States Of G Protein-coupled Receptors Research Articles

Diffusion and Oligomerization States of the Muscarinic M1 Receptor in Live Cells─The Impact of Ligands and Membrane Disruptors.

G protein-coupled receptors (GPCRs) are a major gateway to cellular signaling, which respond to ligands binding at extracellular sites through allosteric conformational changes that modulate their interactions with G proteins and arrestins at intracellular sites. High-resolution structures in different ligand states, together with spectroscopic studies and molecular dynamics simulations, have revealed a rich conformational landscape of GPCRs. However, their supramolecular structure and spatiotemporal distribution is also thought to play a significant role in receptor activation and signaling bias within the native cell membrane environment. Here, we applied single-molecule fluorescence techniques, including single-particle tracking, single-molecule photobleaching, and fluorescence correlation spectroscopy, to characterize the diffusion and oligomerization behavior of the muscarinic M1 receptor (M1R) in live cells. Control samples included the monomeric protein CD86 and fixed cells, and experiments performed in the presence of different orthosteric M1R ligands and of several compounds known to change the fluidity and organization of the lipid bilayer. M1 receptors exhibit Brownian diffusion characterized by three diffusion constants: confined/immobile (∼0.01 μm2/s), slow (∼0.04 μm2/s), and fast (∼0.14 μm2/s), whose populations were found to be modulated by both orthosteric ligands and membrane disruptors. The lipid raft disruptor C6 ceramide led to significant changes for CD86, while the diffusion of M1R remained unchanged, indicating that M1 receptors do not partition in lipid rafts. The extent of receptor oligomerization was found to be promoted by increasing the level of expression and the binding of orthosteric ligands; in particular, the agonist carbachol elicited a large increase in the fraction of M1R oligomers. This study provides new insights into the balance between conformational and environmental factors that define the movement and oligomerization states of GPCRs in live cells under close-to-native conditions.

A method for structure determination of GPCRs in various states.

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the β2-adrenergic receptor (β2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For β2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.

In situ visualization of opioid and cannabinoid drug effects using phosphosite-specific GPCR antibodies

G protein-coupled receptors (GPCRs) are important signal transducers that are phosphorylated upon activation at intracellular serine and threonine residues. Although antibodies that specifically recognize the phosphorylation state of GPCRs have been available for many years, efficient immunolocalization of phosphorylated receptors in their tissues of origin has not been possible. Here, we show that phosphorylation of receptors is highly unstable during routine immunohistochemical procedures, requiring the use of appropriate phosphatase inhibitors particular during tissue perfusion, post-fixation, and cryoprotection but not during immunostaining of tissue sections. We provide proof of concept using phosphorylation state-specific μ-opioid receptor (MOP) and cannabinoid receptor 1 (CB1) antibodies. Indeed, three of four well-characterized phosphosite-specific MOP antibodies, including pS375-MOP, pT376-MOP, and pT379-MOP, showed robust neuronal immunostaining in brain and spinal cord sections of opioid-treated mice only after inclusion of phosphatase inhibitors. We then extended this approach to the CB1 receptor and demonstrated that one of three newly-generated phosphosite-specific CB1 antibodies, namely pS425-CB1, showed striking staining of fibers and varicosities in brain slices from cannabinoid-treated mice. Although subsequent experiments showed that phospho-CB1 immunostaining was less sensitive to phosphatases, we conclude that the use of phosphatase inhibitors should always be considered in the development of immunohistochemical procedures for new phosphosite-specific GPCR antibodies. In summary, we anticipate that this improved protocol will facilitate the widespread use of phosphorylation state-specific antibodies to monitor the activation of endogenous GPCRs under physiological and pharmacological conditions. Our approach may also prove useful to confirm target engagement of GPCR drug candidates in native tissues.

Activity Map and Transition Pathways of G Protein-Coupled Receptor Revealed by Machine Learning.

Approximately, one-third of all U.S. Food and Drug Administration approved drugs target G protein-coupled receptors (GPCRs). However, more knowledge of protein structure-activity correlation is required to improve the efficacy of the drugs targeting GPCRs. In this study, we developed a machine learning model to predict the activation state and activity level of the receptors with high prediction accuracy. Furthermore, we applied this model to thousands of molecular dynamics trajectories to correlate residue-level conformational changes of a GPCR to its activity level. Finally, the most probable transition pathway between activation states of a receptor can be identified using the state-activity information. In addition, with this model, we can associate the contribution of each amino acid to the activation process. Using this method, we can design drugs that mainly target principal amino acids driving the transition between activation states of GPCRs. Our advanced method is generalizable to all GPCR classes and provides mechanistic insight into the activation mechanism in the receptors.

Measuring the rupture force between adenosine A2A G-protein coupled receptors and G-proteins using axial optical tweezers.

G-protein-coupled receptors (GPCRs) are the largest group of membrane receptors. Here we used axial optical tweezers to measure the rupture force between the adenosine A2A receptor in a nanodisc and its associated G-protein. We also measured the interaction between the receptor and an engineered G-protein, mini-Gs, which is a truncated Gs alpha-subunit that can increase agonist affinity and stabilize the active state of GPCRs. These measurements required us to extend the force range accessible with axial optical tweezers to approximately 50 pN. This involved strengthening the DNA handles to which we attach the G-protein or mini-Gs and improving the force calibration of the axial optical tweezers by employing a twistable worm-like chain (tWLC) model. These advances enabled us to measure the rupture force between the adenosine A2A receptor and the G-proteins under various physiological conditions.

Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor.

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.

Molecular Dynamics Simulations for the Determination of the Characteristic Structural Differences between Inactive and Active States of Wild Type and Mutants of the Orexin2 Receptor.

The orexin2 receptor (OX2R), which is classified as a class A G protein-coupled receptor (GPCR), is the target of our study. We performed over 20 several-microsecond-scale molecular dynamics simulations of the wild type and mutants of OX2R to extract the characteristics of the structural changes taking place in the active state. We introduced mutations that exhibited the stable inactive state and the constitutively active state in class A GPCRs. In these simulations, significant characteristic structural changes were observed in the V3096.40Y mutant, which corresponded to a constitutively active mutant. These conformational changes include the outward movement of the transmembrane helix 6 (TM6) and the inward movement of TM7, which are common structural changes in the activation of GPCRs. In addition, we extracted a suitable index for the quantitative evaluation of the active and inactive states of GPCRs, namely, the inter-atomic distance of Cα atoms between x(3.46) and Y(7.53). The structures of the inactive and active states solved by X-ray crystallography and cryo-electron microscopy can be classified using the inter-atomic distance. Furthermore, we clarified that the inward movement of TM7 requires the swapping of M3056.36 on TM6 and L3677.56 on TM7. Finally, we discussed the structural advantages of TM7 inward movement for GPCR activation.

The Open Question of How GPCRs Interact with GPCR Kinases (GRKs).

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR–GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

Allosteric Modulation of GPCRs of Class A by Cholesterol.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

Probing the Conformation States of Neurotensin Receptor 1 Variants by NMR Site-Directed Methyl Labeling.

G protein‐coupled receptors (GPCRs) are key players in mediating signal transduction across the cell membrane. However, due to their intrinsic instability, many GPCRs are not suitable for structural investigations. Various approaches have been developed in recent years to remedy this situation, ranging from the use of more native membrane mimetics to protein‐stabilization methods. The latter approach typically results in GPCRs that contain various numbers of mutations. However, probing the functionality of such variants by in vitro and in vivo assays is often time consuming. In addition, to validate the suitability of such GPCRs for structural investigations, an assessment of their conformation state is required. NMR spectroscopy has been proven to be suitable to probe the conformation state of GPCRs in solution. Here, by using chemical labeling with an isotope‐labeled methyl probe, we show that the activity and the conformation state of stabilized neurotensin receptor 1 variants obtained from directed evolution can be efficiently assayed in 2D NMR experiments. This strategy enables the quantification of the active and inactive conformation states and the derivation of an estimation of the basal as well as agonist‐induced activity of the receptor. Furthermore, this assay can be used as a readout when re‐introducing agonist‐dependent signaling into a highly stabilized, and thus rigidified, receptor by mutagenesis. This approach will be useful in cases where low production yields do not permit the addition of labeled compounds to the growth medium and where 1D NMR spectra of selectively 19F‐labeled receptors are not sufficient to resolve signal overlap for a more detailed analysis.

Unbiased topological parameters to characterize the signatures of GPCR conformational changes

G protein coupled receptors (GPCRs) are integral membrane proteins that allow a cell to convert extracellular stimuli like light, small molecules, peptides, and proteins into intracellular signals. These receptors are conformationally very malleable, which is necessary for their different functional states. Recent developments in the structural biology of membrane proteins and utilization of other biophysical techniques for GPCRs is generating rich evidence of the existence of multiple receptor conformations. Different receptor conformations are thought to be responsible for enabling distinct intracellular signaling outcomes. The structures are now being generated for the inactive, active, and intermediate states for many GPCRs, however, there is no unbiased way to characterize the functional states of GPCRs. We are developing general higher‐order GPCR topology parameters (TMHTOP2) that can find functional‐state specific structural signatures and can be applied to any alpha‐helical transmembrane protein. The newly developed TMHTOP2 topology parameters were able to identify unique conformational signatures of the pre‐active state of the adenosine A2A receptor compared to its inactive and active states, that can now be used to identify the pre‐active states of other receptors as well. This method can be used in identifying and classifying structures from experiments or simulations as active, inactive, and intermediates.

Prediction of Conformation Specific Thermostabilizing Mutations for Class A G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are highly flexible and prone to denaturation during protein extraction in detergents and purification. This poses a huge challenge to purify a conformationally homogeneous solution of GPCRs. Thermostabilizing mutations have been used widely to purify and obtain crystal structures of several GPCRs. However, identifying thermostabilizing mutations for GPCRs remains a tedious and expensive task as they are not transferable even among closely related GPCRs. Additionally, the mutations stabilizing one conformational state of a GPCR do not always stabilize other conformational state(s) of the same GPCR. Previously we developed a computational method, LiticonDesign, for rapid prediction of thermostabilizing mutations for a specific GPCR conformation. In this study, we have used LiticonDesign to predict thermostabilizing mutations for the agonist bound active-intermediate state of the human adenosine receptor (A2AR) using the structure of the inactive state of the same GPCR and vice versa. Our study shows that the thermostable mutation predictions using LiticonDesign, for an active-intermediate state of a GPCR (A2AR in our case), requires a homology model that is derived from an active/active-intermediate state GPCR structure as a template. Similarly, the homology models derived from inactive state GPCR conformations are better in predicting the thermostable mutations for the inactive state of A2AR. Overall, LiticonDesign method is not only efficient in predicting thermostabilizing mutations for a given GPCR sequence but also can recover conformation specific mutations for a state of interest, if a suitable starting structure of desired conformation is chosen.

Mutations in the NPxxY motif stabilize pharmacologically distinct conformational states of the α1B- and β2-adrenoceptors.

G protein-coupled receptors (GPCRs) convert extracellular stimuli to intracellular responses that regulate numerous physiological processes. Crystallographic and biophysical advances in GPCR structural analysis have aided investigations of structure-function relationships that clarify our understanding of these dynamic receptors, but the molecular mechanisms associated with activation and signaling for individual GPCRs may be more complex than was previously appreciated. Here, we investigated the proposed water-mediated, hydrogen-bonded activation switch between the conserved NPxxY motif on transmembrane helix 7 (TMH7) and a conserved tyrosine in TMH5, which contributes to α1B-adrenoceptor (α1B-AR) and β2-AR activation. Disrupting this bond by mutagenesis stabilized the α1B-AR and the β2-AR in inactive-state conformations, which displayed decreased agonist potency for stimulating downstream IP1 and cAMP signaling, respectively. Compared to that for wild-type receptors, agonist-mediated β-arrestin recruitment was substantially reduced or abolished for all α1B-AR and β2-AR inactive-state mutants. However, the inactive-state β2-ARs exhibited decreased agonist affinity, whereas the inactive-state α1B-ARs had enhanced agonist affinity. Conversely, antagonist affinity was unchanged for inactive-state conformations of both α1B-AR and β2-AR. Removing the influence of agonist affinity on agonist potency gave a measure of signaling efficacy, which was markedly decreased for the α1B-AR mutants but little altered for the β2-AR mutants. These findings highlight the pharmacological heterogeneity of inactive-state GPCR conformations, which may facilitate the rational design of drugs that target distinct conformational states of GPCRs.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets 1. Despite detailed structural information however the effects of lipids on the receptors themselves, or on their downstream coupling to G proteins, are ill defined. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We demonstrate preferential binding of phosphatidylinositol 4,5 bisphosphate (PIP2) over related lipids and confirm hotspots for PIP2 binding on the intracellular face of the receptors. Endogenous lipids were also observed bound directly to the entire adenosine A2A receptor (A2AR) trimeric Gαsβγ protein complex in the gas phase. Recruiting engineered Gα subunits (mini-Gs, i, 12) 2 we found that the β1AR: mini-Gαs complex is stabilised by the binding of two PIP2 molecules. By contrast PIP2 did not stabilise coupling between β1AR and other Gα subunits (mini- Gαi, 12) or a high affinity nanobody. Other endogenous lipids that bound to receptors showed no effect on coupling, highlighting the specificity of PIP2. Potential of mean force calculations and increased GTP turnover of the activated neurotensin receptor: trimeric Gαiβγ complex in the presence of PIP2 further support the specific effect of PIP2 on coupling. Together our results identify key residues on cognate Gα subunits that mediate simultaneous PIP2 bridging interactions between basic residues on class A receptors, which do not correspond with those on class B GPCRs. By uncovering the effects of lipid modulation of receptors we highlight consequences for understanding function, G protein selectivity and drug targeting of class A GPCRs.

The Molecular Basis of G Protein-Coupled Receptor Activation.

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

Small-Molecule Positive Allosteric Modulators of the β2-Adrenoceptor Isolated from DNA-Encoded Libraries.

Conventional drug discovery efforts at the β2-adrenoceptor (β2AR) have led to the development of ligands that bind almost exclusively to the receptor’s hormone-binding orthosteric site. However, targeting the largely unexplored and evolutionarily unique allosteric sites has potential for developing more specific drugs with fewer side effects than orthosteric ligands. Using our recently developed approach for screening G protein–coupled receptors (GPCRs) with DNA-encoded small-molecule libraries, we have discovered and characterized the first β2AR small-molecule positive allosteric modulators (PAMs)—compound (Cmpd)-6 [(R)-N-(4-amino-1-(4-(tert-butyl)phenyl)-4-oxobutan-2-yl)-5-(N-isopropyl-N-methylsulfamoyl)-2-((4-methoxyphenyl)thio)benzamide] and its analogs. We used purified human β2ARs, occupied by a high-affinity agonist, for the affinity-based screening of over 500 million distinct library compounds, which yielded Cmpd-6. It exhibits a low micro-molar affinity for the agonist-occupied β2AR and displays positive cooperativity with orthosteric agonists, thereby enhancing their binding to the receptor and ability to stabilize its active state. Cmpd-6 is cooperative with G protein and β-arrestin1 (a.k.a. arrestin2) to stabilize high-affinity, agonist-bound active states of the β2AR and potentiates downstream cAMP production and receptor recruitment of β-arrestin2 (a.k.a. arrestin3). Cmpd-6 is specific for the β2AR compared with the closely related β1AR. Structure–activity studies of select Cmpd-6 analogs defined the chemical groups that are critical for its biologic activity. We thus introduce the first small-molecule PAMs for the β2AR, which may serve as a lead molecule for the development of novel therapeutics. The approach described in this work establishes a broadly applicable proof-of-concept strategy for affinity-based discovery of small-molecule allosteric compounds targeting unique conformational states of GPCRs.

Professor Thue W. Schwartz is the BCPT Nordic Prize Winner 2017 in Basic & Clinical Pharmacology & Toxicology.

Professor Thue W. Schwartz is the BCPT Nordic Prize Winner 2017 in Basic & Clinical Pharmacology & Toxicology.

Molecular modelling of human 5-hydroxytryptamine receptor (5-HT2A) and virtual screening studies towards the identification of agonist and antagonist molecules

The serotonin receptors, also known as 5-hydroxytryptamine (5-HT) receptors, are a group of G protein-coupled receptors (GPCRs) and ligand-gated ion channels found in the central and peripheral nervous systems. GPCRs have a characteristic feature of activating different signalling pathways upon ligand binding and these ligands display several efficacy levels to differentially activate the receptor. GPCRs are primary drug targets due to their central role in several signal transduction pathways. Drug design for GPCRs is also most challenging due to their inherent promiscuity in ligand recognition, which gives rise to several side effects of existing drugs. Here, we have performed the ligand interaction study using the two prominent states of GPCR, namely the active and inactive state of the 5-HT2A receptor. Active state of 5-HT2A receptor model enhances the understanding of conformational difference which influences the ligand-binding site. A 5-HT2A receptor active state model was constructed by homology modelling using active state β2-adrenergic receptor (β2-AR). In addition, virtual screening and docking studies with both active and inactive state models reveal potential small molecule hits which could be considered as agonist-like and antagonist-like molecules. The results from the all-atom molecular dynamics simulations further confirmed that agonists and antagonists interact in different modes with the receptor.

Cell-based and virtual fragment screening for adrenergic α2C receptor agonists

Fragment-based drug discovery has emerged as an alternative to conventional lead identification and optimization strategies generally supported by biophysical detection techniques. Membrane targets like G protein-coupled receptors (GPCRs), however, offer challenges in lack of generic immobilization or stabilization methods for the dynamic, membrane-bound supramolecular complexes. Also modeling of different functional states of GPCRs proved to be a challenging task. Here we report a functional cell-based high concentration screening campaign for the identification of adrenergic α2C receptor agonists compared with the virtual screening of the same ligand set against an active-like homology model of the α2C receptor. The conventional calcium mobilization-based assay identified active fragments with a similar incidence to several other reported fragment screens on GPCRs. 16 out of 3071 screened fragments turned out as specific ligands of α2C, two of which were identified by virtual screening as well and several of the hits possessed surprisingly high affinity and ligand efficiency. Our results indicate that in vitro biological assays can be utilized in the fragment hit identification process for GPCR targets.

Obtaining structural and functional information for GPCRs using the substituted-cysteine accessibility method (SCAM).

G-protein coupled receptors (GPCRs) are proteins of the plasma membrane, which are characterized by seven membrane-spanning segments (TMs). GPCRs play an important role in almost all of our physiological and pathophysiological conditions by interacting with a large variety of ligands and stimulating different G-proteins and signaling cascades. By playing a key role in the function of our body and being involved in the pathophysiology of many disorders, GPCRs are very important therapeutic targets. Determination of the structure and function of GPCRs could advance the design of novel receptor-specific drugs against various diseases. A powerful method to obtain structural and functional information for GPCRs is the cysteine substituted accessibility method (SCAM). SCAM is used to systematically map the TM residues of GPCRs and determine their functional role. SCAM can also be used to determine differences in the structures of the TMs in different functional states of GPCRs.