scholarly journals Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex

2015 ◽  
Vol 112 (5) ◽  
pp. 1601-1606 ◽  
Author(s):  
Marjorie Damian ◽  
Sophie Mary ◽  
Mathieu Maingot ◽  
Céline M'Kadmi ◽  
Didier Gagne ◽  
...  
Keyword(s):  
G Protein ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Active Conformation ◽  
Ghrelin Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Open Question

How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.

scholarly journals Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor

2019 ◽  
Vol 20 (15) ◽  
pp. 3724 ◽  
Author(s):  
Tamara A. M. Mocking ◽  
Maurice C. M. L. Buzink ◽  
Rob Leurs ◽  
Henry F. Vischer
Keyword(s):  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Short Residence Time ◽  
Protein Activation ◽  
G Protein Activation ◽  
Activation Assay ◽  
G Protein Coupled ◽  
Histamine H3

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the ‘effective’ target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R.

scholarly journals Agonist-induced formation of unproductive receptor-G12complexes

2020 ◽  
Vol 117 (35) ◽  
pp. 21723-21730
Author(s):  
Najeah Okashah ◽  
Shane C. Wright ◽  
Kouki Kawakami ◽  
Signe Mathiasen ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Protein Association ◽  
Protein Activation ◽  
Protein Receptor ◽  
G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

scholarly journals Frizzled BRET sensors based on bioorthogonal labeling of unnatural amino acids reveal WNT-induced dynamics of the cysteine-rich domain.

2021 ◽  
Author(s):  
Maria Kowalski-Jahn ◽  
Hannes Schihada ◽  
Ainoleena Turku ◽  
Thomas Huber ◽  
Thomas P. Sakmar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Membrane Receptors ◽  
Rich Domain ◽  
Linker Domain ◽  
Cysteine Rich Domain

Frizzleds (FZD1-10) comprise a class of G protein-coupled receptors containing an extracellular cysteine-rich domain (CRD) that binds lipoglycoproteins of the Wingless/Int-1 family (WNTs). Despite the prominent role of the WNT/FZD system in health and disease, our understanding of how WNT binding to the FZD CRD is translated into receptor activation and transmembrane signaling remains limited. Current hypotheses dispute the roles for conformational dynamics and the involvement of the linker domain connecting the CRD with the seven-helical transmembrane core of FZD. To clarify the mechanism of WNT binding to FZD and to elucidate how WNT/FZD complexes achieve signaling pathway specificity, we devised conformational FZD-CRD biosensors based on bioluminescence-resonance-energy-transfer (BRET). Using FZD engineered with N-terminal nanoluciferase and fluorescently-labeled unnatural amino acids in the linker domain and extracellular loop 3, we show that WNT-3A and WNT-5A induce similar CRD conformational rearrangements despite promoting distinct downstream signaling pathways, and that CRD dynamics are not required for WNT/β-catenin signaling. Thus, the novel FZD-CRD biosensors we report provide insights into the stepwise binding, activation and signaling processes in FZDs. The sensor design is broadly applicable to explore fundamental events in signal transduction mediated by other membrane receptors.

scholarly journals Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Maxime Louet ◽  
Marina Casiraghi ◽  
Marjorie Damian ◽  
Mauricio GS Costa ◽  
Pedro Renault ◽  
...  
Keyword(s):  
G Protein ◽  
Unnatural Amino Acids ◽  
Conformational Dynamics ◽  
Signal Propagation ◽  
Water Molecules ◽  
Growth Hormone Secretagogue Receptor ◽  
Growth Hormone Secretagogue ◽  
Ghrelin Receptor ◽  
Tightly Coupled ◽  
G Protein Coupled

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Kinetics of G-protein-coupled receptor signalling and desensitization

2004 ◽  
Vol 32 (6) ◽  
pp. 1029-1031 ◽  
Author(s):  
C. Krasel ◽  
J.-P. Vilardaga ◽  
M. Bünemann ◽  
M.J. Lohse
Keyword(s):  
G Protein ◽  
Second Messengers ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
G Protein Coupled Receptor ◽  
Protein Subunit ◽  
Protein Activation ◽  
G Protein Coupled ◽  
Kinetics Of

The kinetics of G-protein-coupled receptor activation and deactivation has, so far, been measured only indirectly, most frequently by assessing the production of various second messengers. We have developed methods based on fluorescence resonance energy transfer to quantify the kinetics of receptor activation by agonist (measured as conformational change in the receptor), the kinetics of G-protein activation (measured as G-protein subunit rearrangement) and the kinetics of receptor inactivation by arrestins (measured as receptor–arrestin interaction). Using these methods, we show that receptor activation by agonists and signalling to G-proteins occur on the subsecond time scale, whereas receptor desensitization is limited by receptor phosphorylation and proceeds more slowly.

scholarly journals Allosteric modulation of ghrelin receptor signaling by lipids

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marjorie Damian ◽  
Maxime Louet ◽  
Antoniel Augusto Severo Gomes ◽  
Céline M’Kadmi ◽  
Séverine Denoyelle ◽  
...  
Keyword(s):  
G Protein ◽  
Dependent Manner ◽  
Receptor Signaling ◽  
Ghrelin Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Preferential Binding ◽  
Membrane Components ◽  
G Protein Coupled

AbstractThe membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated.

Monitoring the Structural Dynamics of U2 snRNA Via Single-Molecule Förster Resonance Energy Transfer

2018 ◽  
Author(s):  
Alexander Carl DeHaven
Keyword(s):  
Energy Transfer ◽  
Structural Dynamics ◽  
Single Molecule ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
U2 Snrna ◽  
Folding Dynamics ◽  
The Impact

This thesis contains four topic areas: a review of single-molecule microscropy methods and splicing, conformational dynamics of stem II of the U2 snRNA, the impact of post-transcriptional modifications on U2 snRNA folding dynamics, and preliminary findings on Mango aptamer folding dynamics.

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