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

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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The Molecular Basis of G Protein–Coupled Receptor Activation

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-060614-033910 ◽

2018 ◽

Vol 87 (1) ◽

pp. 897-919 ◽

Cited By ~ 207

Author(s):

William I. Weis ◽

Brian K. Kobilka

Keyword(s):

Ligand Binding ◽

G Protein ◽

Conformational Changes ◽

Cellular Responses ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Active Conformation ◽

Allosteric Coupling ◽

External Stimuli ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) mediate the majority of cellular responses to external stimuli. Upon activation by a ligand, the receptor binds to a partner heterotrimeric G protein and promotes exchange of GTP for GDP, leading to dissociation of the G protein into α and βγ subunits that mediate downstream signals. GPCRs can also activate distinct signaling pathways through arrestins. Active states of GPCRs form by small rearrangements of the ligand-binding, or orthosteric, site that are amplified into larger conformational changes. Molecular understanding of the allosteric coupling between ligand binding and G protein or arrestin interaction is emerging from structures of several GPCRs crystallized in inactive and active states, spectroscopic data, and computer simulations. The coupling is loose, rather than concerted, and agonist binding does not fully stabilize the receptor in an active conformation. Distinct intermediates whose populations are shifted by ligands of different efficacies underlie the complex pharmacology of GPCRs.

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Emerging roles of adhesion G protein-coupled receptors

Biochemical Society Transactions ◽

10.1042/bst20201144 ◽

2021 ◽

Author(s):

Matthew Rosa ◽

Timothy Noel ◽

Matthew Harris ◽

Graham Ladds

Keyword(s):

G Protein ◽

Conformational Changes ◽

Current Knowledge ◽

Receptor Activation ◽

G Protein Coupled Receptors ◽

Extracellular Region ◽

Extracellular Matrix Components ◽

Ligand Interactions ◽

Tethered Ligand ◽

G Protein Coupled

Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles.

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Complexes between photoactivated rhodopsin and transducin: progress and questions

Biochemical Journal ◽

10.1042/bj20100270 ◽

2010 ◽

Vol 428 (1) ◽

pp. 1-10 ◽

Cited By ~ 32

Author(s):

Beata Jastrzebska ◽

Yaroslav Tsybovsky ◽

Krzysztof Palczewski

Keyword(s):

G Protein ◽

Conformational Changes ◽

Structural Changes ◽

Protein Complexes ◽

G Protein Coupled Receptors ◽

Heterotrimeric G Proteins ◽

Α Subunit ◽

Rapid Responses ◽

G Protein Α Subunit ◽

G Protein Coupled

Activation of GPCRs (G-protein-coupled receptors) leads to conformational changes that ultimately initiate signal transduction. Activated GPCRs transiently combine with and activate heterotrimeric G-proteins resulting in GTP replacement of GDP on the G-protein α subunit. Both the detailed structural changes essential for productive GDP/GTP exchange on the G-protein α subunit and the structure of the GPCR–G-protein complex itself have yet to be elucidated. Nevertheless, transient GPCR–G-protein complexes can be trapped by nucleotide depletion, yielding an empty-nucleotide G-protein–GPCR complex that can be isolated. Whereas early biochemical studies indicated formation of a complex between G-protein and activated receptor only, more recent results suggest that G-protein can bind to pre-activated states of receptor or even couple transiently to non-activated receptor to facilitate rapid responses to stimuli. Efficient and reproducible formation of physiologically relevant, conformationally homogenous GPCR–G-protein complexes is a prerequisite for structural studies designed to address these possibilities.

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Ion channels and G protein-coupled receptors as targets for invertebrate pest control: from past challenges to practical insecticides

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab089 ◽

2021 ◽

Author(s):

Yoshihisa Ozoe

Keyword(s):

Ion Channels ◽

G Protein ◽

Pest Control ◽

Gaba Receptors ◽

Chloride Channels ◽

Structural Changes ◽

G Protein Coupled Receptors ◽

Octopamine Receptors ◽

Sites Of Action ◽

G Protein Coupled

Abstract In the late 1970s, we discovered that toxic bicyclic phosphates inhibit the generation of miniature inhibitory junction potentials, implying their antagonism of γ-aminobutyric acid (GABA) receptors (GABARs; GABA-gated chloride channels). This unique mode of action provided a strong incentive for our research on GABARs in later years. Furthermore, minor structural changes conferred insect GABAR selectivity to this class of compounds, convincing us of the possibility of GABARs as targets for insecticides. Forty years later, third-generation insecticides acting as allosteric modulator antagonists at a distinctive site of action in insect GABARs were developed. G protein-coupled receptors (GPCRs) are also promising targets for pest control. We characterized phenolamine receptors functionally and pharmacologically. Of the tested receptors, β-adrenergic-like octopamine receptors were revealed to be the most sensitive to the acaricide/insecticide amitraz. Given the presence of multiple sites of action, ion channels and GPCRs remain potential targets for invertebrate pest control.

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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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Trimethylsilyl reporter groups for NMR studies of conformational changes in G protein‐coupled receptors

FEBS Letters ◽

10.1002/1873-3468.13382 ◽

2019 ◽

Vol 593 (10) ◽

pp. 1113-1121 ◽

Cited By ~ 3

Author(s):

Wanhui Hu ◽

Huixia Wang ◽

Yaguang Hou ◽

Yimei Hao ◽

Dongsheng Liu

Keyword(s):

G Protein ◽

Conformational Changes ◽

G Protein Coupled Receptors ◽

Nmr Studies ◽

G Protein Coupled

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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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