Gi1 Protein Research Articles

Bitter taste TAS2R14 activation by intracellular tastants and cholesterol.

Bitter taste receptors, particularly TAS2R14, play central roles in discerning a wide array of bitter substances, ranging from dietary components to pharmaceutical agents1,2. TAS2R14 is also widely expressed in extragustatory tissues, suggesting its extra roles in diverse physiological processes and potential therapeutic applications3. Here we present cryogenicelectron microscopy structures of TAS2R14 in complex with aristolochic acid, flufenamic acid and compound 28.1, coupling with different G-protein subtypes. Uniquely, a cholesterol molecule is observed occupying what is typically an orthosteric site in class A G-protein-coupled receptors. The three potent agonists bind, individually, to the intracellular pockets, suggesting a distinct activation mechanism for this receptor. Comprehensive structural analysis, combined with mutagenesis and molecular dynamic simulation studies, elucidate the broad-spectrum ligand recognition and activation of the receptor by means of intricate multiple ligand-binding sites. Our study also uncovers the specific coupling modes of TAS2R14 with gustducin and Gi1 proteins. These findings should be instrumental in advancing knowledge of bitter taste perception and its broader implications in sensory biology and drug discovery.

Ligand bias and inverse agonism on 5-HT2A receptor-mediated modulation of G protein activity in post-mortem human brain.

Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics.

Allosteric modulation of G protein-coupled receptors (GPCRs) is a major paradigm in drug discovery. Despite decades of research, a molecular-level understanding of the general principles that govern the myriad pharmacological effects exerted by GPCR allosteric modulators remains limited. The M4 muscarinic acetylcholine receptor (M4 mAChR) is a validated and clinically relevant allosteric drug target for several major psychiatric and cognitive disorders. In this study, we rigorously quantified the affinity, efficacy, and magnitude of modulation of two different positive allosteric modulators, LY2033298 (LY298) and VU0467154 (VU154), combined with the endogenous agonist acetylcholine (ACh) or the high-affinity agonist iperoxo (Ipx), at the human M4 mAChR. By determining the cryo-electron microscopy structures of the M4 mAChR, bound to a cognate Gi1 protein and in complex with ACh, Ipx, LY298-Ipx, and VU154-Ipx, and applying molecular dynamics simulations, we determine key molecular mechanisms underlying allosteric pharmacology. In addition to delineating the contribution of spatially distinct binding sites on observed pharmacology, our findings also revealed a vital role for orthosteric and allosteric ligand-receptor-transducer complex stability, mediated by conformational dynamics between these sites, in the ultimate determination of affinity, efficacy, cooperativity, probe dependence, and species variability. There results provide a holistic framework for further GPCR mechanistic studies and can aid in the discovery and design of future allosteric drugs.

Receptor-specific recognition of NPY peptides revealed by structures of NPY receptors.

In response to three highly conserved neuropeptides, neuropeptide Y (NPY), peptide YY, and pancreatic polypeptide (PP), four G protein–coupled receptors mediate multiple essential physiological processes, such as food intake, vasoconstriction, sedation, and memory retention. Here, we report the structures of the human Y1, Y2, and Y4 receptors in complex with NPY or PP, and the Gi1 protein. These structures reveal distinct binding poses of the peptide upon coupling to different receptors, reflecting the importance of the conformational plasticity of the peptide in recognizing the NPY receptors. The N terminus of the peptide forms extensive interactions with the Y1 receptor, but not with the Y2 and Y4 receptors. Supported by mutagenesis and functional studies, subtype-specific interactions between the receptors and peptides were further observed. These findings provide insight into key factors that govern NPY signal recognition and transduction, and would enable development of selective drugs.

Structural basis of neuropeptide Y signaling through Y1 receptor

Neuropeptide Y (NPY) is highly abundant in the brain and involved in various physiological processes related to food intake and anxiety, as well as human diseases such as obesity and cancer. However, the molecular details of the interactions between NPY and its receptors are poorly understood. Here, we report a cryo-electron microscopy structure of the NPY-bound neuropeptide Y1 receptor (Y1R) in complex with Gi1 protein. The NPY C-terminal segment forming the extended conformation binds deep into the Y1R transmembrane core, where the amidated C-terminal residue Y36 of NPY is located at the base of the ligand-binding pocket. Furthermore, the helical region and two N-terminal residues of NPY interact with Y1R extracellular loops, contributing to the high affinity of NPY for Y1R. The structural analysis of NPY-bound Y1R and mutagenesis studies provide molecular insights into the activation mechanism of Y1R upon NPY binding.

Structural basis of GABAB receptor\u2013Gi protein coupling

G-protein-coupled receptors (GPCRs) have central roles in intercellular communication1,2. Structural studies have revealed how GPCRs can activate G proteins. However, whether this mechanism is conserved among all classes of GPCR remains unknown. Here we report the structure of the class-C heterodimeric GABAB receptor, which is activated by the inhibitory transmitter GABA, in its active form complexed with Gi1 protein. We found that a single G protein interacts with the GB2 subunit of the GABAB receptor at a site that mainly involves intracellular loop 2 on the side of the transmembrane domain. This is in contrast to the G protein binding in a central cavity, as has been observed with other classes of GPCR. This binding mode results from the active form of the transmembrane domain of this GABAB receptor being different from that of other GPCRs, as it shows no outside movement of transmembrane helix 6. Our work also provides details of the inter- and intra-subunit changes that link agonist binding to G-protein activation in this heterodimeric complex.

Cryo-EM structures of inactive and active GABAB receptor

Metabotropic GABAB G protein-coupled receptor functions as a mandatory heterodimer of GB1 and GB2 subunits and mediates inhibitory neurotransmission in the central nervous system. Each subunit is composed of the extracellular Venus flytrap (VFT) domain and transmembrane (TM) domain. Here we present cryo-EM structures of full-length human heterodimeric GABAB receptor in the antagonist-bound inactive state and in the active state complexed with an agonist and a positive allosteric modulator in the presence of Gi1 protein at a resolution range of 2.8-3.0 Å. Our structures reveal that agonist binding stabilizes the closure of GB1 VFT, which in turn triggers a rearrangement of TM interfaces between the two subunits from TM3-TM5/TM3-TM5 in the inactive state to TM6/TM6 in the active state and finally induces the opening of intracellular loop 3 and synergistic shifting of TM3, 4 and 5 helices in GB2 TM domain to accommodate the α5-helix of Gi1. We also observed that the positive allosteric modulator anchors at the dimeric interface of TM domains. These results provide a structural framework for understanding class C GPCR activation and a rational template for allosteric modulator design targeting the dimeric interface of GABAB receptor.

Preferential Coupling of Dopamine D2S and D2L Receptor Isoforms with Gi1 and Gi2 Proteins-In Silico Study.

The dopamine D2 receptor belongs to rhodopsin-like G protein-coupled receptors (GPCRs) and it is an important molecular target for the treatment of many disorders, including schizophrenia and Parkinson’s disease. Here, computational methods were used to construct the full models of the dopamine D2 receptor short (D2S) and long (D2L) isoforms (differing with 29 amino acids insertion in the third intracellular loop, ICL3) and to study their coupling with Gi1 and Gi2 proteins. It was found that the D2L isoform preferentially couples with the Gi2 protein and D2S isoform with the Gi1 protein, which is in accordance with experimental data. Our findings give mechanistic insight into the interplay between isoforms of dopamine D2 receptors and Gi proteins subtypes, which is important to understand signaling by these receptors and their mediation by pharmaceuticals, in particular psychotic and antipsychotic agents.

Ligand-Induced Coupling between Oligomers of the M2 Receptor and the Gi1 Protein in Live Cells

Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M2 muscarinic receptor and of Gi1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M2 and mCherry-M2) and G proteins (i.e., eGFP-Gαi1β1γ2 and mCherry-Gαi1β1γ2) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and Gi1 can exist as homo-oligomers. Oligomerization of differently tagged Gi1 decreased upon the activation of co-expressed wild-type M2 receptor by an agonist. Measurements on a tagged M2 receptor (M2-mCherry) and eGFP-Gαi1β1γ2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M2(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gαi1β1γ2, indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M2 receptor on the one hand and of Gi1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.

The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors

The Gi/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of Gi/o-GPCR coupling and the high rate of Gi/o signal transduction have been hypothesized to be enabled by the existence of stable associates between Gi/o proteins and their cognate GPCRs in the inactive state (Gi/o-GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gαi1 subunits labeled with fluorescent proteins and four GPCRs: the α2A-adrenergic receptor, GABAB, cannabinoid receptor type 1 (CB1R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gαi1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gαi1 subunits and α2A-adrenergic receptor, GABAB, or dopamine receptor type 2 receptors did not reveal any interaction between the Gi1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB1R exhibited an interaction with the Gi1 protein. Further experiments revealed that this interaction is caused solely by CB1R basal activity; no preassembly between CB1R and the Gi1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active Gi1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and Gi1 (basal coupling). These findings suggest that Gi1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs.

Single-Molecule Study of the Oligomeric States of the M2 Muscarinic Receptor, the Gi1 Protein and the M2-Gi1 Complex

Inconsistent results obtained in the identification of oligomers of rhodopsin-like GPCRs by various means and in various preparations has generated much debate on the role of oligomers. Estimates of size has largely focused on oligomers of the receptor yet it is the size of the receptor coupled to the G protein that is the most relevant but remains unknown. Using the photo-bleaching of fusions of single-molecules of eGFP, we evaluated the oligomeric size of the muscarinic M2 receptor, the Gi1 protein and various multimeric controls. The relationship between the number of photo-bleaching steps within homogeneous population of oligomeric controls of known size gave a distribution of bleaching steps instead of discrete steps. Statistical analysis of the distribution in terms of the binomial model yielded estimates of the oligomeric size. Using this approach, we examined the oligomeric size of both the receptor and the G protein in extracts that represent different stages of the signaling cycle: in the basal state, following activation by ligands, during and after coupling of the complex. The data indicate that whereas the receptor remains as a tetramer throughout the cycle, the oligomeric size of the G protein is dynamic. G proteins exist as hexamers in the basal state, couple as tetramers and uncouple from the receptor (i.e., fully activated) as dimers. Measurements with a receptor mimic to the wild-type G protein and a constitutively active mutant also yielded dimers. The spatial feasibility of a super-complex for the di-tetrameric arrangement of the receptor and G protein is presented using experimentally derived constraints.

Interaction of different third intracellular loop fragments of human dopamine d2l receptor with a-subunit of gi1 protein - prospective therapeutic application

In order to find the essential structural motif of the D2L dopamine receptor necessary for the interaction with a-subunit of Gi1 protein, four fragments of the third cytoplasmic loop (CPL3) of this receptor were cloned, expressed in E. coli and purified. After that, fusion proteins with glutathione-S-transferase (GST) were prepared and the interactions quantified by a colorimetric assay for GST activity determination. The presence of D2L-CPL3 fragment-Gia1 complexes was detected by SDS-polyacrylamide gel electrophoresis (PAGE). Kd values for the interaction of the three fragments with Gia1 were similar and in nmol/L range of concentrations, while the peptide representing the insert in the long form of the dopamine D2 receptor expressed about 10-fold lower binding affinity. These results could serve to design new therapeutic agents that might act at the level of receptor/G protein interaction rather than at the level of ligand-receptor binding.

AMPA receptor activation induces association of G‐beta protein with the alpha subunit of the sodium channel in neurons

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine-nucleotide binding- (G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), which has been shown to be physically associated with the alpha-subunit of Gi1 protein and activates this G-protein. Here, we report that, in addition to a Ca2+ influx, AMPA induces the mobilization of Ca2+ from the mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes required the activation of tetrodotoxin-sensitive Na+ channels. AMPA receptor activation modified the gating properties of the Na+ channel, independently of the AMPA current, suggesting a G-protein-mediated process. Indeed, co-immunoprecipitation experiments indicated that AMPA receptor activation induced the association of Gbeta with the alpha-subunit of the Na+ channel. These results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G-proteins and that this novel metabotropic function is involved in the control of neuronal excitability.