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The zebrafish, Danio rerio, is a widely adopted in vivo model that conserves organs such as the liver, kidney, stomach, and brain, being, therefore, suitable for studying human diseases, drug discovery and toxicology. The brain aminergic systems are also conserved and the histamine H1, H2 and H3 receptors were previously cloned and identified in the zebrafish brain. Genome studies identified another putative H2 receptor (Hrh2) with ∼50% sequence identity with H2 receptor orthologs. In this study, we recombinantly expressed both zebrafish H2 receptor paralogs (hrh2a and hrh2b) and compared their pharmacology with the human H2 receptor ortholog. Our results showed that both zebrafish receptors conserve all the class A GPCR motifs. However, in contrast with the Hrh2a paralog, the Hrh2b does not possess all the amino acid residues shown to participate in histamine binding. The zebrafish Hrh2a receptor displays high affinity for [3H]-tiotidine with a binding profile for H2 receptor ligands similar to that of the human H2 receptor. The zebrafish Hrh2a receptor couples to GαS and Gαq/11 proteins, resulting in cAMP accumulation and activation of several reporter genes linked to the Gαq/11 pathway. Additionally, this receptor shows high constitutive activity, with histamine potency in the low nanomolar range for cAMP accumulation and the micromolar range for the activation of the NFAT response element. Moreover, dimaprit and amthamine seem to preferentially activate GαS over Gαq/11 proteins via the zebrafish Hrh2a receptor. These results can contribute to clarifying the functional roles of the H2 receptor in zebrafish.

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The serotonin type 6 receptor (5-HT6R) displays a strong constitutive activity, suggesting it participates largely in the physiological and pathological processes controlled by the receptor. The active states of 5-HT6R engage particular signal transduction pathways that lead to different biological responses. In this study, we present the development of 5-HT6R neutral antagonists at Gs signaling built upon the 2-phenylpyrrole scaffold. Using molecular dynamics simulations, we outline the relationship between the exposure of the basic center of the molecules and their ability to target the agonist-activated state of the receptor. Our study identifies compound 30 as a potent and selective neutral antagonist at 5-HT6R-operated Gs signaling. Furthermore, we demonstrate the cytoprotective effects of 30 and structurally diverse 5-HT6R neutral antagonists at Gs signaling in C8-D1A cells and human astrocytes exposed to rotenone. This effect is not observed for 5-HT6R agonists or inverse agonists. In light of these findings, we propose compound 30 as a valuable molecular probe to study the biological effects associated with the agonist-activated state of 5-HT6R and provide insight into the glioprotective properties of 5-HT6R neutral antagonists at Gs signaling.

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BACKGROUND AND PURPOSE: Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. EXPERIMENTAL APPROACH: Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. KEY RESULTS: MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. CONCLUSION AND IMPLICATIONS: The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

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Neurotransmitter ligands electrically excite neurons by activating ionotropic glutamate receptor (iGluR) ion channels. Knowledge of the iGluR amino acid residues that dominate ligand-induced activation would enable the prediction of function from sequence. We therefore explored the molecular determinants of activity in rat N-methyl-D-aspartate (NMDA)-type iGluRs (NMDA receptors), complex heteromeric iGluRs comprising two glycine-binding GluN1 and two glutamate-binding GluN2 subunits, using amino acid sequence analysis, mutagenesis, and electrophysiology. We find that a broadly conserved aspartate residue controls both ligand potency and channel activity, to the extent that certain substitutions at this position bypass the need for ligand binding in GluN1 subunits, generating NMDA receptors activated solely by glutamate. Furthermore, we identify a homomeric iGluR from the placozoan Trichoplax adhaerens that has utilized native mutations of this crucial residue to evolve into a leak channel that is inhibited by neurotransmitter binding, pointing to a dominant role of this residue throughout the iGluR superfamily.

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The histamine H3 receptor (H3R) regulates as a presynaptic G protein-coupled receptor the release of histamine and other neurotransmitters in the brain, and is consequently a potential therapeutic target for neuronal disorders. The human H3R encodes for seven splice variants that vary in the length of intracellular loop 3 and/or the C-terminal tail but are all able to induce heterotrimeric Gi protein signaling. The last two decades H3R drug discovery and lead optimization has been exclusively focused on the 445 amino acids-long reference isoform H3R-445. In this study, we pharmacologically characterized for the first time all seven H3R isoforms by determining their binding affinities for reference histamine H3 receptor agonists and inverse agonists. The H3R-453, H3R-415, and H3R-413 isoforms display similar binding affinities for all ligands as the H3R-445. However, increased agonist binding affinities were observed for the three shorter isoforms H3R-329, H3R-365, and H3R-373, whereas inverse agonists such as the approved anti-narcolepsy drug pitolisant (Wakix®) displayed significantly decreased binding affinities for the latter two isoforms. This opposite change in binding affinity of agonist versus inverse agonists on H3R-365 and H3R-373 is associated with their higher constitutive activity in a cAMP biosensor assay as compared to the other five isoforms. The observed differences in pharmacology between longer and shorter H3R isoforms should be considered in future drug discovery programs.

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GPR101 is an orphan G protein-coupled receptor that promotes growth hormone secretion in the pituitary. The microduplication of the GPR101 gene has been linked with the X-linked acrogigantism, or X-LAG, syndrome. This disease is characterized by excessive growth hormone secretion and abnormal rapid growth beginning early in life. Mechanistically, GPR101 induces growth hormone secretion through constitutive activation of multiple heterotrimeric G proteins. However, the full scope of GPR101 signaling remains largely elusive. Herein, we investigated the association of GPR101 to multiple transducers and uncovered an important basal interaction with Arrestin 2 (ß-arrestin 1) and Arrestin 3 (ß-arrestin 2). By using a GPR101 mutant lacking the C-terminus and cell lines with an Arrestin 2/3 null background, we show that the arrestin association leads to constitutive clathrin- and dynamin-mediated GPR101 internalization. To further highlight GPR101 intracellular fate, we assessed the colocalization of GPR101 with Rab protein markers. Internalized GPR101 was mainly colocalized with the early endosome markers, Rab5 and EEA-1, and to a lesser degree with the late endosome marker Rab7. However, GPR101 was not colocalized with the recycling endosome marker Rab11. These findings show that the basal arrestin recruitment by GPR101 C-terminal tail drives the receptor constitutive clathrin-mediated internalization. Intracellularly, GPR101 concentrates in the endosomal compartment and is degraded through the lysosomal pathway. In conclusion, we uncovered a constitutive intracellular trafficking of GPR101 that potentially represents an important layer of regulation of its signaling and function.

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BACKGROUND: Many hypertension therapeutics were developed prior to major advances in drug receptor theory. Moreover, newer drugs may take advantage of some of the newly understood modalities of receptor function. GOAL: The goal of this review is to provide an up-to-date summary of drug receptor theory. This is followed by a discussion of the drug classes recognized for treating hypertension to which new concepts in receptor theory apply. RESULTS: We raise ideas for mechanisms of potential new antihypertensive drugs and whether they may take advantage of new theories in drug-receptor interaction.

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Pulmonary veins (PV) are the main source of ectopy, triggering atrial fibrillation. This study investigated the roles of G protein-coupled inwardly rectifying potassium (GIRK) channels in the PV and the left atrium (LA) of the rat. Simultaneous intracellular microelectrode recording from the LA and the PV of the rat found that in the presence or absence of acetylcholine, the GIRK channel blocker tertiapin-Q induced AP duration elongation in the LA and the loss of over-shooting AP in the PV, suggesting the presence of constitutively active GIRK channels in these tissues. Patch-clamp recordings from isolated myocytes showed that tertiapin-Q inhibited a basal inwardly rectified background current in PV cells with little effect in LA cells. Experiments with ROMK1 and KCa1.1 channel blockers ruled out the possibility of an off-target effect. Western blot showed that GIRK4 subunit expression was greater in PV cardiomyocytes, which may explain the differences observed between PV and LA in response to tertiapin-Q. In conclusion, GIRK channels blockade abolishes AP only in the PV, providing a molecular target to induce electrical disconnection of the PV from the LA.

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The melanocortin-4 receptor (MC4R) is essential for the modulation of energy balance and reproduction in both fish and mammals. Rainbow trout (Oncorhynchus mykiss) has been extensively studied in various fields and provides a unique opportunity to investigate divergent physiological roles of paralogues. Herein we identified four trout mc4r (mc4ra1, mc4ra2, mc4rb1, and mc4rb2) genes. Four trout Mc4rs (omMc4rs) were homologous to those of teleost and mammalian MC4Rs. Multiple sequence alignments, a phylogenetic tree, chromosomal synteny analyses, and pharmacological studies showed that trout mc4r genes may have undergone different evolutionary processes. All four trout Mc4rs bound to two peptide agonists and elevated intracellular cAMP levels dose-dependently. High basal cAMP levels were observed at two omMc4rs, which were decreased by Agouti-related peptide. Only omMc4rb2 was constitutively active in the ERK1/2 signaling pathway. Ipsen 5i, ML00253764, and MCL0020 were biased allosteric modulators of omMc4rb1 with selective activation upon ERK1/2 signaling. ML00253764 behaved as an allosteric agonist in Gs-cAMP signaling of omMc4rb2. This study will lay the foundation for future physiological studies of various mc4r paralogs and reveal the evolution of MC4R in vertebrates.

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GPR82 is an orphan G protein-coupled receptor (GPCR) that has been implicated in lipid storage in mouse adipocytes. However, the intracellular signaling as well as the specific ligands of GPR82 remain unknown. GPR82 is closely related to GPR34, a GPCR for the bioactive lipid molecule lysophosphatidylserine. In this study, we screened a lipid library using GPR82-transfected cells to search for ligands that act on GPR82. By measuring cyclic adenosine monophosphate levels, we found that GPR82 is an apparently constitutively active GPCR that leads to Gi protein activation. In addition, edelfosine (1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine), an artificial lysophospholipid with a cationic head group that exerts antitumor activity, inhibited the Gi protein activation by GPR82. Two endogenous lysophospholipids with cationic head groups, lysophosphatidylcholine (1-oleoyl-sn-glycero-3-phosphocholine) and lysophosphatidylethanolamine (1-oleoyl-sn-glycero-3-phosphoethanolamine), also exhibited GPR82 inhibitory activity, albeit weaker than edelfosine. Förster resonance energy transfer imaging analysis consistently demonstrated that Gi protein-coupled GPR82 has an apparent constitutive activity that is edelfosine-sensitive. Consistent data were obtained from GPR82-mediated binding analysis of guanosine-5'-O-(3-thiotriphosphate) to cell membranes. Furthermore, in GPR82-transfected cells, edelfosine inhibited insulin-induced extracellular signal-regulated kinase activation, like compounds that function as inverse agonists at other GPCRs. Therefore, edelfosine is likely to act as an inverse agonist of GPR82. Finally, GPR82 expression inhibited adipocyte lipolysis, which was abrogated by edelfosine. Our findings suggested that the cationic lysophospholipids edelfosine, lysophosphatidylcholine and lysophosphatidylethanolamine are novel inverse agonists for Gi-coupled GPR82, which is apparently constitutively active, and has the potential to exert lipolytic effects through GPR82.

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Rhodopsin is a G protein-coupled receptor (GPCR) present in the rod outer segment (ROS) of photoreceptor cells that initiates the phototransduction cascade required for scotopic vision. Due to the remarkable advancements in technological tools, the chemistry of rhodopsin has begun to unravel especially over the past few decades, but mostly at the ensemble scale. Atomic force microscopy (AFM) is a tool capable of providing critical information from a single-molecule point of view. In this regard, to bolster our understanding of rhodopsin at the nanoscale level, AFM-based imaging, force spectroscopy, and nano-indentation techniques were employed on ROS disc membranes containing rhodopsin, isolated from vertebrate species both in normal and diseased states. These AFM studies on samples from native retinal tissue have provided fundamental insights into the structure and function of rhodopsin under normal and dysfunctional states. We review here the findings from these AFM studies that provide important insights on the supramolecular organization of rhodopsin within the membrane and factors that contribute to this organization, the molecular interactions stabilizing the structure of the receptor and factors that can modify those interactions, and the mechanism underlying constitutive activity in the receptor that can cause disease.

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The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CRE-driven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight.

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While most therapeutic research on G-protein-coupled receptors (GPCRs) focuses on receptor activation by (endogenous) agonists, significant therapeutic potential exists through agonist-independent intrinsic constitutive activity that can occur in various physiological and pathophysiological settings. For example, inhibiting the constitutive activity of 5-HT6R-a receptor that is found almost exclusively in the brain and mediates excitatory neurotransmission-has demonstrated a therapeutic effect on cognitive/memory impairment associated with several neuropsychiatric disorders. However, the structural basis of such constitutive activity remains unclear. Here, we present a cryo-EM structure of serotonin-bound human 5-HT6R-Gs heterotrimer at 3.0-Å resolution. Detailed analyses of the structure complemented by comprehensive interrogation of signaling illuminate key structural determinants essential for constitutive 5-HT6R activity. Additional structure-guided mutagenesis leads to a nanobody mimic Gαs for 5-HT6R that can reduce its constitutive activity. Given the importance of 5-HT6R for a large number of neuropsychiatric disorders, insights derived from these studies will accelerate the design of more effective medications, and shed light on the molecular basis of constitutive activity.

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Diabetic neuropathy is often associated with chronic pain. Serotonin type 6 (5-HT6) receptor ligands, particularly inverse agonists, have strong analgesic potential and may be new candidates for treating diabetic neuropathic pain and associated co-morbid cognitive deficits. The current study addressed the involvement of 5-HT6 receptor constitutive activity and mTOR signaling in an experimental model of diabetic neuropathic pain induced by streptozocin (STZ) injection in the rat. Here, we show that mechanical hyperalgesia and associated cognitive deficits are suppressed by the administration of 5-HT6 receptor inverse agonists or rapamycin. The 5-HT6 receptor ligands also reduced tactile allodynia in traumatic and toxic neuropathic pain induced by spinal nerve ligation and oxaliplatin injection. Furthermore, both painful and co-morbid cognitive symptoms in diabetic rats are reduced by intrathecal delivery of a cell-penetrating peptide that disrupts 5-HT6 receptor-mTOR physical interaction. These findings demonstrate the deleterious influence of the constitutive activity of spinal 5-HT6 receptors upon painful and cognitive symptoms in diabetic neuropathic pains of different etiologies. They suggest that targeting the constitutive activity of 5-HT6 receptors with inverse agonists or disrupting the 5-HT6 receptor-mTOR interaction might be valuable strategies for the alleviation of diabetic neuropathic pain and cognitive co-morbidities.

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BACKGROUND AND PURPOSE: CaV 3.1-3 currents differentially contribute to neuronal firing patterns. CaV 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about CaV 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D5 recpetor constitutive activity, a GPCR with high levels of basal activity, on CaV 3 functionality. D5 recpetor and CaV 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy. EXPERIMENTAL APPROACH: Our study models were HEK293T cells heterologously expressing D1 or D5 receptor and CaV 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D1 /D5 inverse agonist) and a D5 receptor mutant lacking constitutive activity as experimental tools. We measured CaV 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique. KEY RESULTS: We found a higher sensitivity to TTA-P2 (CaV 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that CaV 3.2 and CaV 3.3-but not CaV 3.1-are targets of D5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled CaV 3 currents levels. CONCLUSIONS AND IMPLICATIONS: Native hippocampal CaV 3 and recombinant CaV 3.2-3 are sensitive to D5 receptor constitutive activity. Manipulation of D5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy.

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The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

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Selatogrel is a potent inhibitor of adenosine diphosphate (ADP) binding to the P2Y12 receptor, preventing platelet activation. We have previously shown that the P2Y12 receptor constitutively activates Gi- and Go-protein-mediated signaling in human platelets. Here, we report that selatogrel acts as an inverse agonist of the P2Y12 receptor. Specifically, using bioluminescence resonance energy transfer2 (BRET2) probes, selatogrel, ticagrelor, and elinogrel were shown to stabilize the inactive form of the Gαi/o-Gßγ complex in cells with recombinant expression of the P2Y12 receptor. In dose-response experiments, while selatogrel exhibited a maximal efficacy similar to ticagrelor, selatogrel was approximately 100-fold more potent than ticagrelor. Quantification of relative cyclic adenosine monophosphate (cAMP) levels in cells expressing the cAMP BRET1 sensor (CAMYEL probe) confirmed that selatogrel completely abolished the constitutive activity of the P2Y12 receptor. In agreement, selatogrel increased basal cAMP levels in human platelets, confirming inverse agonism on the endogenous human platelet P2Y12 receptor. In agreement with the biochemical phenotype of inverse agonism efficacy of selatogrel, the 2.8 Angstrom resolution cocrystal structure of selatogrel bound to the P2Y12 receptor confirmed that selatogrel stabilizes the inactive, basal state of the receptor. Selatogrel bound to pocket 1, spanning helix III to VII. Furthermore, the binding mode of selatogrel, suggesting steric overlap with the proposed binding site of ADP and the ADP analog 2-methylthioadenosine diphosphate (2MeSADP), agrees with the functional characterization of selatogrel preventing platelet activation by blocking ADP binding to the P2Y12 receptor.

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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.

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RNA editing is a process by which post-transcriptional changes of mRNA nucleotides alter protein function through modification of the amino acid content. The 5HT2C serotonin receptor, which undergoes 32 distinct RNA-editing events leading to 24 protein isoforms, is a notable example of this process. These 5HT2C isoforms display differences in constitutive activity, agonist/inverse agonist potencies, and efficacies. To elucidate the molecular mechanisms responsible for these effects of RNA editing, we present four active-state 5HT2C-transducer-coupled structures of three representative isoforms (INI, VGV, and VSV) with the selective drug lorcaserin (Belviq) and the classic psychedelic psilocin. We also provide a comprehensive analysis of agonist activation and constitutive activity across all 24 protein isoforms. Collectively, these findings reveal a unique hydrogen-bonding network located on intracellular loop 2 that is subject to RNA editing, which differentially affects GPCR constitutive and agonist signaling activities.

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The receptor RORγ belongs to the nuclear receptor superfamily that senses small signaling molecules and regulates at the gene transcription level. Since RORγ has a high basal activity and plays an important role in immune responses, inhibitors targeting this receptor have been a focus for many studies. The receptor-ligand interaction is complex, and often subtle differences in ligand structure can determine its role as an inverse agonist or an agonist. We examined more than 130 existing RORγ crystal structures that have the same receptor complexed with different ligands. We reported the features of receptor-ligand interaction patterns and the differences between agonist and inverse agonist binding. Specific changes in the contact interaction map are identified to distinguish active and inactive conformations. Further statistical analysis of the contact interaction patterns using principal component analysis reveals a dominant mode which separates allosteric binding vs. canonical binding and a second mode which may indicate active vs. inactive structures. We also studied the nature of constitutive activity by performing a 100-ns computer simulation of apo RORγ. Using constitutively active nuclear receptor CAR as a comparison, we identified a group of conserved contacts that have similar contact strength between the two receptors. These conserved contact interactions, especially a couple key contacts in H11-H12 interaction, can be considered essential to the constitutive activity of RORγ. These protein-ligand and internal protein contact interactions can be useful in the development of new drugs that direct receptor activity.