromolecular complexes containing ACE2 and AT1R, AT2R or MasR in adult but not fetal mouse lung tissue. These findings highlight the relevance of RAS in SARS-CoV-2 infection and the role of ACE2-containing complexes as potential therapeutic targets The Molecular Neurobiology Laboratory of the University of Barcelona is considered a research group of excellence by the Regional Catalonian Government (grup consolidat #2017 SGR 1497) SI

Background Recent approved medicines whose active principles are Δ9Tetrahidrocannabinol (Δ9-THC) and/or cannabidiol (CBD) open novel perspectives for other phytocannabinoids also present in Cannabis sativa L. varieties. Furthermore, solid data on the potential benefits of acidic and varinic phytocannabinoids in a variety of diseases are already available. Mode of action of cannabigerol (CBG), cannabidiolic acid (CBDA), cannabigerolic acid (CBGA), cannabidivarin (CBDV) and cannabigerivarin (CBGV) is, to the very least, partial. Hypothesis/Purpose Cannabinoid CB1 or CB2 receptors, which belong to the G-protein-coupled receptor (GPCR) family, are important mediators of the action of those cannabinoids. Pure CBG, CBDA, CBGA, CBDV and CBGV from Cannabis sativa L. are differentially acting on CB1 or CB2 cannabinoid receptors. Study Design Determination of the affinity of phytocannabinoids for cannabinoid receptors and functional assessment of effects promoted by these compounds when interacting with cannabinoid receptors. Methods A heterologous system expressing the human versions of CB1 and/or CB2 receptors was used. Binding to membranes was measured using radioligands and binding to living cells using a homogenous time resolved fluorescence resonance energy transfer (HTRF) assay. Four different functional outputs were assayed: determination of cAMP levels and of extracellular-signal-related-kinase phosphorylation, label-free dynamic mass redistribution (DMR) and s-arrestin recruitment. Results Affinity of cannabinoids depend on the ligand of reference and may be different in membranes and in living cells. All tested phytocannabinoids have agonist-like behavior but behaved as inverse-agonists in the presence of selective receptor agonists. CBGV displayed enhanced potency in many of the functional outputs. However, the most interesting result was a biased signaling that correlated with differential affinity, i.e. the overall results suggest that the binding mode of each ligand leads to specific receptor conformations underlying biased signaling outputs. Conclusion Results here reported and the recent elucidation of the three-dimensional structure of CB1 and CB2 receptors help understanding the mechanism of action that might be protective and the molecular drug-receptor interactions underlying biased signaling.