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Recent breakthroughs in the determination of atomic resolution 3-D cryo-electron microscopy structures of membrane proteins present an unprecedented opportunity for drug discovery. Structure-based drug discovery utilizing in silico methods enables the study of dynamic connectivity of stable conformations induced by the drug in achieving its effect. With the ever-expanding computational power, simulations of this type reveal protein dynamics in the nano-, micro-, and even millisecond time scales. In the present study, aiming to characterize the protein dynamics of the 5HT2A receptor stimulated by ligands (agonist/antagonist), we performed 1 µs MD simulations on 5HT2A/DOI (agonist), 5HT2A/GSK215083 (antagonist), and 5HT2A (APO, no ligand) systems. The crystal structure of 5HT2A/zotepine (antagonist) (PDB: 6A94) was used to set up the simulation systems in a lipid bilayer environment. We found the monitoring of the ionic lock residue pair (R3.50-E6.30) of 5HT2A in MD simulations to be a good approximation of the effects of agonists (ionic lock breakage) or antagonists (ionic lock formation) on receptor activation. We further performed analyses of the MD trajectories, including Principal Component Analysis (PCA), hydrogen bond, salt bridge, and hydrophobic interaction network analyses, and correlation between residues to identify key elements of receptor activation. Our results suggest that in order to trigger receptor activation, DOI must interact with 5HT2A through residues V5.39, G5.42, S5.43, and S5.46 on TM5, inducing significant conformational changes in the backbone angles of G5.42 and S5.43. DOI also interacted with residues W6.48 (toggle switch) and F6.51 on TM6, causing major conformational shifts in the backbone angles of F6.44 and V6.45. These structural changes were transmitted to the intracellular ends of TM5, TM6, and ICL3, resulting in the breaking of the ionic lock and subsequent G protein activation. The studies could be helpful in future design of selective agonists/antagonists for various serotonin receptors (5HT1A, 5HT2A, 5HT2B, 5HT2C, and 5HT7) involved in detrimental disorders, such as addiction and schizophrenia. [ABSTRACT FROM AUTHOR]

Responding to global standards and legislative updates in Canada, including Bill S-5 (2023), toxicity testing is shifting towards more ethical, in vitro methods. Traditional two-dimensional (2D) monolayer cell cultures, limited in replicating the complex in vivo environment, have prompted the development of more relevant three-dimensional (3D) spheroidal hepatocyte cultures. This study introduces the first 3D spheroid model for McA-RH7777 cells, assessing xenobiotic receptor activation, cellular signaling, and toxicity against dexamethasone and naphthenic acid (NA)-fraction components; NAFCs. Our findings reveal that 3D McA-RH7777 spheroids demonstrate enhanced sensitivity and more uniform dose–response patterns in gene expression related to xenobiotic metabolism (AhR and PPAR) for both single compounds and complex mixtures. Specifically, 3D cultures showed significant gene expression changes upon dexamethasone exposure and exhibited varying degrees of sensitivity and resistance to the apoptotic effects induced by NAFCs, in comparison to 2D cultures. The optimization of 3D culture conditions enhances the model's physiological relevance and enables the identification of genomic signatures under varied exposures. This study highlights the potential of 3D spheroid cultures in providing a more accurate representation of the liver's microenvironment and advancing our understanding of cellular mechanisms in toxicity testing. [ABSTRACT FROM AUTHOR]

Somatostatin receptor 5 (SSTR5) is an important G protein--coupled receptor and drug target for neuroendocrine tumors and pituitary disorders. This study presents two high-resolution cryogenicelectron microscope structures of the SSTR5-Gi complexes bound to the cyclic neuropeptide agonists, cortistatin-17 (CST17) and octreotide, with resolutions of 2.7 Å and 2.9 Å, respectively. The structures reveal that binding of these peptides causes rearrangement of a "hydrophobic lock", consisting of residues from transmembrane helices TM3 and TM6. This rearrangement triggers outward movement of TM6, enabling Gαi protein engagement and receptor activation. In addition to hydrophobic interactions, CST17 forms conserved polar contacts similar to somatostatin-14 binding to SSTR2, while further structural and functional analysis shows that extracellular loops differently recognize CST17 and octreotide. These insights elucidate agonist selectivity and activation mechanisms of SSTR5, providing valuable guidance for structure-based drug development targeting this therapeutically relevant receptor. [ABSTRACT FROM AUTHOR]

Abstract Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/β5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/β5 loop, the α4 helix, and the α4/β6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment.

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/β5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/β5 loop, the α4 helix, and the α4/β6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment. [ABSTRACT FROM AUTHOR]

Signal transduction through G protein-coupled receptors (GPCRs) has been a major focus in cell biology for decades. Numerous disorders are associated with GPCRs that utilize Gi proteins to inhibit adenylyl cyclase (AC) as well as regulate other effectors. Several early studies have successfully defined the AC-interacting domains of several members of Gαi by measuring the loss of activity upon homologous replacements of putative regions of constitutive active Gαi mutants. However, whether such findings can indeed be translated into the context of a receptor-activated Gαi have not been rigorously verified. To address this issue, an array of known and new chimeric mutations was introduced into GTPase-deficient Q204L (QL) and R178C (RC) mutants of Gαi1, followed by examinations on their ability to inhibit AC. Surprisingly, most chimeras failed to abolish the constitutive activity brought on by the QL mutation, while some were able to eliminate the inhibitory activity of RC mutants. Receptor-mediated inhibition of AC was similarly observed in the same chimeric constructs harbouring the pertussis toxin (PTX)-resistant C351I mutation. Moreover, RC-bearing loss-of-function chimeras appeared to be hyper-deactivated by endogenous RGS protein. Molecular docking revealed a potential interaction between AC and the α3/β5 loop of Gαi1. Subsequent cAMP assays support a cooperative action of the α3/β5 loop, the α4 helix, and the α4/β6 loop in mediating AC inhibition by Gαi1-i3. Our results unveiled a notable functional divergence between constitutively active mutants and receptor-activated Gαi1 to inhibit AC, and identified a previously unknown AC-interacting domain of Gαi subunits. These results collectively provide valuable insights on the mechanism of AC inhibition in the cellular environment. [ABSTRACT FROM AUTHOR]

Recent breakthroughs in the determination of atomic resolution 3-D cryo-electron microscopy structures of membrane proteins present an unprecedented opportunity for drug discovery. Structure-based drug discovery utilizing in silico methods enables the study of dynamic connectivity of stable conformations induced by the drug in achieving its effect. With the ever-expanding computational power, simulations of this type reveal protein dynamics in the nano-, micro-, and even millisecond time scales. In the present study, aiming to characterize the protein dynamics of the 5HT2A receptor stimulated by ligands (agonist/antagonist), we performed 1 µs MD simulations on 5HT2A/DOI (agonist), 5HT2A/GSK215083 (antagonist), and 5HT2A (APO, no ligand) systems. The crystal structure of 5HT2A/zotepine (antagonist) (PDB: 6A94) was used to set up the simulation systems in a lipid bilayer environment. We found the monitoring of the ionic lock residue pair (R3.50-E6.30) of 5HT2A in MD simulations to be a good approximation of the effects of agonists (ionic lock breakage) or antagonists (ionic lock formation) on receptor activation. We further performed analyses of the MD trajectories, including Principal Component Analysis (PCA), hydrogen bond, salt bridge, and hydrophobic interaction network analyses, and correlation between residues to identify key elements of receptor activation. Our results suggest that in order to trigger receptor activation, DOI must interact with 5HT2A through residues V5.39, G5.42, S5.43, and S5.46 on TM5, inducing significant conformational changes in the backbone angles of G5.42 and S5.43. DOI also interacted with residues W6.48 (toggle switch) and F6.51 on TM6, causing major conformational shifts in the backbone angles of F6.44 and V6.45. These structural changes were transmitted to the intracellular ends of TM5, TM6, and ICL3, resulting in the breaking of the ionic lock and subsequent G protein activation. The studies could be helpful in future design of selective agonists/antagonists for various serotonin receptors (5HT1A, 5HT2A, 5HT2B, 5HT2C, and 5HT7) involved in detrimental disorders, such as addiction and schizophrenia.

Receptor tyrosine kinases (RTKs) are a class of protein kinases that play crucial roles in various cellular processes, including cell migration, morphological differentiation, cell growth, and angiogenesis. In humans, 58 RTKs have been identified and categorized into 20 distinct families based on the composition of their extracellular regions. RTKs are primarily activated by specific ligands that bind to their extracellular region. They not only regulate tumor transformation, proliferation, metastasis, drug resistance, and angiogenesis, but also initiate and maintain the self‐renewal and cloning ability of cancer stem cells. Accurate diagnosis and grading of tumors with dysregulated RTKs are essential in clinical practice. There is a growing body of evidence supporting the benefits of RTKs‐targeted therapies for cancer patients, and researchers are actively exploring new targets and developing targeted agents. However, further optimization of RTK inhibitors is necessary to effectively target the diverse RTK alterations observed in human cancers. This review provides insights into the classification, structure, activation mechanisms, and expression of RTKs in tumors. It also highlights the research advances in RTKs targeted anticancer therapy and emphasizes their significance in optimizing cancer diagnosis and treatment strategies. [ABSTRACT FROM AUTHOR]

Recent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision. [ABSTRACT FROM AUTHOR]

Abstract Receptor tyrosine kinases (RTKs) are a class of protein kinases that play crucial roles in various cellular processes, including cell migration, morphological differentiation, cell growth, and angiogenesis. In humans, 58 RTKs have been identified and categorized into 20 distinct families based on the composition of their extracellular regions. RTKs are primarily activated by specific ligands that bind to their extracellular region. They not only regulate tumor transformation, proliferation, metastasis, drug resistance, and angiogenesis, but also initiate and maintain the self‐renewal and cloning ability of cancer stem cells. Accurate diagnosis and grading of tumors with dysregulated RTKs are essential in clinical practice. There is a growing body of evidence supporting the benefits of RTKs‐targeted therapies for cancer patients, and researchers are actively exploring new targets and developing targeted agents. However, further optimization of RTK inhibitors is necessary to effectively target the diverse RTK alterations observed in human cancers. This review provides insights into the classification, structure, activation mechanisms, and expression of RTKs in tumors. It also highlights the research advances in RTKs targeted anticancer therapy and emphasizes their significance in optimizing cancer diagnosis and treatment strategies.

Objective: The aim of the present study was to assess if the endocannabinoid system is involved differently in patients with recurrent and non-recurrent periodontal disease and if in sites that have a predisposition for reactivation, levels of anandamide (AEA) change after periodontal therapy. Background: Periodontal disease (PD) may be due to a dysregulation of the endocannabinoid system. Methods: Periodontal patients were recruited, treated for PD and monitored. Gingival samples from these patients with recurrent (n = 10) and non-recurrent (n = 10) periodontal disease were harvested before and after treatment and compared to those of periodontally healthy (n = 10) subjects. Levels of CB1 and CB2, AEA and CBs receptor activation were assessed in healthy and inflamed samples using immunohistochemistry, chromatography and autoradiography. In healed sites, AEA levels were also assessed. Results: The number of CBs in inflamed sites of recurrent patients was significantly higher than in those with non-recurrent disease and also higher than those in healthy subjects. Inflamed sites of recurrent patients had significantly lower CBs receptor activation than those of healthy subjects. Levels of AEA in inflamed sites of non-recurrent patients were significantly higher than those found both in inflamed recurrent sites and in healthy sites. Otherwise, the amount of AEA in healthy subjects and recurrent inflamed sites was similar. After periodontal therapy, levels of AEA were significantly lower in both periodontal groups. In recurrent sites, they resulted significantly lower than in non-recurrent and even in healthy subjects. Conclusions: The endocannabinoid system seems involved differently in subjects with recurrent and non-recurrent periodontal disease. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) are the essential regulators of cell signal transduction pathways and play important roles in biological processes. RTK dimerization is generally considered the first step in receptor activation and cell communication. And the abnormal expression of RTK dimers is closely related to the occurrence and development of many diseases. Therefore, the visualization of RTK dimerization is of great significance for monitoring physiological processes. The genetic and nongenetic imaging strategies have attracted widespread attention due to their high efficiency and high sensitivity. In this review, the RTKs and their dimers as well as the advances in strategies for imaging RTK dimers are introduced. Furthermore, we analyze the limitations of existing imaging strategies and put forward suggestions for the future development of imaging probes. We expect that this review will inspire more in-depth investigation of RTK dimers, which will also broaden the application of strategies of RTK dimers in biomedical areas. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM 2.5)-induced metabolic disorders have attracted increasing attention, however, the underlying molecular mechanism of PM2.5-induced hepatic bile acid disorder remains unclear. In this study, we investigated the effects of PM 2.5 components on the disruption of bile acid in hepatocytes through farnesoid X receptor (FXR) pathway. The receptor binding assays showed that PM 2.5 extracts bound to FXR directly, with half inhibitory concentration (IC 50) value of 21.7 μg/mL. PM 2.5 extracts significantly promoted FXR-mediated transcriptional activity at 12.5 μg/mL. In mouse primary hepatocytes, we found PM 2.5 extracts (100 μg/mL) significantly decreased the total bile acid levels, inhibited the expression of bile acid synthesis gene (Cholesterol 7 alpha-hydroxylase, Cyp7a1), and increased the expression of bile acid transport genes (Multidrug resistance associated protein 2, Abcc2; and Bile salt export pump, Abcb11). Moreover, these alterations were significantly attenuated by knocking down FXR in hepatocytes. We further divided the organic components and water-soluble components from PM 2.5 , and found that two components bound to and activated FXR, and decreased the bile acid levels in hepatocytes. In addition, benzo[ a ]pyrene (B[ a ]P) and cadmium (Cd) were identified as two bioactive components in PM 2.5 -induced bile acid disorders through FXR signaling pathway. Overall, we found PM 2.5 components could bind to and activate FXR, thereby disrupting bile acid synthesis and transport in hepatocytes. These new findings also provide new insights into PM 2.5 -induced toxicity through nuclear receptor pathways. [Display omitted] • PM 2.5 components could bind to and activate farnesoid X receptor. • PM 2.5 components disrupt bile acid synthesis and transport in hepatocytes. • B[a]P and Cd might be two bioactive components. [ABSTRACT FROM AUTHOR]

Integrins and discoidin domain receptors (DDRs) 1 and 2 promote cell adhesion and migration on both fibrillar and non fibrillar collagens. Collagen I contains DDR and integrin selective binding motifs; however, the relative contribution of these two receptors in regulating cell migration is unclear. DDR1 has five isoforms (DDR1a-e), with most cells expressing the DDR1a and DDR1b isoforms. We show that human embryonic kidney 293 cells expressing DDR1b migrate more than DDR1a expressing cells on DDR selective substrata as well as on collagen I in vitro. In addition, DDR1b expressing cells show increased lung colonization after tail vein injection in nude mice. DDR1a and DDR1b differ from each other by an extra 37 amino acids in the DDR1b cytoplasmic domain. Interestingly, these 37 amino acids contain an NPxY motif which is a central control module within the cytoplasmic domain of β integrins and acts by binding scaffold proteins, including talin. Using purified recombinant DDR1 cytoplasmic tail proteins, we show that DDR1b directly binds talin with higher affinity than DDR1a. In cells, DDR1b, but not DDR1a, colocalizes with talin and integrin β1 to focal adhesions and enhances integrin β1-mediated cell migration. Moreover, we show that DDR1b promotes cell migration by enhancing Rac1 activation. Mechanistically DDR1b interacts with the GTPase-activating protein (GAP) Breakpoint cluster region protein (BCR) thus reducing its GAP activity and enhancing Rac activation. Our study identifies DDR1b as a major driver of cell migration and talin and BCR as key players in the interplay between integrins and DDR1b in regulating cell migration.

The P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor. [ABSTRACT FROM AUTHOR]

Many studies over the past decade have reported that internalized membrane receptors can trigger distinct signal activation, rather than being desensitized inside the cell. Here, we propose the concept of 'internalized activation' as a distinctive component of the receptor theory framework and exhibit its significance and role in diseases. [ABSTRACT FROM AUTHOR]

SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a "sequential" or "bypassing" mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations. [Display omitted] • SUCNR1 attracts and binds succinate through a cluster of arginines around TM-VI • The arginines constitute two low-energy succinate binding sites • SUCNR1 is activated by simultaneous binding of two succinates at high concentrations • Binding of succinate, but not antagonist, unlocks constrained ECL-2 via water cluster Extracellular succinate is an auto- and paracrine metabolic stress signal sensed by SUCNR1. Using MD simulations, Shenol et al. found that SUCNR1 has two low-energy binding sites, and its selective activation by high, local concentrations of succinate requires simultaneous occupancy of both high-affinity sites. [ABSTRACT FROM AUTHOR]

The finger loop in the central crest of the receptor‐binding site of arrestins engages the cavity between the transmembrane helices of activated G‐protein‐coupled receptors. Therefore, it was hypothesized to serve as the sensor that detects the activation state of the receptor. We performed comprehensive mutagenesis of the finger loop in bovine visual arrestin‐1, generated mutant radiolabeled proteins by cell‐free translation, and determined the effects of mutations on the in vitro binding of arrestin‐1 to purified phosphorylated light‐activated rhodopsin. This interaction is driven by two factors, rhodopsin activation and rhodopsin‐attached phosphates. Therefore, the binding of arrestin‐1 to light‐activated unphosphorylated rhodopsin is low. To evaluate the role of the finger loop specifically in the recognition of the active receptor conformation, we tested the effects of these mutations in the context of truncated arrestin‐1 that demonstrates much higher binding to unphosphorylated activated and phosphorylated inactive rhodopsin. The majority of finger loop residues proved important for arrestin‐1 binding to light‐activated rhodopsin, with six mutations affecting the binding exclusively to this form. Thus, the finger loop is the key element of arrestin‐1 activation sensor. The data also suggest that arrestin‐1 and its enhanced mutant bind various functional forms of rhodopsin differently. [ABSTRACT FROM AUTHOR]

The P2X4 receptor (P2X4R) is an ATP‐gated cation channel. Here, we used fast‐scan atomic force microscopy (AFM) to visualize changes in the structure and mobility of individual P2X4Rs in response to activation. P2X4Rs were purified from detergent extracts of transfected cells and integrated into lipid bilayers. Activation resulted in a rapid (2 s) and substantial (10–20 nm2) increase in the cross‐sectional area of the extracellular region of the receptor and a corresponding decrease in receptor mobility. Both effects were blocked by the P2X4R antagonist 5‐BDBD. Addition of cholesterol to the bilayer reduced receptor mobility, although the ATP‐induced reduction in mobility was still observed. We suggest that the observed responses to activation may have functional consequences for purinergic signalling. [ABSTRACT FROM AUTHOR]

Receptors in the tumor necrosis factor receptor superfamily (TNFRSF) regulate proliferation of immune cells or induce programmed cell death, and many of them are candidates for antibody-based immunotherapy. Previous studies on several death receptors in the TNFRSF including Fas, p75NTR, and DR5 showed that the transmembrane helix (TMH) of these receptors can specifically oligomerize and their oligomeric states have direct consequences on receptor activation, suggesting a much more active role of TMH in receptor signaling than previously appreciated. Here, we report the structure of the TMH of TNFR1, another well studied member of the TNFRSF, in neutral bicelles that mimic a lipid bilayer. We find that TNFR1 TMH forms a defined trimeric complex in bicelles, and no evidences of higher-order clustering of trimers have been detected. Unexpectedly, a conserved proline, which is critical for Fas TMH trimerization, does not appear to play an important role in TNFR1 TMH trimerization, which is instead mediated by a glycine near the middle of the TMH. Further, TNFR1 TMH trimer shows a larger hydrophobic core than that of Fas or DR5, with four layers of hydrophobic interaction along the threefold axis. Comparison of the TNFR1 TMH structure with that of Fas and DR5 reveals reassuring similarities that have functional implications but also significant structural diversity that warrants systematic investigation of TMH oligomerization property for other members of the TNFRSF.

Much of the early work on Nuclear Hormone Receptors (NHRs) focused on their essential roles as mediators of sex steroid hormone signaling in reproductive development and function, and thyroid hormone-dependent formation of the central nervous system. However, as NHRs display tissue-specific distributions and activities, it is not surprising that they are involved and vital in numerous aspects of human development and essential for homeostasis of all organ systems. Much attention has recently been focused on the role of NHRs in energy balance, metabolism, and lipid homeostasis. Dysregulation of NHR function has been implicated in numerous pathologies including cancers, metabolic obesity and syndrome, Type II diabetes mellitus, cardiovascular disease, hyperlipidemia, male and female infertility and other reproductive disorders. This review will discuss the dysregulation of NHR function by environmental endocrine disrupting chemicals (EDCs), and the associated pathological consequences of exposure in numerous tissues and organ systems, as revealed by experimental, clinical, and epidemiological studies. [ABSTRACT FROM AUTHOR]

We used a range of computational techniques to reveal an increased histamine affinity for its H2 receptor upon deuteration, which was interpreted through altered hydrogen bonding interactions within the receptor and the aqueous environment preceding the binding. Molecular docking identified the area between third and fifth transmembrane α-helices as the likely binding pocket for several histamine poses, with the most favorable binding energy of −7.4 kcal mol−1 closely matching the experimental value of −5.9 kcal mol−1. The subsequent molecular dynamics simulation and MM-GBSA analysis recognized Asp98 as the most dominant residue, accounting for 40% of the total binding energy, established through a persistent hydrogen bonding with the histamine −NH3+ group, the latter further held in place through the N–H∙∙∙O hydrogen bonding with Tyr250. Unlike earlier literature proposals, the important role of Thr190 is not evident in hydrogen bonds through its −OH group, but rather in the C–H∙∙∙π contacts with the imidazole ring, while its former moiety is constantly engaged in the hydrogen bonding with Asp186. Lastly, quantum-chemical calculations within the receptor cluster model and utilizing the empirical quantization of the ionizable X–H bonds (X = N, O, S), supported the deuteration-induced affinity increase, with the calculated difference in the binding free energy of −0.85 kcal mol−1, being in excellent agreement with an experimental value of −0.75 kcal mol−1, thus confirming the relevance of hydrogen bonding for the H2 receptor activation.

G protein-coupled receptors (GPCRs) are cellular master regulators that translate extracellular stimuli such as light, small molecules or peptides into a cellular response. Upon ligand binding, they bind intracellular proteins such as G proteins or arrestins, modulating intracellular signaling cascades. Here, we use a protein-fragment complementation approach based on nanoluciferase (split luciferase assay) to assess interaction of all four known human arrestins with four different GPCRs (two class A and two class B receptors) in live cells. Besides directly tagging the 11S split-luciferase subunit to the receptor, we also could demonstrate that membrane localization of the 11S subunit with a CAAX-tag allowed us to probe arrestin recruitment by endogenously expressed GPCRs. Varying the expression levels of our reporter constructs changed the dynamic behavior of our assay, which we addressed with an advanced baculovirus-based multigene expression system. Our detection assay allowed us to probe the relevance of each of the two arrestin binding sites in the different GPCRs for arrestin binding. We observed remarkable differences between the roles of each arresting binding site in the tested GPCRs and propose that the distinct advantages of our system for probing receptor interaction with effector proteins will help elucidate the molecular basis of GPCR signaling efficacy and specificity in different cell types.

We used a combination of density functional theory (DFT) calculations and the implicit quantization of the acidic N−H and O−H bonds to assess the effect of deuteration on the binding of agonists (2-methylhistamine and 4-methylhistamine) and antagonists (cimetidine and famotidine) to the histamine H2 receptor. The results show that deuteration significantly increases the affinity for 4-methylhistamine and reduces it for 2-methylhistamine, while leaving it unchanged for both antagonists, which is found in excellent agreement with experiments. The revealed trends are interpreted in the light of the altered strength of the hydrogen bonding upon deuteration, known as the Ubbelohde effect, which affects ligand interactions with both active sites residues and solvent molecules preceding the binding, thus providing strong evidence for the relevance of hydrogen bonding for this process. In addition, computations further underline an important role of the Tyr250 residue for the binding. The obtained insight is relevant for the therapy in the context of (per)deuterated drugs that are expected to enter therapeutic practice in the near future, while this approach may contribute towards understanding receptor activation and its discrimination between agonists and antagonists.

Elucidating the physiological roles and modes of action of the recently discovered ligands (designated ALKAL1,2 or AUG-α,β) of the receptor tyrosine kinases Anaplastic Lymphoma Kinase (ALK) and Leukocyte Tyrosine Kinase (LTK) has been limited by difficulties in producing sufficient amounts of the two ligands and their poor stability. Here we describe procedures for expression and purification of AUG-α and a deletion mutant lacking the N-terminal variable region. Detailed biochemical characterization of AUG-α by mass spectrometry shows that the four conserved cysteines located in the augmentor domain (AD) form two intramolecular disulfide bridges while a fifth, primate-specific cysteine located in the N-terminal variable region mediates dimerization through formation of a disulfide bridge between two AUG-α molecules. In contrast to AUG-α, the capacity of AUG-α AD to undergo dimerization is strongly compromised. However, full-length AUG-α and the AUG-α AD deletion mutant stimulate similar tyrosine phosphorylation of cells expressing either ALK or LTK. Both AUG-α and AUG-α AD also stimulate a similar profile of MAP kinase response in L6 cells and colony formation in soft agar by autocrine stimulation of NIH 3T3 cells expressing ALK. Moreover, both AUG-α and AUG-α AD stimulate neuronal differentiation of human neuroblastoma NB1 and PC12 cells in a similar dose-dependent manner. Taken together, these experiments show that deletion of the N-terminal variable region minimally affects the activity of AUG-α toward LTK or ALK stimulation in cultured cells. Reduced dimerization might be compensated by high local concentration of AUG-α AD bound to ALK at the cell membrane and by potential ligand-induced receptor-receptor interactions. [ABSTRACT FROM AUTHOR]

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca 2+ , inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function. [ABSTRACT FROM AUTHOR]

Plant-unique membrane receptor kinases with leucine-rich repeat ectodomains (LRR-RKs) can sense small molecule, peptide, and protein ligands. Many LRR-RKs require SERK-family coreceptor kinases for high-affinity ligand binding and receptor activation. How one coreceptor can contribute to the specific binding of distinct ligands and activation of different LRR-RKs is poorly understood. Here we quantitatively analyze the contribution of SERK3 to ligand binding and activation of the brassinosteroid receptor BRI1 and the peptide hormone receptor HAESA. We show that while the isolated receptors sense their respective ligands with drastically different binding affinities, the SERK3 ectodomain binds the ligand-associated receptors with very similar binding kinetics. We identify residues in the SERK3 N-terminal capping domain, which allow for selective steroid and peptide hormone recognition. In contrast, residues in the SERK3 LRR core form a second, constitutive receptor-coreceptor interface. Genetic analyses of protein chimera between BRI1 and SERK3 define that signaling-competent complexes are formed by receptor-coreceptor heteromerization in planta. A functional BRI1-HAESA chimera suggests that the receptor activation mechanism is conserved among different LRR-RKs, and that their signaling specificity is encoded in the kinase domain of the receptor. Our work pinpoints the relative contributions of receptor, ligand, and coreceptor to the formation and activation of SERK-dependent LRR-RK signaling complexes regulating plant growth and development. [ABSTRACT FROM AUTHOR]

The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC 50 ). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy = 81%, sensitivity = 85% and specificity = 76%. The 3D QSAR model developed to predict EC 50 of PPARγ full agonists had the following statistical parameters: q 2 cv = 0.610, N opt = 7, SEP cv = 0.505, r 2 pr = 0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development. [ABSTRACT FROM AUTHOR]

To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions. [Display omitted] • Long-timescale MD simulations of AMPAR LBD dimer activation and deactivation • The dimer transitioned from active to putative desensitized conformations • Identified a linker region whose structural rearrangements affected the transitions • These conformations, and transitions among them, may underlie AMPAR desensitization Aittoniemi et al. report long-timescale molecular dynamics simulations of AMPAR ligand-binding domain dimers in which activation, deactivation, and transitions to and among putative desensitized conformations were observed. The authors identify and experimentally validate a linker region whose dynamics affect these functional transitions. [ABSTRACT FROM AUTHOR]

This research reports on the membrane interactions of orexin A (OXA), an α-helical and amphipathic neuropeptide that contains 33 residues and two disulfide bonds in the N-terminal region. OXA, which activates the orexins 1 and 2 receptors in neural and immune cell membranes, has essential pleiotropic physiological effects, including at the levels of arousal, sleep/wakefulness, energy balance, neuroprotection, lipid signaling, the inflammatory response, and pain. As a result, the orexin system has become a prominent target to treat diseases such as sleep disorders, drug addiction, and inflammation. While the high-resolution structure of OXA has been investigated in water and bound to micelles, there is a lack of information about its conformation bound to phospholipid membranes and its receptors. NMR is a powerful method to investigate peptide structures in a membrane environment. To facilitate the NMR structural studies of OXA exposed to membranes, we present a novel synthetic scheme, leading to the production of isotopically-labeled material at high purity. A receptor activation assay shows that the 15N-labeled peptide is biologically active. Biophysical studies are performed using surface plasmon resonance, circular dichroism, and NMR to investigate the interactions of OXA with phospholipid bilayers. The results demonstrate a strong interaction between the peptide and phospholipids, an increase in α-helical content upon membrane binding, and an in-plane orientation of the C-terminal region critical to function. This new knowledge about structure-activity relationships in OXA could inspire the design of novel therapeutics that leverage the anti-inflammatory and neuro-protective functions of OXA, and therefore could help address neuroinflammation, a major issue associated with neurological disorders such as Alzheimer's disease. [Display omitted] • Orexin A, a neuropeptide involved in multiple vital functions, activates G-protein coupled receptors in neural and immune cell membranes. • Structure-activity relationship studies of orexin A provide knowledge essential to designing novel therapeutics for human health. • Isotopically-labeled orexin A was synthesized using pseudo-proline dipeptides and an orthogonal strategy for disulfide bond formation. • Biophysical studies investigating orexin A bound to phospholipid membranes indicate partial α-helical content and an orientation parallel to the bilayer surface. • Knowledge of the membrane-bound conformation of orexin A as obtained here could provide important information about its bioactive state. [ABSTRACT FROM AUTHOR]

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.

Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-bind- ing site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp³ and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on "conformationally constrained" GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function. [ABSTRACT FROM AUTHOR]

The extracellular loops (ECLs) of G protein-coupled receptors (GPCRs) can bind directly to docked orthosteric or allosteric ligands, they can contain transient contact points for ligand entry into the transmembrane (TM) bundle and they can regulate the activation of the receptor signalling pathways. Of the three ECLs, ECL2 is the largest and most structurally diverse reflecting its functional importance. This has been shown through biochemical techniques and has been supported by the many subsequent crystal structures of GPCRs bound to both agonists and antagonists. ECL2 shares common structural features between (and sometimes across) receptor sub-families and can facilitate ligand entry to the TM core or act directly as a surface of the ligand-binding pocket. Structural similarities seem to underpin common binding mechanisms; however, where these exist, variations in primary sequence ensure ligand-binding specificity. This review will compare current understanding of the structural themes and main functional roles of ECL2 in ligand binding, activation and regulation of the major families of GPCRs. [ABSTRACT FROM AUTHOR]

Chemokines and their cell surface G protein-coupled receptors are critical for cell migration, not only in many fundamental biological processes but also in inflammatory diseases and cancer. Recent X-ray structures of two chemokines complexed with full-length receptors provided unprecedented insight into the atomic details of chemokine recognition and receptor activation, and computational modeling informed by new experiments leverages these insights to gain understanding of many more receptor:chemokine pairs. In parallel, chemokine receptor structures with small molecules reveal the complicated and diverse structural foundations of small molecule antagonism and allostery, highlight the inherent physicochemical challenges of receptor:chemokine interfaces, and suggest novel epitopes that can be exploited to overcome these challenges. The structures and models promote unique understanding of chemokine receptor biology, including the interpretation of two decades of experimental studies, and will undoubtedly assist future drug discovery endeavors. [ABSTRACT FROM AUTHOR]

We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active ( K act ) and inactive ( K inact ) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor ( K q-obs ), and 3) the sensitivity constant of the signaling pathway ( K E-obs ). The latter two parameters define the output response of the receptor, and hence, their ratio ( K q-obs / K E ) is a useful measure of system bias. If the cellular system is reasonably stable and the K q-obs and K E-obs values of the signaling pathway are known, the K act and K inact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias. [ABSTRACT FROM AUTHOR]

Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools. [ABSTRACT FROM AUTHOR]