Publications related to G protein-coupled receptor

Micropatterned immobilisation of a G protein-coupled receptor and direct detection of G protein activation

EPFL2000

Computational rewiring of allosteric pathways reprograms GPCR selective responses to ligands

Patrick Daniel Barth, Mahdi Hijazi, Dániel Kéri, Aurélien Laurent Jean-Charles Oggier

G-protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and drug targets, and respond to a wide variety of chemical stimuli to activate diverse cellular functions. Understanding and predicting how ligand binding triggers a specific signaling response is critical for drug discovery and design but remains a major challenge. Here, computational design of GPCR allosteric functions is used to uncover the mechanistic relationships between agonist ligand chemistry, receptor sequence, structure, dynamics and allosteric signaling in the dopamine D2 receptor. Designed gain of function D2 variants for dopamine displayed very divergent G-protein signaling responses to other ligand agonists that strongly correlated with ligand structural similarity. Consistent with these observations, computational analysis revealed distinct topologies of allosteric signal transduction pathways for each ligand-bound D2 pair that were perturbed differently by the designs. We leveraged these findings by rewiring ligand-specific pathways and designed receptors with highly selective ligand responses. Overall, our study suggests that distinct ligand agonists can activate a given signaling effector through specific “allosteric activator” moieties that engage partially independent signal transmission networks in GPCRs. The results provide a mechanistic framework for understanding and predicting the impact of sequence polymorphism on receptor pharmacology, informing selective drug design and rationally designing receptors with highly selective ligand responses for basic and therapeutic applications.

bioRxiv2022

Investigation of the neurokinin-1 receptor by fluorescence techniques

Bruno Meyer

G protein-coupled receptors (GPCR) constitute by far the largest family of transmembrane cell-surface proteins involved in signal transduction and are the most important targets for the development of novel therapeutic compounds. Many processes mediated by GPCR signal transduction are still not well understood at the molecular level and novel methods for their investigation are of general interest. The thesis presented here describes investigations on the neurokinin-1 receptor (NK1R), a member of the GPCR family, using fluorescence techniques. Fluorescence resonance energy transfer (FRET), the technique of preference in this work, is used to measure ligand-protein and protein-protein interactions in living cells. In order to reach this goal, the NK1R has been labelled with either fluorescent proteins or by using the novel post-translational ACP labelling technique. A FRET-based method for monitoring specific ligand binding to NK1R is described. In this context, a fluorescent agonist for NK1R was synthesized and characterized. The ACP labelling technique was shown to be superior for this kind of experiments compared to labelling with fluorescent proteins. The same ACP labelling approach was shown to be ideally suited to study oligomerization of membrane proteins by FRET. It was shown, that the NK1R does not form homo-dimers, as many other GPCRs do. From these experiments we furthermore conclude, that the NK1R is present in small microdomains in the membrane. Interactions between the NK1R and the different subunits of the heterotrimeric Gαqβ1γ2-complex were investigated by FRET using different instrumentation. Proximity of the NK1R and the G protein could be detected, but no change in FRET was observed after activation of the receptor. Total internal reflection fluorescence (TIRF), was used to develop an assay for studying ligand binding in vitro. Here, the NK1R was immobilized directly on a surface using a cell membrane preparation. This method was shown to be highly sensitive.

EPFL2005

Reprogramming G Protein-Coupled Receptor Structure, Function And Signaling By Computational Design

Dániel Kéri

G protein-coupled receptors (GPCRs) are 7-transmembrane alpha-helical integral membrane proteins on which cells heavily rely to receive information regarding their external environment. These receptors are able to transfer information to intracellular downstream effectors upon stimulation from their cognate ligands, which can be considerably diverse. They can include light, small molecules, peptides, protons; Due to its evolutionary success, over millions of years of evolution, this protein family has expanded to include over 800 members. Despite the large size of the family and the diverse inputs they accept, the structure and the mechanism for relaying signals to their downstream partners, G proteins and B-arrestins, remains largely the same. As these receptors are involved in a great deal of biological processes such as sight (rhodopsin), cognition (dopamine, serotonin, opioid receptors), immunity (chemokine receptors), heart function (adrenergic, angiotensin receptors), etc.; a great deal of effort has gone into understanding their structure and function. Our efforts have gone into understanding their signaling properties and rationally modifying them. Most proteins are fairly flexible entities and their structures tend to oscillate and move around, sampling a number of conformations. Nature has taken advantage of these natural motions to regulate protein function from distant binding sites, in a phenomenon called allostery. GPCRs are unique in the sense that their primary function relies on allostery. By tweaking the allosteric signaling of GPCRs, we hope to have a better understand allostery, use the principles we learn to create designer GPCR biosensors with the desired sensitivity, and to eventually create de novo allosteric proteins. In this work, I present our progress in this effort, which has been significant. We have created a highly accurate in silico method to allosterically alter relative stabilities of the active and inactive states of the GPCR dopamine D2 receptor, but also to independently control its allosteric signaling strength. With our methodology we have been able to create receptors which have high basal activities by selectively stabilizing the active state of the receptor. This has already facilitated the structure determination of the active, G protein-bound dopamine D2 receptor; a first for this GPCR. Additionally, we have created a number of dopamine D2 variants with roughly 100 times higher sensitivity to dopamine than the WT receptor. These efforts continue to characterize the functional effects of these allosteric mutations. Furthermore, we are also working on a collaboration to rewire the signaling pathway of the adenosine A2A receptor. Adenosine is a common immunosuppresant in solid tumor micro-environments (TMEs). By rewiring the downstream signaling of the A2A receptor, we hope to reverse the response of T-cells in these TMEs to activate and attack the solid tumor.

EPFL2021

Activation and Spatial Organization of the Adenosine A2A Receptor in Supported Plasma Membrane Sheets

Sophie Roizard

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signal transduction across cell membranes and constitute a very important class of therapeutic targets. The main mechanisms involving GPCR activation and signal transduction to the cell interior are understood but many of the processes which fine-tune GPCRs signalling need to be unraveled. The work performed in this thesis intended to develop new methods to study GPCRs in their native environment but in a simplified system compared to live cells. In this context, the tethering GPCR-containing membrane fragments on solid surfaces appeared to be interesting: it enabled studying the receptors in their real native environment with several high resolution imaging techniques and with the possibility to engineer and control the downstream intracellular signaling components. Two procedures have been established to produce plasma membrane supported on micrometer-sized porous beads or on microscope grids for fluorescence or electron microscopy studies. They have been applied in this thesis to study the Adenosine A2A receeptor (A2A) as a prototypical GPCR. The first procedure enabled the transfer of native membrane patches from live cells with an inside-out orientation onto lectin-coated beads. Using heterologously expressed A2ARs carrying a yellow fluorescent protein (A2AR-Citrine) we showed that the tethered membranes were simultaneously accessible from both sides and comprised fully functional receptors in terms of ligand and G protein binding. These beads were investigated by confocal (Chapter 2) and single molecule (Chapter 3) fluorescence microscopies to study agonist and antagonist binding to the A2AR and the assembly and disassembly of the ternary complexes agonist-A2AR-Gαβγ proteins. The interactions between the different signalling partners could be observed in real time using multicolor fluorescence microscopy. These beads were also used in collaboration with colleagues for screening ligand binding to other transmembrane receptors. The second procedure produced GPCRs-containing plasma membrane fragments ripped-off on electron microscope grids for investigation by transmission electron microscopy (TEM) (Chapter 4). This procedure was improved from an existing protocol in order to produce reproducibly and with a high yield large membrane sheets and to preserve the membrane-associated cytoskeleton during the ripping-off. A2AR-Citrine were specifically gold immuno-labelled on their extracellular terminus using the FLAG-tag or their intracellular side by targeting the protein Citrine. The high resolution of TEM showed that the gold immuno-labelled A2AR-Citrine clustered and compartmentalized on actin-based cytoskeletton. This approach provided the ability to sample multiple single cells on a single grid and presented excellent potential to probe and unravel the organization of protein signalling networks in direct association with the plasma membrane-associated cytoskeleton.

EPFL2013

The role of metal ions in G protein-coupled receptor signalling and drug discovery

Horst Vogel, Xueying Wang, Shuguang Yuan

Metal ions, such as Na+, Mg2+, Ca2+, and Zn2+, are highly abundant in biological systems. For many G protein-coupled receptors (GPCRs), metal ions, especially Na+, have been found to stabilize the receptors' inactivate state preventing receptor activation. Besides this role, recent studies indicated that metal ions can modulate ligand-GPCR interactions allosterically. These findings on ion-mediated GPCR signaling open new vistas for understanding the functioning of GPCRs and further could help in designing potent therapeutic compounds targeting GPCRs. Here we report on recent findings about role of orthosteric and allosteric metal ions in GPCR signaling and drug discovery. This article is categorized under: Structure and Mechanism > Molecular Structures Molecular and Statistical Mechanics > Molecular Mechanics Molecular and Statistical Mechanics > Molecular Interactions

WILEY2021

Computational design of G Protein-Coupled Receptor allosteric signal transductions

Patrick Daniel Barth, Dániel Kéri

Membrane receptors sense and transduce extracellular stimuli into intracellular signaling responses but the molecular underpinnings remain poorly understood. We report a computational approach for designing protein allosteric signaling functions. By combining molecular dynamics simulations and design calculations, the method engineers amino-acid ‘microswitches’ at allosteric sites that modulate receptor stability or long-range coupling, to reprogram specific signaling properties. We designed 36 dopamine D2 receptor variants, whose constitutive and ligand-induced signaling agreed well with our predictions, repurposed the D2 receptor into a serotonin biosensor and predicted the signaling effects of more than 100 known G-protein-coupled receptor (GPCR) mutations. Our results reveal the existence of distinct classes of allosteric microswitches and pathways that define an unforeseen molecular mechanism of regulation and evolution of GPCR signaling. Our approach enables the rational design of allosteric receptors with enhanced stability and function to facilitate structural characterization, and reprogram cellular signaling in synthetic biology and cell engineering applications.

2019

G Protein coupled receptors (GPCRs) constitute an abundant family of membrane proteins which play a central role in cellular signaling and are important targets for modern medicine. Despite intensive research, central questions of the molecular mechanism of GPCR mediated signal transduction remain unresolved. This thesis concerns α1b-adrenergic receptor (α1b-AR) as a prototypic GPCR. The question whether ARs form homo- or hetero-oligomers was investigated using fluorescence resonance energy transfer (FRET). An important finding was that both α1a and α1b-AR subtypes form homo-oligomers. Also hetero-oligomers have been observed between the α1b- and the α1a-AR subtypes, but not with less related receptors. Another interesting finding concerns α1-AR co-internalization which correlates with the ability of the receptor to hetero-oligomerize. Investigating particular processes in living cells is often complicated by the complex cellular environment. Here we have developed a rather simple approach to study transmembrane signaling by using supported cell membrane sheets. They were prepared by pressing a glass coverslip on the apical part of cultured cells, ripping off large regions of the native plasma membrane. For the α1b-AR we still could observe ligand binding to such sheets, indicating that GPCR functionality was at least partly conserved. Due to the absence of cytosolic autofluorescence of the cells, supported membrane sheets were also found to be suited for single-molecule microscopy. Because both leaflets of the supported membranes remained fluid, it was possible to follow the diffusion of certain membrane proteins. For example using fluorescence recovery after photobleaching (FRAP) and single-molecule microscopy, we investigated how G proteins diffused along the membrane and compartmentalized. The predominant role of lipid anchors in G protein localization was highlighted by measuring the diffusion of two Gαq proteins fused with citrine at two different positions and citrine based constructs designed to mimic the monomeric and the trimeric form of a G protein. Finally, first results were obtained showing the feasibility to form supported cell membrane sheets on microstructured devices consisting of a planar silicon support perforated with arrays of holes. This geometry provides a full accessibility to both intra- and extracellular sides of the bilayer. This concept could potentially be applied for the development of chip-based screening assays.

EPFL2005

Fluorescent labeling of NK2 receptor at specific sites in vivo and fluorescence energy transfer analysis of NK2 ligand-receptor complexes

Jonathan Knowles, Gerardo Turcatti, Horst Vogel

A fluorescent unnatural amino acid was introduced biosynthetically at known sites into the G protein-coupled neurokinin (tachykinin) NK2 receptor by suppression of UAG nonsense codons with the aid of a chem. misacylated synthetic tRNA specifically designed for the incorporation of unnatural amino acids during heterologous expression in Xenopus oocytes. A systematic UAG-scanning mutagenesis in NK2 extra- or intracellular loops and proximal transmembrane domains established that read-through at some UAG sites may represent a limitation to the range of applicability of the nonsense suppression methodol. Fluorescence-labeled NK2 mutants contg. an unique fluorescent nitrobenzoxadiazoyl-diaminopropionic acid residue at known sites were shown to be functionally active. Intermol. distances were detd. by measuring the fluorescence resonance energy transfer (FRET) between the fluorescent unnatural amino acid and a fluorescently labeled NK2 heptapeptide antagonist in a native membrane environment. These distances confirmed the seven transmembrane topol. for G protein-coupled receptors and detd. a structural model for NK2 ligand-receptor interactions. The peptide is inserted between the fifth and sixth transmembrane domains, thus suggesting that antagonism may be caused by preventing correct packing of the helixes required for receptor function. [on SciFinder (R)]

1997

The Principles of Ligand Specificity on beta-2-adrenergic receptor

Shuguang Yuan

G protein-coupled receptors are recognized as one of the largest families of membrane proteins. Despite sharing a characteristic seven-transmembrane topology, G protein-coupled receptors regulate a wide range of cellular signaling pathways in response to various physical and chemical stimuli, and prevail as an important target for drug discovery. Notably, the recent progress in crystallographic methods led to a breakthrough in elucidating the structures of membrane proteins. The structures of beta(2)-adrenergic receptor bound with a variety of ligands provide atomic details of the binding modes of agonists, antagonists and inverse agonists. In this study, we selected four representative molecules from each functional class of ligands and investigated their impacts on beta(2)-adrenergic receptor through a total of 12 x 100 ns molecular dynamics simulations. From the obtained trajectories, we generated molecular fingerprints exemplifying propensities of protein-ligand interactions. For each functional class of compounds, we characterized and compared the fluctuation of the protein backbone, the volumes in the intracellular pockets, the water densities in the receptors, the domain interaction networks as well as the movements of transmembrane helices. We discovered that each class of ligands exhibits a distinct mode of interactions with mainly TM5 and TM6, altering the shape and eventually the state of the receptor. Our findings provide insightful prospective into GPCR targeted structure-based drug discoveries.

Nature Publishing Group2016