Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.

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Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by Free Energy simulations

10.1101/2021.06.09.447724 ◽

2021 ◽

Author(s):

Hugo Gutierrez de Teran ◽

Willem Jespers ◽

Laura H. Heitman ◽

Adriaan P. IJzerman ◽

Eddy Sotelo ◽

...

Keyword(s):

Free Energy ◽

G Protein ◽

Point Mutations ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

A2a Adenosine Receptor ◽

Thermodynamic Cycles ◽

Energy Perturbation ◽

Energy Simulations ◽

G Protein Coupled

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G-protein-coupled-receptor activation of the smooth muscle calponin gene

Biochemical Journal ◽

10.1042/bj3570587 ◽

2001 ◽

Vol 357 (2) ◽

pp. 587-592 ◽

Cited By ~ 9

Author(s):

Nickolai O. DULIN ◽

Sergei N. ORLOV ◽

Chad M. KITCHEN ◽

Tatyana A. VOYNO-YASENETSKAYA ◽

Joseph M. MIANO

Keyword(s):

Smooth Muscle ◽

Protein Kinase ◽

G Protein ◽

Transcriptional Activation ◽

Fold Increase ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Mitogen Activated Protein Kinase ◽

G Protein Coupled

A hallmark of cultured smooth muscle cells (SMCs) is the rapid down-regulation of several lineage-restricted genes that define their in vivo differentiated phenotype. Identifying factors that maintain an SMC differentiated phenotype has important implications in understanding the molecular underpinnings governing SMC differentiation and their subversion to an altered phenotype in various disease settings. Here, we show that several G-protein coupled receptors [α-thrombin, lysophosphatidic acid and angiotensin II (AII)] increase the expression of smooth muscle calponin (SM-Calp) in rat and human SMC. The increase in SM-Calp protein appears to be selective for G-protein-coupled receptors as epidermal growth factor was without effect. Studies using AII showed a 30-fold increase in SM-Calp protein, which was dose- and time-dependent and mediated by the angiotensin receptor-1 (AT1 receptor). The increase in SM-Calp protein with AII was attributable to transcriptional activation of SM-Calp based on increases in steady-state SM-Calp mRNA, increases in SM-Calp promoter activity and complete abrogation of protein induction with actinomycin D. To examine the potential role of extracellular signal-regulated kinase (Erk1/2), protein kinase B, p38 mitogen-activated protein kinase and protein kinase C in AII-induced SM-Calp, inhibitors to each of the signalling pathways were used. None of these signalling molecules appears to be crucial for AII-induced SM-Calp expression, although Erk1/2 may be partially involved. These results identify SM-Calp as a target of AII-mediated signalling, and suggest that the SMC response to AII may incorporate a novel activity of SM-Calp.

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Disabling Gβγ SNARE interaction in transgenic mice disrupts GPCR-mediated presynaptic inhibition leading to physiological and behavioral phenotypes

10.1101/280347 ◽

2018 ◽

Cited By ~ 2

Author(s):

Zack Zurawski ◽

Analisa D. Thompson Gray ◽

Lillian J. Brady ◽

Brian Page ◽

Emily Church ◽

...

Keyword(s):

Transgenic Mice ◽

Calcium Channels ◽

G Protein ◽

Presynaptic Inhibition ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Voltage Gated ◽

Voltage Gated Calcium Channels ◽

Crucial Component ◽

G Protein Coupled

ABSTRACTGi/o-coupled G-protein coupled receptors modulate neurotransmission presynaptically through inhibition of exocytosis. Release of Gβγ subunits decreases the activity of voltage-gated calcium channels (VGCC), decreasing excitability. A less understood Gβγ–mediated mechanism downstream of calcium entry is the binding of Gβγ to SNARE complexes. Here, we create a mouse partially deficient in this interaction. SNAP25Δ3 homozygote animals are developmentally normalbut impaired gait and supraspinal nociception. They also have elevated stress-induced hyperthermia and impaired inhibitory postsynaptic responses to α2A-AR, but normal inhibitory postsynaptic responses to Gi/o-coupled GABAB receptor activation. SNAP25Δ3 homozygotes have deficits in inhibition of hippocampal postsynaptic responses to 5 HT1b agonists that affect hippocampal learning. These data suggest that Gi/o-coupled GPCR inhibition of exocytosis through the Gβγ-SNARE interaction is a crucial component of numerous physiological and behavioral processes.

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Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806077115 ◽

2018 ◽

Vol 115 (37) ◽

pp. 9252-9257 ◽

Cited By ~ 17

Author(s):

Jamie L. Lahvic ◽

Michelle Ammerman ◽

Pulin Li ◽

Megan C. Blair ◽

Emma R. Stillman ◽

...

Keyword(s):

Fatty Acids ◽

G Protein ◽

Cell Lines ◽

Saturated Fatty Acids ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Hydroxy Fatty Acids ◽

High Affinity ◽

G Protein Coupled

Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling molecules with cardioprotective and vasodilatory actions. We recently showed that 11,12-EET enhances hematopoietic induction and engraftment in mice and zebrafish. EETs are known to signal via G protein-coupled receptors, with evidence supporting the existence of a specific high-affinity receptor. Identification of a hematopoietic-specific EET receptor would enable genetic interrogation of EET signaling pathways, and perhaps clinical use of this molecule. We developed a bioinformatic approach to identify an EET receptor based on the expression of G protein-coupled receptors in cell lines with differential responses to EETs. We found 10 candidate EET receptors that are expressed in three EET-responsive cell lines, but not expressed in an EET-unresponsive line. Of these, only recombinant GPR132 showed EET-responsiveness in vitro, using a luminescence-based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b prevented EET-induced hematopoiesis, and marrow from GPR132 knockout mice showed decreased long-term engraftment capability. In contrast to high-affinity EET receptors, GPR132 is reported to respond to additional hydroxy-fatty acids in vitro, and we found that these same hydroxy-fatty acids enhance hematopoiesis in the zebrafish. We conducted structure–activity relationship analyses using both cell culture and zebrafish assays on diverse medium-chain fatty acids. Certain oxygenated, unsaturated free fatty acids showed high activation of GPR132, whereas unoxygenated or saturated fatty acids had lower activity. Absence of the carbon-1 position carboxylic acid prevented activity, suggesting that this moiety is required for receptor activation. GPR132 responds to a select panel of oxygenated polyunsaturated fatty acids to enhance both embryonic and adult hematopoiesis.

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Molecular mechanisms of GABAB receptor activation: new insights from the mechanism of action of CGP7930, a positive allosteric modulator

Biochemical Society Transactions ◽

10.1042/bst0320871 ◽

2004 ◽

Vol 32 (5) ◽

pp. 871-872 ◽

Cited By ~ 15

Author(s):

V. Binet ◽

C. Goudet ◽

C. Brajon ◽

L. Le Corre ◽

F. Acher ◽

...

Keyword(s):

G Protein ◽

Mechanism Of Action ◽

Molecular Mechanisms ◽

Gabab Receptor ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Class Iii ◽

G Protein Coupled ◽

New Strategies

The GABAB (γ-aminobutyric acid-B) receptor is composed of two subunits, GABAB1 and GABAB2. Both subunits share structural homology with other class-III G-protein-coupled receptors. They contain two main domains, a heptahelical domain typical of all G-protein-coupled receptors and a large ECD (extracellular domain). It has not been demonstrated whether the association of these two subunits is always required for function. However, GABAB2 plays a major role in coupling with G-proteins, and GABAB1 has been shown to bind GABA. To date, only ligands interacting with GABAB1-ECD have been identified. In the present study, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABAB receptor. We have shown that it can weakly activate the wild-type GABAB receptor, but also the GABAB2 expressed alone, thus being the first described agonist of GABAB2. CGP7930 retains its weak agonist activity on a GABAB2 subunit deleted of its ECD. Thus the heptahelical domain of GABAB2 behaves similar to a rhodopsin-like receptor. These results open new strategies for studying the mechanism of activation of GABAB receptor and examine any possible role of GABAB2.

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Dissecting the allosteric networks governing agonist efficacy and potency in G protein-coupled receptors

10.1101/2021.09.14.460253 ◽

2021 ◽

Author(s):

Franziska Marie Heydenreich ◽

Maria Marti-Solano ◽

Manbir Sandhu ◽

Brian K Kobilka ◽

Michel Bouvier ◽

...

Keyword(s):

G Protein ◽

Conformational Changes ◽

Structural Changes ◽

Receptor Activation ◽

Residue Level ◽

G Protein Coupled Receptors ◽

Maximal Response ◽

Pharmacological Properties ◽

Receptor Ligand ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) translate binding of extracellular ligands into intracellular responses through conformational changes. Ligand properties are described by the maximum response (efficacy) and the agonist concentration at half-maximal response (potency). Integrating structural changes with pharmacological properties remains challenging and has not yet been performed at the resolution of individual amino acids. We use epinephrine and β2-adrenergic receptor as a model to integrate residue-level pharmacology data with intramolecular residue contact data describing receptor activation. This unveils the allosteric networks driving ligand efficacy and potency. We provide detailed insights into how structural rearrangements are linked to fundamental pharmacological properties at single-residue level in a receptor-ligand system. Our approach can be used to determine such pharmacological networks for any receptor-ligand complex.

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Decision letter: Computational design of thermostabilizing point mutations for G protein-coupled receptors

10.7554/elife.34729.018 ◽

2018 ◽

Keyword(s):

G Protein ◽

Point Mutations ◽

Computational Design ◽

G Protein Coupled Receptors ◽

G Protein Coupled

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G Protein-Coupled Receptors in Taste Physiology and Pharmacology

Frontiers in Pharmacology ◽

10.3389/fphar.2020.587664 ◽

2020 ◽

Vol 11 ◽

Author(s):

Raise Ahmad ◽

Julie E. Dalziel

Keyword(s):

Binding Site ◽

G Protein ◽

Molecular Mechanisms ◽

Taste Receptor ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Type I ◽

Taste Receptors ◽

Agonist Binding ◽

G Protein Coupled

Heterotrimeric G protein-coupled receptors (GPCRs) comprise the largest receptor family in mammals and are responsible for the regulation of most physiological functions. Besides mediating the sensory modalities of olfaction and vision, GPCRs also transduce signals for three basic taste qualities of sweet, umami (savory taste), and bitter, as well as the flavor sensation kokumi. Taste GPCRs reside in specialised taste receptor cells (TRCs) within taste buds. Type I taste GPCRs (TAS1R) form heterodimeric complexes that function as sweet (TAS1R2/TAS1R3) or umami (TAS1R1/TAS1R3) taste receptors, whereas Type II are monomeric bitter taste receptors or kokumi/calcium-sensing receptors. Sweet, umami and kokumi receptors share structural similarities in containing multiple agonist binding sites with pronounced selectivity while most bitter receptors contain a single binding site that is broadly tuned to a diverse array of bitter ligands in a non-selective manner. Tastant binding to the receptor activates downstream secondary messenger pathways leading to depolarization and increased intracellular calcium in TRCs, that in turn innervate the gustatory cortex in the brain. Despite recent advances in our understanding of the relationship between agonist binding and the conformational changes required for receptor activation, several major challenges and questions remain in taste GPCR biology that are discussed in the present review. In recent years, intensive integrative approaches combining heterologous expression, mutagenesis and homology modeling have together provided insight regarding agonist binding site locations and molecular mechanisms of orthosteric and allosteric modulation. In addition, studies based on transgenic mice, utilizing either global or conditional knock out strategies have provided insights to taste receptor signal transduction mechanisms and their roles in physiology. However, the need for more functional studies in a physiological context is apparent and would be enhanced by a crystallized structure of taste receptors for a more complete picture of their pharmacological mechanisms.

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A Microdomain Formed by the Extracellular Ends of the Transmembrane Domains Promotes Activation of the G Protein-Coupled α-Factor Receptor

Molecular and Cellular Biology ◽

10.1128/mcb.24.5.2041-2051.2004 ◽

2004 ◽

Vol 24 (5) ◽

pp. 2041-2051 ◽

Cited By ~ 18

Author(s):

Jennifer C. Lin ◽

Ken Duell ◽

James B. Konopka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Transmembrane Domain ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Transmembrane Domains ◽

Consecutive Series ◽

Subsequent Step ◽

G Protein Coupled ◽

Specific Reagent

ABSTRACT The α-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.

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