Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation

2021 ◽  
Author(s):  
Rebeca Diez-Alarcia ◽  
Yuji Odagaki ◽  
Patricia Miranda-Azpiazu ◽  
Ane M. Gabilondo ◽  
J. Javier Meana ◽  
...  
Keyword(s):  
Brain Tissue ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Postmortem Brain ◽  
Binding Assays ◽  
Postmortem Brain Tissue ◽  
Gtpγs Binding ◽  
G Protein Coupled

scholarly journals Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

2003 ◽  
Vol 8 (5) ◽  
pp. 500-510 ◽  
Author(s):  
Anke Niedernberg ◽  
Sorin Tunaru ◽  
Andree Blaukat ◽  
Bruce Harris ◽  
Evi Kostenis
Keyword(s):  
G Protein ◽  
Partial Agonist ◽  
A Priori ◽  
G Protein Coupled Receptors ◽  
Binding Assays ◽  
Functional Assays ◽  
Gtpγs Binding ◽  
Sphingosine 1 Phosphate ◽  
Intracellular Ca ◽  
G Protein Coupled

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P5 receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPγS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca2+ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC50 values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPγS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPγS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. ( Journal of Biomolecular Screening 2003:500-510)

scholarly journals Cell-Free Assay of G-Protein-Coupled Receptors Using Fluorescence Polarization

2008 ◽  
Vol 13 (5) ◽  
pp. 424-429 ◽  
Author(s):  
Jessi Wildeson Jones ◽  
Tiffani A. Greene ◽  
Christine A. Grygon ◽  
Benjamin J. Doranz ◽  
Martha P. Brown
Keyword(s):  
G Protein ◽  
Fluorescence Polarization ◽  
G Protein Coupled Receptors ◽  
Peptide Ligand ◽  
Binding Assays ◽  
Free System ◽  
Molecular Binding ◽  
Soluble Cell ◽  
G Protein Coupled ◽  
Peptide Interaction

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). ( Journal of Biomolecular Screening 2008:424-429)

GPR31 and GPR151 are activated under acidic conditions

2019 ◽  
Vol 166 (4) ◽  
pp. 317-322 ◽  
Author(s):  
Misaki Mashiko ◽  
Aya Kurosawa ◽  
Yuki Tani ◽  
Takashi Tsuji ◽  
Shigeki Takeda
Keyword(s):  
G Protein ◽  
Cho Cells ◽  
Fusion Proteins ◽  
Narrow Range ◽  
G Protein Coupled Receptors ◽  
Cellular Assays ◽  
Gtpγs Binding ◽  
Acidic Conditions ◽  
G Protein Coupled

Abstract Recent studies have revealed that not only proton-sensing channels, but also one family of G protein-coupled receptors (GPCRs) comprising OGR1, GPR4, G2A and TDAG8 are responsible for the sensing of extracellular protons, or pH. Here, we report that two other GPCRs, GPR31 and GPR151, were also activated in acidic condition. Elevated pH of assay mixtures resulted in a remarkable increase in [35S]GTPγS binding by GPR31–Giα and GPR151–Giα fusion proteins in a narrow range between pH 6 and 5. Our reporter gene assays with CHO cells expressing recombinant GPR31 or GPR151 also showed that activation was maximal at pH ∼5.8. Although these results from in vitro and cellular assays revealed slightly different pH sensitivities, all of our results indicated that GPR31 and GPR151 sensed extracellular protons equally well as other proton-sensing GPCRs.

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