Prototypical G Protein-coupled Receptors Research Articles

Ligand chain length drives activation of lipid G protein-coupled receptors

Sphingosine-1-phosphate (S1P) is a lipid mediator that can activate five cell membrane G protein-coupled receptors (GPCRs) which carry a variety of essential functions and are promising drug targets. S1P is composed of a polar zwitterionic head-group and a hydrophobic alkyl chain. This implies an activation mechanism of its cognate receptor that must be significantly different from what is known for prototypical GPCRs (ie receptor to small hydrophilic ligands). Here we aim to identify the structural features responsible for S1P agonism by combining molecular dynamics simulations and functional assays using S1P analogs of different alkyl chain lengths. We propose that high affinity binding involves polar interactions between the lipid head-group and receptor side chains while activation is due to hydrophobic interactions between the lipid tail and residues in a distinct binding site. We observe that ligand efficacy is directly related to alkyl chain length but also varies with receptor subtypes in correlation with the size of this binding pocket. Integrating experimental and computational data, we propose an activation mechanism for the S1P receptors involving agonist-induced conformational events that are conserved throughout class A GPCRs.

Membrane cholesterol access into a G-protein-coupled receptor

Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol’s impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

The Effect of Membrane Polyunsaturated Fatty Acids on Receptor Partitioning to Ordered Domains

Numerous G protein-coupled receptors (GPCRs) contain cholesterol binding sites, and are therefore assumed to partition into cholesterol-rich ordered membrane domains to function properly. Neurotransmitter receptors - many of which are GPCRs and located mainly in the nervous system - are responsible for regulating our behavior and mood, and their compromised signaling often results in neurodegenerative diseases. Interestingly, many such diseases are associated with a decrease in polyunsaturated fatty acid (PUFA) levels in the brain.The PUFAs, usually paired with a saturated lipid tail in hybrid lipids, are known to favorably interact with receptors. This might - in addition to modulating the activation of individual receptors - also affect larger scale GPCR organization, including their partitioning between ordered and disordered membrane domains. However, such effects are very tedious to be studied experimentally, and therefore the determinants of membrane protein partitioning remain currently elusive.Here, using multi-scale computer simulations and free energy calculations, we show that the association of the adenosine A2A receptor, a prototypical GPCR, with the PUFA tail of a hybrid lipid drastically affects its partitioning. The strong PUFA-receptor interaction results in the formation of a well defined lipid corona, whose outer shell is consists of the saturated tails of a hybrid lipid. This cushion softens the membrane perturbation caused by the rough receptor surface and hence makes it more compatible with ordered membrane domains. Interestingly, this favorable solvation by PUFAs seems to be characteristic only for membrane proteins consisting of helical bundles.Our results reveal an unexpected connection between PUFAs and disease: A decrease in the brain PUFA levels might affect GPCR partitioning, thereby compromising their function and perhaps eventually leading to disease. This finding, together with our previous study revealing the role of PUFAs on GPCR oligomerization, highlights the complex interplay of brain membrane lipid composition and neurotransmitter receptor function.

Kinetics of G Protein-Coupled Receptor Dimerization from Markov State Model Analysis of Coarse-Grained Simulations

Experimental and computational studies published over the past decade have mostly focused on the structural and thermodynamic properties of putative G Protein-Coupled Receptor(GPCR) dimers. Although equally crucial for addressing the role of dimerization in GPCR function, a thorough description of the timescales required to form different dimeric interfaces has yet to be provided. In this study, we approach this question by applying Markov State Model analysis to coarse grained simulations of a prototypic GPCR, the mu-opioid receptor (MOR), carried out in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol membrane model using an adaptive sampling strategy. We identify several dimerization interfaces of MOR grouped in four macrostates with significantly different dimerization rates, and characterize the role of lipid dynamics, protein-lipid and specific protein-protein interactions in modulating the kinetic properties of receptor dimerization. Furthermore, we discuss the interplay between receptor activation and dimer formation by comparing the results obtained for the activated and inactive receptor conformations of MOR, and observe substantial differences in the dimerization kinetics for the two conformations.

Regulation of α2B-Adrenergic Receptor Cell Surface Transport by GGA1 and GGA2.

The molecular mechanisms that control the targeting of newly synthesized G protein-coupled receptors (GPCRs) to the functional destinations remain poorly elucidated. Here, we have determined the role of Golgi-localized, γ-adaptin ear domain homology, ADP ribosylation factor-binding proteins 1 and 2 (GGA1 and GGA2) in the cell surface transport of α2B-adrenergic receptor (α2B-AR), a prototypic GPCR, and studied the underlying mechanisms. We demonstrated that knockdown of GGA1 and GGA2 by shRNA and siRNA significantly reduced the cell surface expression of inducibly expressed α2B-AR and arrested the receptor in the perinuclear region. Knockdown of each GGA markedly inhibited the dendritic expression of α2B-AR in primary cortical neurons. Consistently, depleting GGA1 and GGA2 attenuated receptor-mediated signal transduction measured as ERK1/2 activation and cAMP inhibition. Although full length α2B-AR associated with GGA2 but not GGA1, its third intracellular loop was found to directly interact with both GGA1 and GGA2. More interestingly, further mapping of interaction domains showed that the GGA1 hinge region and the GGA2 GAE domain bound to multiple subdomains of the loop. These studies have identified an important function and revealed novel mechanisms of the GGA family proteins in the forward trafficking of a cell surface GPCR.

Endosomal Phosphatidylinositol 3-Kinase Is Essential for Canonical GPCR Signaling.

G protein-coupled receptors (GPCRs), the largest family of signaling receptors, are critically regulated by endosomal trafficking, suggesting that endosomes might provide new strategies for manipulating GPCR signaling. Here we test this hypothesis by focusing on class III phosphatidylinositol 3-kinase (Vps34), which is an essential regulator of endosomal trafficking. We verify that Vps34 is required for recycling of the β2-adrenoceptor (β2AR), a prototypical GPCR, and then investigate the effects of Vps34 inhibition on the canonical cAMP response elicited by β2AR activation. Vps34 inhibition impairs the ability of cells to recover this response after prolonged activation, which is in accord with the established role of recycling in GPCR resensitization. In addition, Vps34 inhibition also attenuates the short-term cAMP response, and its effect begins several minutes after initial agonist application. These results establish Vps34 as an essential determinant of both short-term and long-term canonical GPCR signaling, and support the potential utility of the endosomal system as a druggable target for signaling.

A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

Conformationally selective RNA aptamers allosterically modulate the β2-adrenoceptor.

G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs.

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other1. Upon activation by extracellular agonists, these seven transmembrane domain (7TM)-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades1. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide binding pocket of the G protein α-subunit to catalyze GDP release, the key step required for GTP binding and activation of G proteins2. The structure also offers hints on how G protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G protein coupling to β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone binding site. Surprisingly, the effects of G protein on the hormone binding site can be observed in the absence of a bound agonist, where G protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G protein-mediated enhancement of agonist affinity, which has been observed for many GPCR-G protein pairs. Our studies also suggest that in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G protein-mediated enhancement of agonist affinity.

Understanding the Effect of Polyunsaturated Fatty Acids on Rhodopsin using All-Atom Molecular Dynamics Simulations

G protein-coupled receptors (GPCRs) are membrane proteins that can transduce external stimuli across lipid bilayers via conformational changes, which tune intermolecular interactions with their cognate G protein. This mechanism and their involvement in the mediation of many signaling pathways make them important drug targets. The functional versatility of these receptors is thought to be governed by their dynamics and conformational plasticity, which can be modulated by several factors including the membrane surrounding them. Here, we analyze microsecond-scale all-atom molecular dynamics simulations of rhodopsin (∼150 μs in aggregate) to investigate how the conformational dynamics of this prototypical GPCR can be modulated by its lipid environment. We observe that polyunsaturated fatty acids interact differently with active and inactive ensembles of the receptor and thus may play a role in stabilzing different protein states.

Comparative analyses of downstream signal transduction targets modulated after activation of the AT1 receptor by two β-arrestin-biased agonists.

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with β-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or β-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known β-arrestin-biased agonists, such as [Sar1, Ile4, Ile8]-AngII (SII), and [Sar1, D-Ala8]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned β-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between “G protein-dependent” and “β-arrestin-dependent” signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of β-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization “G protein-dependent” vs. “β-arrestin-dependent” signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines.

Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses.

The G protein-coupled receptor rhodopsin: a historical perspective.

Rhodopsin is a key light-sensitive protein expressed exclusively in rod photoreceptor cells of the retina. Failure to express this transmembrane protein causes a lack of rod outer segment formation and progressive retinal degeneration, including the loss of cone photoreceptor cells. Molecular studies of rhodopsin have paved the way to understanding a large family of cell-surface membrane proteins called G protein-coupled receptors (GPCRs). Work started on rhodopsin over 100 years ago still continues today with substantial progress made every year. These activities underscore the importance of rhodopsin as a prototypical GPCR and receptor required for visual perception-the fundamental process of translating light energy into a biochemical cascade of events culminating in vision.

The role of membrane curvature elastic stress for function of rhodopsin-like G protein-coupled receptors.

The human genome encodes about 800 different G protein-coupled receptors (GPCR). They are key molecules in signal transduction pathways that transmit signals of a variety of ligands such as hormones and neurotransmitters to the cell interior. Upon ligand binding, the receptors undergo structural transitions that either enhance or inhibit transmission of a specific signal to the cell interior. Here we discuss results which indicate that transmission of such signals can be strongly modulated by the composition of the lipid matrix into which GPCR are imbedded. Experimental results have been obtained on rhodopsin, a prototype GPCR whose structure and function is representative for the great majority of GPCR in humans. The data shed light on the importance of curvature elastic stress in the lipid domain for function of GPCR.

Crystal structure of a common GPCR-binding interface for G protein and arrestin.

G-protein-coupled receptors (GPCRs) transmit extracellular signals to activate intracellular heterotrimeric G proteins (Gαβγ) and arrestins. For G protein signalling, the Gα C-terminus (GαCT) binds to a cytoplasmic crevice of the receptor that opens upon activation. A consensus motif is shared among GαCT from the Gi/Gt family and the ‘finger loop’ region (ArrFL1–4) of all four arrestins. Here we present a 2.75 Å crystal structure of ArrFL-1, a peptide analogue of the finger loop of rod photoreceptor arrestin, in complex with the prototypical GPCR rhodopsin. Functional binding of ArrFL to the receptor was confirmed by ultraviolet-visible absorption spectroscopy, competitive binding assays and Fourier transform infrared spectroscopy. For both GαCT and ArrFL, binding to the receptor crevice induces a similar reverse turn structure, although significant structural differences are seen at the rim of the binding crevice. Our results reflect both the common receptor-binding interface and the divergent biological functions of G proteins and arrestins.

Activation of G-protein-coupled receptors correlates with the formation of a continuous internal water pathway.

Recent crystal structures of G-protein-coupled receptors (GPCRs) have revealed ordered internal water molecules, raising questions about the functional role of those waters for receptor activation that could not be answered by the static structures. Here, we used molecular dynamics simulations to monitor--at atomic and high temporal resolution--conformational changes of central importance for the activation of three prototypical GPCRs with known crystal structures: the adenosine A2A receptor, the β2-adrenergic receptor and rhodopsin. Our simulations reveal that a hydrophobic layer of amino acid residues next to the characteristic NPxxY motif forms a gate that opens to form a continuous water channel only upon receptor activation. The highly conserved tyrosine residue Y(7.53) undergoes transitions between three distinct conformations representative of inactive, G-protein activated and GPCR metastates. Additional analysis of the available GPCR crystal structures reveals general principles governing the functional roles of internal waters in GPCRs.

Functional water molecules in rhodopsin activation.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular response to an extensive variety of extracellular stimuli. Studies of rhodopsin, a prototype GPCR, have suggested that water plays an important role in mediating the activation of family A GPCRs. However, our understanding of the function of water molecules in the GPCR activation is still rather limited because resolving the functional water molecules solely based on the results from existing experiments is challenging. Using all-atom molecular dynamics simulations in combination with inhomogeneous fluid theory, we identify in this work the positioning of functional water molecules in the inactive state, the Meta II state, and the constitutive active state of rhodopsin, basing on the thermodynamic signatures of the water molecules. We find that one hydration site likely functions as a switch to regulate the distance between Glu181 and the Schiff base in the rhodopsin activation. We observe that water molecules adjacent to the "NpxxY" motif are not as stable in the Meta II state as in the inactive state as indicated by the thermodynamics signatures, and we rationalize that the behaviors of these water molecules are closely correlated with the rearrangement of the water-mediated hydrogen-bond network in the "NPxxY" motif, which is essential for mediating the activation of the receptor. We thereby propose a hypothesis of the water-mediated rhodopsin activation pathway.

Divergent Transducer-specific Molecular Efficacies Generate Biased Agonism at a G Protein-coupled Receptor (GPCR)

The concept of "biased agonism" arises from the recognition that the ability of an agonist to induce a receptor-mediated response (i.e. "efficacy") can differ across the multiple signal transduction pathways (e.g. G protein and β-arrestin (βarr)) emanating from a single GPCR. Despite the therapeutic promise of biased agonism, the molecular mechanism(s) whereby biased agonists selectively engage signaling pathways remain elusive. This is due in large part to the challenges associated with quantifying ligand efficacy in cells. To address this, we developed a cell-free approach to directly quantify the transducer-specific molecular efficacies of balanced and biased ligands for the angiotensin II type 1 receptor (AT1R), a prototypic GPCR. Specifically, we defined efficacy in allosteric terms, equating shifts in ligand affinity (i.e. KLo/KHi) at AT1R-Gq and AT1R-βarr2 fusion proteins with their respective molecular efficacies for activating Gq and βarr2. Consistent with ternary complex model predictions, transducer-specific molecular efficacies were strongly correlated with cellular efficacies for activating Gq and βarr2. Subsequent comparisons across transducers revealed that biased AT1R agonists possess biased molecular efficacies that were in strong agreement with the signaling bias observed in cellular assays. These findings not only represent the first measurements of the thermodynamic driving forces underlying differences in ligand efficacy between transducers but also support a molecular mechanism whereby divergent transducer-specific molecular efficacies generate biased agonism at a GPCR.

Role of reactive oxygen species as signal transducers in β2 adrenergic receptor mediated β‐arrestin signaling (662.2)

Reactive oxygen species (ROS) have previously been linked to deleterious effects caused by oxidative stress as well as to inflammatory disorders, degenerative disorders and cancers. More recently, ROS have been recognized as important signaling molecules, and have been shown to be involved in G protein‐coupled receptor (GPCR) signal transduction. Our laboratory has previously shown that agonism of the prototypical GPCR, the β2 adrenergic receptor (β2AR), generates intracellular ROS and that β2AR‐associated G protein signaling is dependent on ROS. Here, we demonstrate that ROS are critical mediators of β2AR signaling to β‐arrestins. Using coimmunoprecipitation and BRET, we reveal that the physical interaction of β2AR and β‐arrestins are dependent on ROS. Furthermore, using phosphorylation of ERK1/2 as an endpoint for both G‐protein and β‐arrestin signaling, we demonstrate that ROS are required for both G protein and β‐arrestin dependent ERK1/2 phosphorylation. These results are presented in light of data, which demonstrate that β2AR agonism can lead to receptor S‐Sulfenation, a process that is dependent on ROS, and which suggests that ROS may serve a purposeful role in stabilizing β2AR in signal‐capable conformations.Grant Funding Source: Internal funding

Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light.