Prototypical G Protein-coupled Receptors Research Articles

Biased Signaling in Mutated Variants of β2-Adrenergic Receptor: Insights from Molecular Dynamics Simulations.

The molecular basis of receptor bias in G protein-coupled receptors (GPCRs) caused by mutations that preferentially activate specific intracellular transducers over others remains poorly understood. Two experimentally identified biased variants of β2-adrenergic receptors (β2AR), a prototypical GPCR, are a triple mutant (T68F, Y132A, and Y219A) and a single mutant (Y219A); the former bias the receptor toward the β-arrestin pathway by disfavoring G protein engagement, while the latter induces G protein signaling explicitly due to selection against GPCR kinases (GRKs) that phosphorylate the receptor as a prerequisite of β-arrestin binding. Though rigorous characterizations have revealed functional implications of these mutations, the atomistic origin of the observed transducer selectivity is not clear. In this study, we investigated the allosteric mechanism of receptor bias in β2AR using microseconds of all-atom Gaussian accelerated molecular dynamics (GaMD) simulations. Our observations reveal distinct rearrangements in transmembrane helices, intracellular loop 3, and critical residues R1313.50 and Y3267.53 in the conserved motifs D(E)RY and NPxxY for the mutant receptors, leading to their specific transducer interactions. Moreover, partial dissociation of G protein from the receptor core is observed in the simulations of the triple mutant in contrast to the single mutant and wild-type receptor. The reorganization of allosteric communications from the extracellular agonist BI-167107 to the intracellular receptor-transducer interfaces drives the conformational rearrangements responsible for receptor bias in the single and triple mutants. The molecular insights into receptor bias of β2AR presented here could improve the understanding of biased signaling in GPCRs, potentially opening new avenues for designing novel therapeutics with fewer side-effects and superior efficacy.

Target immobilization on glass microfiber membranes as a label-free strategy for hit identification.

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V2R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V2R were immobilized on the surface of a glass microfiber (GMF) coated with O6-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V2R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V2R ligand treated with SNAP-V2R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the Ki value determined by displacement assays. Furthermore, a Ki prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V2R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V2R@GMF-BG was incubated with the total extract, the ligand that binds to V2R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V2R antagonist. This V2R-immobilized GMF platform can help determine the affinity of V2R-binding hit compounds and screen the compounds efficiently and accurately.

Elucidation of a dynamic interplay between a beta-2 adrenergic receptor, its agonist, and stimulatory G protein

G protein-coupled receptors (GPCRs) represent the largest group of membrane receptors for transmembrane signal transduction. Ligand-induced activation of GPCRs triggers G protein activation followed by various signaling cascades. Understanding the structural and energetic determinants of ligand binding to GPCRs and GPCRs to G proteins is crucial to the design of pharmacological treatments targeting specific conformations of these proteins to precisely control their signaling properties. In this study, we focused on interactions of a prototypical GPCR, beta-2 adrenergic receptor (β2AR), with its endogenous agonist, norepinephrine (NE), and the stimulatory G protein (Gs). Using molecular dynamics (MD) simulations, we demonstrated the stabilization of cationic NE, NE(+), binding to β2AR by Gs protein recruitment, in line with experimental observations. We also captured the partial dissociation of the ligand from β2AR and the conformational interconversions of Gs between closed and open conformations in the NE(+)-β2AR-Gs ternary complex while it is still bound to the receptor. The variation of NE(+) binding poses was found to alter Gs α subunit (Gsα) conformational transitions. Our simulations showed that the interdomain movement and the stacking of Gsα α1 and α5 helices are significant for increasing the distance betweenthe Gsαand β2AR, which may indicate apartial dissociation ofGsαThe distance increase commences when Gsα is predominantly in an open state and can be triggered by the intracellular loop 3 (ICL3) of β2AR interacting with Gsα, causing conformational changes of the α5 helix. Our results help explain molecular mechanisms of ligand and GPCR-mediated modulation of G protein activation.

Elucidation of a dynamic interplay between guanine nucleotide and the ternary complex of a beta-adrenergic receptor, its agonist and stimulatory G protein.

Heterotrimeric G proteins, composed of α, β, and γ subunits, are molecular switches which can be regulated to either turn on or turn off based on their binding to transmembrane G-protein-coupled receptors (GPCRs) and guanine nucleotides, GTP or GDP. Coupled with their receptors, G proteins play a critical role in determining the specificity and temporal characteristics of the cellular responses of a diverse array of extracellular stimuli. G proteins decouple from GPCRs upon GTP binding to their Gα subunits, allowing both Gα and Gβγ to interact with downstream effector proteins. What is the role of GDP and GTP binding in the activation and dissociation of G proteins and what Gα conformational changes does GTP/GDP binding trigger? Answering these questions requires detailed time-resolved structural information. In this study we focused on interactions of GTP and GDP with stimulatory G protein (Gs), which is coupled with a prototypical GPCR, the beta-adrenergic receptor (βAR) using Rosetta modeling and all-atom molecular dynamics simulations. We found that GTP/GDP binding indirectly alters Gsα conformation by causing inter-domain rearrangement while directly causing α5 helix tilting, thus favoring Gsα dissociation. GTP/GDP binding also enhances correlations between Gsα inter-domain arrangement and Gsα conformational changes. Specifically, we found negative correlation between α5 titling angle and α1–α5 stacking which was different from the apo Gs protein case we observed previously. In summary, we analyzed conformational specificities of Gs protein–guanine nucleotide interactions and found correlations between Gs dissociation and its internal domain rearrangement. This provides intrinsic molecular insights into the understanding of G protein activation and may be beneficial for the design of safe and efficacious pharmaceuticals in precisely controlling the signaling properties of βARs.

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonizts and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

A Chemical Biological Approach to Study G Protein-Coupled Receptors: Labeling the Adenosine A1 Receptor Using an Electrophilic Covalent Probe.

G protein-coupled receptors (GPCRs) have been known for decades as attractive drug targets. This has led to the development and approval of many ligands targeting GPCRs. Although ligand binding effects have been studied thoroughly for many GPCRs, there are multiple aspects of GPCR signaling that remain poorly understood. The reasons for this are the difficulties that are encountered upon studying GPCRs, for example, a poor solubility and low expression levels. In this work, we have managed to overcome some of these issues by developing an affinity-based probe for a prototypic GPCR, the adenosine A1 receptor (A1AR). Here, we show the design, synthesis, and biological evaluation of this probe in various biochemical assays, such as SDS-PAGE, confocal microscopy, and chemical proteomics.

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α2A-adrenergic receptor (α2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β2-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.

AKAP12 Modulates β‐arrestin2 Recruitment to the β2‐Adrenergic Receptors

BackgroundG‐Protein Coupled Receptors (GPCRs) are central mediators of signaling cascades in many biological systems. GPCRs internalization rates vary based on the presence/absence of its ligand and the type of the receptor. β2‐Adrenergic Receptor (β2AR) is a prototypical GPCR, reaching its maximum in internalization within ~10 minutes after ligand treatment. One regulator of β2AR internalization is β‐arrestin2. GPCR–β‐arrestin binding is biphasic where the phosphorylated carboxyl terminus of GPCRs docks to the N‐domain of β‐arrestin first, and then the seven transmembrane core of the receptor engages with β‐arrestin. However, it has been discovered that the core interaction is dispensable for receptor endocytosis. AKAP12 is a large scaffolding protein that was reported to be essential in agonist‐induced internalization and resensitization of β2AR. Our central hypothesis is that higher expression levels of AKAP12 may enhance β‐arrestin2 recruitment to the β2AR and subsequently increase β2AR internalization.MethodsHEK293 cells were grouped into three groups: [1] Controls: endogenous AKAP12 expression, [2] Overexpression (OX): transiently overexpressing human AKAP12, and [3] Knockdown (KD): treated with human AKAP12 siRNA. β‐arrestin2 recruitment to the receptor and β2AR internalization was evaluated using the Nanobit luciferase system. For each assay, we tested four β2AR‐β‐arrestin2 or Early Endosome‐β2AR orientations. Orientation pairs with the highest luminescence signals were used for the experiments to obtain maximum sensitivity. At 48 hours post‐transfection, Nanobit assays were performed by recording baseline luminescence for 10 minutes. Next, 10µM epinephrine was added and luminescence measurements were recorded immediately and then once every minute for 1 hour. Results are represented as Luminescence Fold Induction (LFI). Receptor trafficking to early endosomes post epinephrine treatment was further confirmed using the Proximity Ligation Assay (PLA).ResultsHEK cells, OX AKAP12 [9 fold higher] had a significantly lower β‐arrestin2 recruitment to the β2AR (2.26±0.39, LFI) as compared to controls (7.47±1.12, LFI; p=0.0004). AKAP12 KD [AKAP12 ~80% silenced] had a significantly higher β‐arrestin2 recruitment to the β2AR (10.35±0.56, LFI) as compared to both control and OX AKAP12 groups (p=0.035 and p<0.0001), respectively. At 15 minutes post epinephrine addition, β2AR trafficking to early endosomes was significantly lower in the OX AKAP12 group (1.82±0.22, LFI), as compared to the control (2.47±0.09, LFI; p=0.01), while the KD AKAP12 group had a significantly higher internalization (2.98±0.14, LFI), as compared to control and OX AKAP12 groups (p=0.005 and p=0.0003), respectively. Our PLA data further confirmed that the OX AKAP12 group displayed significantly lower β2AR trafficking as defined by its decreased interaction with Early Endosome Antigen 1 (EEA1), as compared to the control (p= 0.0451).ConclusionsContrary to previously thought, higher AKAP12 expression levels may interfere with β2AR trafficking to early endosomes partially through inhibiting β‐arrestin2 recruitment to the β2AR.

Arrestin Facilitates Rhodopsin Dephosphorylation in Vivo.

Deactivation of G-protein-coupled receptors (GPCRs) involves multiple phosphorylations followed by arrestin binding, which uncouples the GPCR from G-protein activation. Some GPCRs, such as rhodopsin, are reused many times. Arrestin dissociation and GPCR dephosphorylation are key steps in the recycling process. In vitro evidence suggests that visual arrestin (ARR1) binding to light-activated, phosphorylated rhodopsin hinders dephosphorylation. Whether ARR1 binding also affects rhodopsin dephosphorylation in vivo is not known. We investigated this using both male and female mice lacking ARR1. Mice were exposed to bright light and placed in darkness for different periods of time, and differently phosphorylated species of rhodopsin were assayed by isoelectric focusing. For WT mice, rhodopsin dephosphorylation was nearly complete by 1 h in darkness. Surprisingly, we observed that, in the Arr1 KO rods, rhodopsin remained phosphorylated even after 3 h. Delayed dephosphorylation in Arr1 KO rods cannot be explained by cell stress induced by persistent signaling, since it is not prevented by the removal of transducin, the visual G-protein, nor can it be explained by downregulation of protein phosphatase 2A, the putative rhodopsin phosphatase. We further show that cone arrestin (ARR4), which binds light-activated, phosphorylated rhodopsin poorly, had little effect in enhancing rhodopsin dephosphorylation, whereas mice expressing binding-competent mutant ARR1-3A showed a similar time course of rhodopsin dephosphorylation as WT. Together, these results reveal a novel role of ARR1 in facilitating rhodopsin dephosphorylation in vivo.SIGNIFICANCE STATEMENT G-protein-coupled receptors (GPCRs) are transmembrane proteins used by cells to receive and respond to a broad range of extracellular signals that include neurotransmitters, hormones, odorants, and light (photons). GPCR signaling is terminated by two sequential steps: phosphorylation and arrestin binding. Both steps must be reversed when GPCRs are recycled and reused. Dephosphorylation, which is required for recycling, is an understudied process. Using rhodopsin as a prototypical GPCR, we discovered that arrestin facilitated rhodopsin dephosphorylation in living mice.

Implications of ligand-receptor binding kinetics on GLP-1R signalling

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins and in recent years there has been a growing appreciation of the importance in understanding temporal aspects of GPCR behaviour, including the kinetics of ligand binding and downstream receptor mediated signalling. Class B1 GPCRs are activated by peptide agonists and are validated therapeutic targets for numerous diseases. However, the kinetics of ligand binding and how this is linked to downstream activation of signalling cascades is not routinely assessed in development of peptide agonists for this receptor class. The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical class B1 GPCR and a validated target for treatment of global health burdens, including type 2 diabetes and obesity. In this study we examined the kinetics of different steps in GLP-1R activation and subsequent cAMP production mediated by a series of GLP-1R peptide agonists, including the ligand-receptor interaction, ligand-receptor-mediated G protein engagement and conformational change and cAMP production. Our results revealed GLP-1R peptide agonist dissociation kinetics (Koff), but not association kinetics (Kon), were positively correlated with the onset of receptor-G protein coupling/conformational change, onset of cAMP production and duration of cAMP signalling. Thus, this study advances the understanding of molecular events that couple GLP-1R ligand binding to intracellular signaling, with the findings likely to have implications for mechanistic understanding of agonist action at other related class B1 GPCRs.

Biphasic activation of \u03b2-arrestin 1 upon interaction with a GPCR revealed by methyl-TROSY NMR

β-arrestins (βarrs) play multifaceted roles in the function of G protein-coupled receptors (GPCRs). βarrs typically interact with phosphorylated C-terminal tail (C tail) and transmembrane core (TM core) of GPCRs. However, the effects of the C tail- and TM core-mediated interactions on the conformational activation of βarrs have remained elusive. Here, we show the conformational changes for βarr activation upon the C tail- and TM core-mediated interactions with a prototypical GPCR by nuclear magnetic resonance (NMR) spectroscopy. Our NMR analyses demonstrated that while the C tail-mediated interaction alone induces partial activation, in which βarr exists in equilibrium between basal and activated conformations, the TM core- and the C tail-mediated interactions together completely shift the equilibrium toward the activated conformation. The conformation-selective antibody, Fab30, promotes partially activated βarr into the activated-like conformation. This plasticity of βarr conformation in complex with GPCRs engaged in different binding modes may explain the multifunctionality of βarrs.

Genome Mining-Based Discovery of Blenny Fish-Derived Peptides Targeting the Mouse κ-Opioid Receptor.

Over the past years, peptides have attracted increasing interest for G protein-coupled receptor (GPCR) drug discovery and development. Peptides occupy a unique chemical space that is not easily accessible for small molecules and antibodies and provide advantages over these ligand classes such as lower toxicity and higher selectivity. The κ-opioid receptor (KOR) is a prototypic GPCR and an appealing therapeutic target for the development of safer and more effective analgesics. Recently, peptides have emerged as analgesic drug candidates with improved side effect profiles. We have previously identified plant-derived peptides, which activate KOR. Based on this precedent, here we relied on publicly available databases to discover novel KOR peptide ligands by genome mining. Using human preprodynorphin as a query, we identified blenny fish-derived peptides, referred to as blenniorphins, capable of binding to and activating KOR with nanomolar affinity and potency, respectively. Additionally, the blenniorphins altered β-arrestin-2 recruitment at the KOR. Our study demonstrates the utility of genome mining to identify peptide GPCR ligands with intriguing pharmacological properties and unveils the potential of blenny fishes as a source for novel KOR ligands.

Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

Effects of Soft Matter on G-Protein-Coupled Receptor Activation

G protein-coupled receptors (GPCRs) constitute both the largest class of medicinal targets and the largest family of human membrane proteins. Current three-dimensional structures have shown the existence of many water molecules immersed inside GPCRs. However, the functional role of these water molecules has remained uncertain. Our previous work has shown that water modulates the activation of visual rhodopsin, a prototypical GPCR [1]. Here we expanded our approach to further investigate how the conformational energetics of GPCR activation are modulated by water using a series of osmolytes that included polyethylene glycols (PEGs), dextrans, polyvinylpyrrolidones (PVPs), and their monomers. We hypothesized that osmotic pressure affects the thermodynamic stability of conformational substates of photoactivated rhodopsin. Accordingly, we changed the osmotic stress on rhodopsin using different concentrations of various polymer solutes. Shifting of the metarhodopsin equilibrium due to the osmotic pressure was probed using UV-visible spectroscopy. The larger osmolytes (e.g., PEG 1500-6000) promote the inactive metarhodopsin-I (MI) state, while the small organic osmolytes such as smaller molar mass PEGs, their monomers, and small sugars increase the active metarhodopsin-II (MII) fraction. These size effects are a result of the degree of penetration of osmolytes into the protein core of rhodopsin. Small polymers penetrate the protein core and favor the open MII state of rhodopsin, while the large polymers behave like ideal osmolytes and dehydrate rhodopsin. Most interestingly, PEGs and dextrans with extremely large molar masses deviated in their trends from other large polymers (PEGs), which we attribute to a combined result of crowding and penetration effects. The results provide us with new insights into the interplay between osmotic stress, penetration, and crowding effects. Our findings further the understanding of the vital role of water in the GPCR activation mechanisms. [1] U. Chawla et al. (2020) Angew.Chem.Int.Ed. doi:10.1002/anie.202003342.

Structures of the glucocorticoid-bound adhesion receptor GPR97-Go complex.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available1-3. Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97)4-6, a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-Go complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W6.53, which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the Go protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the Go protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

Spatiotemporal Characterization of GPCR Activity and Function during Endosomal Trafficking Pathway.

G protein-coupled receptor (GPCR) is activated by extracellular signals. After their function at plasma membrane, GPCRs are internalized to be desensitized, while emerging evidence suggests that some GPCRs maintain their activity even after internalization. The endosomal trafficking pathway of a prototypic GPCR, β adrenergic receptor 2 (B2AR), is in the range of several hours, however, spatiotemporal B2AR activity during this long-term endosomal trafficking pathway has not been characterized yet. Here, we analyze an agonist-induced real-time B2AR activity and its downstream function at the level of individual vesicles, utilizing a fluorescence resonance energy transfer (FRET)-based B2AR biosensor and cAMP reporters tethered at different trafficking stages of endosomes. Our results report that the internalized B2ARs sustain the activity and maintain the production of cAMP for several hours during the endosomal trafficking pathway. Temporal kinetics of B2AR activity is mathematically well explained by our active-vesicle population model modified from the Ricker model. Therefore, our GPCR monitoring system and a new kinetics model can be applied to understand the spatiotemporal GPCR activity and its downstream function during the endosomal trafficking pathway.

Heterodimerization With 5-HT2BR Is Indispensable for β2AR-Mediated Cardioprotection.

The β2-adrenoceptor (β2-AR), a prototypical GPCR (G protein-coupled receptor), couples to both Gs and Gi proteins. Stimulation of the β2-AR is beneficial to humans and animals with heart failure presumably because it activates the downstream Gi-PI3K-Akt cell survival pathway. Cardiac β2-AR signaling can be regulated by crosstalk or heterodimerization with other GPCRs, but the physiological and pathophysiological significance of this type of regulation has not been sufficiently demonstrated. Here, we aim to investigate the potential cardioprotective effect of β2-adrenergic stimulation with a subtype-selective agonist, (R,R')-4-methoxy-1-naphthylfenoterol (MNF), and to decipher the underlying mechanism with a particular emphasis on the role of heterodimerization of β2-ARs with another GPCR, 5-hydroxytryptamine receptors 2B (5-HT2BRs). Using pharmacological, genetic and biophysical protein-protein interaction approaches, we studied the cardioprotective effect of the β2-agonist, MNF, and explored the underlying mechanism in both in vivo in mice and cultured rodent cardiomyocytes insulted with doxorubicin, hydrogen peroxide (H2O2) or ischemia/reperfusion. In doxorubicin (Dox)-treated mice, MNF reduced mortality and body weight loss, while improving cardiac function and cardiomyocyte viability. MNF also alleviated myocardial ischemia/reperfusion injury. In cultured rodent cardiomyocytes, MNF inhibited DNA damage and cell death caused by Dox, H2O2 or hypoxia/reoxygenation. Mechanistically, we found that MNF or another β2-agonist zinterol markedly promoted heterodimerization of β2-ARs with 5-HT2BRs. Upregulation of the heterodimerized 5-HT2BRs and β2-ARs enhanced β2-AR-stimulated Gi-Akt signaling and cardioprotection while knockdown or pharmacological inhibition of the 5-HT2BR attenuated β2-AR-stimulated Gi signaling and cardioprotection. These data demonstrate that the β2-AR-stimulated cardioprotective Gi signaling depends on the heterodimerization of β2-ARs and 5-HT2BRs.

C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gβγ and β-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

A combination of machine learning and infrequent metadynamics to efficiently predict kinetic rates, transition states, and molecular determinants of drug dissociation from G protein-coupled receptors.

Determining the drug-target residence time (RT) is of major interest in drug discovery given that this kinetic parameter often represents a better indicator of in vivo drug efficacy than binding affinity. However, obtaining drug-target unbinding rates poses significant challenges, both computationally and experimentally. This is particularly palpable for complex systems like G Protein-Coupled Receptors (GPCRs) whose ligand unbinding typically requires very long timescales oftentimes inaccessible by standard molecular dynamics simulations. Enhanced sampling methods offer a useful alternative, and their efficiency can be further improved by using machine learning tools to identify optimal reaction coordinates. Here, we test the combination of two machine learning techniques, automatic mutual information noise omission and reweighted autoencoded variational Bayes for enhanced sampling, with infrequent metadynamics to efficiently study the unbinding kinetics of two classical drugs with different RTs in a prototypic GPCR, the μ-opioid receptor. Dissociation rates derived from these computations are within one order of magnitude from experimental values. We also use the simulation data to uncover the dissociation mechanisms of these drugs, shedding light on the structures of rate-limiting transition states, which, alongside metastable poses, are difficult to obtain experimentally but important to visualize when designing drugs with a desired kinetic profile.

Sodium ions allosterically modulate the M2 muscarinic receptor

G protein coupled receptors (GPCRs) play a key role in the vast majority of cellular signal transduction processes. Previous experimental evidence has shown that sodium ion (Na+) allosterically modulate several class A GPCRs and theoretical studies suggested that the same also holds true for muscarinic receptors. Here we examined, using Xenopus oocytes as an expression system, the effect of Na+ on a prototypical GPCR, the M2 muscarinic receptor (M2R). We found that removal of extracellular Na+ resulted in a decrease in the potency of ACh toward the M2R and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. We further show that this allosteric effect of Na+ does not underlie the voltage-dependence of this receptor.