Browse
Submit Data
Databases
API
Help

Dataset Information

8 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Lifetime of muscarinic receptor-G-protein complexes determines coupling efficiency and G-protein subtype selectivity.

ABSTRACT: G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M3-R interactions but rather the affinities of Gq and Go proteins to M3-Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

SUBMITTER: Ilyaskina OS

PROVIDER: S-EPMC5948956 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

DHHC5-mediated palmitoylation of S1P receptor subtype 1 determines G-protein coupling.

Project description:Sphingosine 1-phosphate (S1P) is a pleiotropic lipid mediator involved in the regulation of immune cell trafficking and vascular permeability acting mainly through G-protein-coupled S1P receptors (S1PRs). However, mechanism underlying how S1PRs are coupled with G-proteins remains unknown. Here we have uncovered that palmitoylation of a prototypical subtype S1P1R is prerequisite for subsequent inhibitory G-protein (Gi) coupling. We have identified DHHC5 as an enzyme for palmitoylation of S1P1R. Under basal conditions, S1P1R was functionally associated with DHHC5 in the plasma membranes (PM) and was fully palmitoylated, enabling Gi coupling. Upon stimulation, the receptor underwent internalisation leaving DHHC5 in PM, resulting in depalmitoylation of S1P1R. We also revealed that while physiological agonist S1P-induced endocytosed S1P1R readily recycled back to PM, pharmacological FTY720-P-induced endocytosed S1P1R-positive vesicles became associated with DHHC5 in the later phase, persistently transmitting Gi signals there. This indicates that FTY720-P switches off the S1P signal in PM, while switching on its signal continuously inside the cells. We propose that DHHC5-mediated palmitoylation of S1P1R determines Gi coupling and its signalling in a spatio/temporal manner.

| S-EPMC5707436 | biostudies-literature

Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes.

Project description:Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/ 11 family, whereas M2R and M4R couple to the Gi/ o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.

| S-EPMC7034192 | biostudies-literature

Influence of the accessory protein SET on M3 muscarinic receptor phosphorylation and G protein coupling.

Project description:The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.

| S-EPMC3382831 | biostudies-literature

The coupling of the M2 muscarinic receptor to its G protein is voltage dependent.

Project description:G protein coupled receptors (GPCRs) participate in the majority of signal transduction processes in the body. Specifically, the binding of an external agonist promotes coupling of the GPCR to its G protein and this, in turn, induces downstream signaling. Recently, it was shown that agonist binding to the M2 muscarinic receptor (M2R) and to other GPCRs is voltage dependent. Here we examine, whether the coupling of the M2R to its G protein is also voltage-dependent. We first show, in Xenopus oocytes, that the activity of the M2R in the absence of agonist (constitutive activity) can be used to report the coupling. We then show that the coupling is, by itself, voltage dependent. This novel finding is of physiological importance, as it shows that the actual signal transduction, whose first step is the coupling of the GPCR to its cognate G protein, is voltage dependent.

| S-EPMC6822938 | biostudies-literature

Ligand recognition and G-protein coupling selectivity of cholecystokinin A receptor.

Project description:Cholecystokinin A receptor (CCKAR) belongs to family A G-protein-coupled receptors and regulates nutrient homeostasis upon stimulation by cholecystokinin (CCK). It is an attractive drug target for gastrointestinal and metabolic diseases. One distinguishing feature of CCKAR is its ability to interact with a sulfated ligand and to couple with divergent G-protein subtypes, including Gs, Gi and Gq. However, the basis for G-protein coupling promiscuity and ligand recognition by CCKAR remains unknown. Here, we present three cryo-electron microscopy structures of sulfated CCK-8-activated CCKAR in complex with Gs, Gi and Gq heterotrimers, respectively. CCKAR presents a similar conformation in the three structures, whereas conformational differences in the 'wavy hook' of the Gα subunits and ICL3 of the receptor serve as determinants in G-protein coupling selectivity. Our findings provide a framework for understanding G-protein coupling promiscuity by CCKAR and uncover the mechanism of receptor recognition by sulfated CCK-8.

| S-EPMC8604728 | biostudies-literature

On homology modeling of the M₂ muscarinic acetylcholine receptor subtype.

Project description:Twelve homology models of the human M2 muscarinic receptor using different sets of templates have been designed using the Prime program or the modeller program and compared to crystallographic structure (PDB:3UON). The best models were obtained using single template of the closest published structure, the M3 muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led to worse models. Data document a key role of the template in homology modeling. The models differ substantially. The quality checks built into the programs do not correlate with the RMSDs to the crystallographic structure and cannot be used to select the best model. Re-docking of the antagonists present in crystallographic structure and relative binding energy estimation by calculating MM/GBSA in Prime and the binding energy function in YASARA suggested it could be possible to evaluate the quality of the orthosteric binding site based on the prediction of relative binding energies. Although estimation of relative binding energies distinguishes between relatively good and bad models it does not indicate the best one. On the other hand, visual inspection of the models for known features and knowledge-based analysis of the intramolecular interactions allows an experimenter to select overall best models manually.

| S-EPMC3717152 | biostudies-literature

Polyarginine molecular weight determines transfection efficiency of calcium condensed complexes.

Project description:Cell penetrating peptides (CPPs) have been extensively studied in polyelectrolyte complexes as a means to enhance the transfection efficiency of plasmid DNA (pDNA). Increasing the molecular weight of CPPs often enhances gene expression but poses a risk of increased cytotoxicity and immunogenicity compared to low molecular weight CCPs. Conversely, low molecular weight CPPs typically have low transfection efficiency due to large complex size. Complexes made using low molecular weight CPPs were found to be condensed to a small size by adding calcium. In this study, complexes of low molecular weight polyarginine and pDNA were condensed with calcium. These complexes showed high transfection efficiency and low cytotoxicity in A549 carcinomic human alveolar basal epithelial cells. The relationships between transfection efficiency and polyarginine size (5, 7, 9, or 11 amino acids), polyarginine/pDNA charge ratios, and calcium concentrations were studied. Polyarginine 7 was significantly more effective than other polyarginines under most formulation conditions, suggesting a link between cell penetration ability and transfection efficiency.

| S-EPMC4207646 | biostudies-literature

Solvent exchange in preformed photocatalyst-donor precursor complexes determines efficiency.

Project description:In homogeneous photocatalytic reduction of CO2, it is widely assumed that the primary electron transfer from the sacrificial donor to the catalyst is diffusion controlled, thus little attention has been paid to optimizing this step. We present spectroscopic evidence that the precursor complex is preformed, driven by preferential solvation, and two-dimensional infrared spectroscopy reveals triethanolamine (donor)/tetrahydrofuran (solvent) exchange in the photocatalyst's solvation shell, reaching greatest magnitude at the known optimal concentration (?20% v/v TEOA in THF) for catalytically reducing CO2 to CO. Transient infrared absorption shows the appearance of the singly reduced catalyst on an ultrafast (<70 ps) time scale, consistent with non-diffusion controlled electron transfer within the preformed precursor complex. Identification of preferential catalyst-cosolvent interactions suggests a revised paradigm for the primary electron transfer, while illuminating the pivotal importance of solvent exchange in determining the overall efficiency of the photocycle.

| S-EPMC5887230 | biostudies-literature

Structural basis for the ligand recognition and G protein subtype selectivity of kisspeptin receptor.

Project description:Kisspeptin receptor (KISS1R), belonging to the class A peptide-GPCR family, plays a key role in the regulation of reproductive physiology after stimulation by kisspeptin and is regarded as an attractive drug target for reproductive diseases. Here, we demonstrated that KISS1R can couple to the Gi/o pathway besides the well-known Gq/11 pathway. We further resolved the cryo-electron microscopy (cryo-EM) structure of KISS1R-Gq and KISS1R-Gi complexes bound to the synthetic agonist TAK448 and structure of KISS1R-Gq complex bound to the endogenous agonist KP54. The high-resolution structures provided clear insights into mechanism of KISS1R recognition by its ligand and can facilitate the design of targeted drugs with high affinity to improve treatment effects. Moreover, the structural and functional analyses indicated that conformational differences in the extracellular loops (ECLs), intracellular loops (ICLs) of the receptor, and the "wavy hook" of the Gα subunit may account for the specificity of G protein coupling for KISS1R signaling.

| S-EPMC11328905 | biostudies-literature

Muscarinic acetylcholine receptor signaling generates OFF selectivity in a simple visual circuit.

Project description:ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either light increments or decrements. Despite lacking the anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on investigating the physiological properties and behavioral relevance of larval visual interneurons. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes a cholinergic interneuron (cha-lOLP) and hyperpolarizes a glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Gαo signaling produces the sign-inversion required for OFF detection in glu-lOLP, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify the molecular and circuit mechanisms underlying ON vs. OFF discrimination in the Drosophila larval visual system.

| S-EPMC6733798 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data