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 Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

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 Identification of key phosphorylation sites in PTH1R that determine arrestin3 binding and fine-tune receptor signaling

2016 ◽  
Vol 473 (22) ◽  
pp. 4173-4192 ◽  
Author(s):  
Diana Zindel ◽  
Sandra Engel ◽  
Andrew R. Bottrill ◽  
Jean-Philippe Pin ◽  
Laurent Prézeau ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Mutational Analysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Phosphorylation Sites ◽  
Parathyroid Hormone Receptor ◽  
Extracellular Signal ◽  
G Protein Coupled

The parathyroid hormone receptor 1 (PTH1R) is a member of family B of G-protein-coupled receptors (GPCRs), predominantly expressed in bone and kidney where it modulates extracellular Ca2+ homeostasis and bone turnover. It is well established that phosphorylation of GPCRs constitutes a key event in regulating receptor function by promoting arrestin recruitment and coupling to G-protein-independent signaling pathways. Mapping phosphorylation sites on PTH1R would provide insights into how phosphorylation at specific sites regulates cell signaling responses and also open the possibility of developing therapeutic agents that could target specific receptor functions. Here, we have used mass spectrometry to identify nine sites of phosphorylation in the C-terminal tail of PTH1R. Mutational analysis revealed identified two clusters of serine and threonine residues (Ser489–Ser495 and Ser501–Thr506) specifically responsible for the majority of PTH(1–34)-induced receptor phosphorylation. Mutation of these residues to alanine did not affect negatively on the ability of the receptor to couple to G-proteins or activate extracellular-signal-regulated kinase 1/2. Using fluorescence resonance energy transfer and bioluminescence resonance energy transfer to monitor PTH(1–34)-induced interaction of PTH1R with arrestin3, we show that the first cluster Ser489–Ser495 and the second cluster Ser501–Thr506 operated in concert to mediate both the efficacy and potency of ligand-induced arrestin3 recruitment. We further demonstrate that Ser503 and Thr504 in the second cluster are responsible for 70% of arrestin3 recruitment and are key determinants for interaction of arrestin with the receptor. Our data are consistent with the hypothesis that the pattern of C-terminal tail phosphorylation on PTH1R may determine the signaling outcome following receptor activation.

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.

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