scholarly journals Over-expression of the Regulator of the Signaling of G Protein-Coupled Receptors RGS9-2 reduces the signaling elicited by the human histamine H3 receptor in HEK-293T cells

2021 ◽  
Author(s):  
Gustavo Nieto-Alamilla ◽  
Juan Escamilla-Sánchez ◽  
José Antonio Arias-Montaño
Keyword(s):  
G Protein ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Protein Activation ◽  
Over Expression ◽  
G Protein Activation ◽  
Significant Difference ◽  
Gtpγs Binding ◽  
G Protein Coupled ◽  
Human Histamine

In HEK-293T cells transiently transfected with the human histamine H3 receptor (hH3R), we studied the effect of over-expressing the human RGS9-2 protein on H3R-mediated stimulation of [35S]-GTPγS binding and inhibition of forskolin-induced cAMP formation. Maximal specific binding (Bmax) of [3H]-N-methyl-histamine to cell membranes was 468 ± 12 and 442 ± 38 fmol/mg protein for HEK-293T-hH3R and HEK-293T-hH3R/hRGS9-2 cells, respectively, with dissociation constants (Kd) 2.57 nM and 3.38 nM. The H3R agonist immepip stimulated [35S]-GTPγS binding with similar potency and efficacy (Emax 146.3 ± 4.4 % and 150.0 ± 5.3 % of basal, pEC50 8.57 ± 0.26 and 9.00 ± 0.33, respectively), but was significantly less efficacious to inhibit forskolin-induced cAMP accumulation in HEK-293T-hH3R/hRGS9 cells (-19.2 ± 5.3 versus -37.7 ± 5.1 % in HEK-293T-hH3R cells) with no significant difference in potency (pEC50 9.60 ± 0.14 and 9.07 ± 0.29, respectively). These results indicate that in HEK-293T cells hRGS9-2 regulates hH3R445 signaling downstream G protein activation

scholarly journals Novel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations

2020 ◽  
Vol 295 (51) ◽  
pp. 17486-17496
Author(s):  
Christopher T. Schafer ◽  
Anthony Shumate ◽  
David L. Farrens
Keyword(s):  
G Protein ◽  
Protein Activation ◽  
Canonical Class ◽  
Equilibrium Binding ◽  
C Terminus ◽  
G Protein Activation ◽  
Ligand Discovery ◽  
Pharmaceutical Agents ◽  
G Protein Coupled ◽  
Covalently Bound

Rhodopsin is a canonical class A photosensitive G protein–coupled receptor (GPCR), yet relatively few pharmaceutical agents targeting this visual receptor have been identified, in part due to the unique characteristics of its light-sensitive, covalently bound retinal ligands. Rhodopsin becomes activated when light isomerizes 11-cis-retinal into an agonist, all-trans-retinal (ATR), which enables the receptor to activate its G protein. We have previously demonstrated that, despite being covalently bound, ATR can display properties of equilibrium binding, yet how this is accomplished is unknown. Here, we describe a new approach for both identifying compounds that can activate and attenuate rhodopsin and testing the hypothesis that opsin binds retinal in equilibrium. Our method uses opsin-based fluorescent sensors, which directly report the formation of active receptor conformations by detecting the binding of G protein or arrestin fragments that have been fused onto the receptor's C terminus. We show that these biosensors can be used to monitor equilibrium binding of the agonist, ATR, as well as the noncovalent binding of β-ionone, an antagonist for G protein activation. Finally, we use these novel biosensors to observe ATR release from an activated, unlabeled receptor and its subsequent transfer to the sensor in real time. Taken together, these data support the retinal equilibrium binding hypothesis. The approach we describe should prove directly translatable to other GPCRs, providing a new tool for ligand discovery and mutant characterization.

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 activation of p42 and p44 mitogen-activated protein kinases following expression of the mouse δ opioid receptor in Rat-1 fibroblasts: effects of receptor expression levels and comparisons with G-protein activation

1996 ◽  
Vol 320 (1) ◽  
pp. 227-235 ◽  
Author(s):  
Andrew R BURT ◽  
I. Craig CARR ◽  
Ian MULLANEY ◽  
Neil G. ANDERSON ◽  
Graeme MILLIGAN
Keyword(s):  
Membrane Protein ◽  
G Protein ◽  
Opioid Receptor ◽  
Gtpase Activity ◽  
Protein Activation ◽  
G Protein Activation ◽  
Mitogen Activated Protein ◽  
Δ Opioid Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

Rat-1 fibroblasts were transfected with a cDNA encoding the mouse Δ opioid receptor. Two separate clones, D2 (which expressed some 6 pmol of the receptor/mg of membrane protein) and DOE (which expressed some 0.2 pmol/mg of membrane protein), were examined in detail. With membranes from both clones, the opioid agonist [D-Ala2]leucine enkephalin (DADLE) caused stimulation of high-affinity GTPase activity and of the binding of guanosine 5´-[γ-[35S]thio]triphosphate, and inhibition of forskolin-amplified adenylate cyclase activity. DADLE also induced phosphorylation and activation of both the p42MAPK (42 kDa isoform) and p44MAPK (44 kDa isoform) members of the mitogen-activated protein kinase (MAP kinase) family. All of these effects of DADLE were prevented in both clones by pretreatment of the cells with pertussis toxin. The maximal response that could be produced by DADLE in direct assays of G-protein activation were substantially greater in clone D2 than in clone DOE, but in both clones essentially full phosphorylation of both p42MAPK and p44MAPK could be achieved. EC50 values for DADLE stimulation of GTPase activity and for activation of p44MAPK were substantially lower in clone D2 than in clone DOE. Moreover, in both clones the EC50 value for DADLE stimulation of p44MAPK was substantially lower than that for stimulation of GTPase activity, and the Hill coefficients for agonist activation of p44MAPK (h > 1) displayed marked co-operativity whereas those for G-protein activation did not (h 0.8–1.0). DADLE activation of p44MAPK showed more sustained kinetics in clone D2 than in clone DOE. By contrast, lysophosphatidic acid, acting at an endogenously expressed G-protein-coupled receptor, also activated p44MAPK in both clones in a pertussis toxin-sensitive manner, but both the kinetics and the concentration–response curve for activation of p44MAPK by this ligand were similar. As with other systems, maintained cellular levels of a cAMP analogue prevented the effects of both G-protein-coupled receptors on activation of p44MAPK. These results demonstrate for the first time that an opioid receptor, at least when expressed in Rat-1 fibroblasts, is able to initiate activation of the MAP kinase cascade in a Gi-dependent manner, and show that only a very small proportion of the cellular Gi population is required to be activated to result in full phosphorylation of the p42MAPK and p44MAPK MAP kinases.

scholarly journals Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Junke Liu ◽  
Zongyong Zhang ◽  
David Moreno-Delgado ◽  
James AR Dalton ◽  
Xavier Rovira ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cell Communication ◽  
Allosteric Modulators ◽  
Protein Activation ◽  
Metabotropic Glutamate ◽  
Allosteric Control ◽  
G Protein Activation ◽  
Gpcr Dimer ◽  
G Protein Coupled

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

scholarly journals Vibrational spectroscopy analysis of ligand efficacy in M2R

2021 ◽  
Author(s):  
Kota Katayama ◽  
Kohei Suzuki ◽  
Ryoji Suno ◽  
Ryoji Kise ◽  
Hirokazu Tsujimoto ◽  
...  
Keyword(s):  
G Protein ◽  
Conformational Changes ◽  
Muscarinic Acetylcholine Receptor ◽  
Physiological Effect ◽  
Attenuated Total Reflection ◽  
Strongly Correlated ◽  
Protein Activation ◽  
G Protein Activation ◽  
Conformational Alteration ◽  
G Protein Coupled

Abstract The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.

scholarly journals C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Anthony W Azevedo ◽  
Thuy Doan ◽  
Hormoz Moaven ◽  
Iza Sokal ◽  
Faiza Baameur ◽  
...  
Keyword(s):  
G Protein ◽  
Single Photon ◽  
Low Noise ◽  
Tight Control ◽  
G Protein Coupled Receptor ◽  
Rod Photoreceptors ◽  
Protein Activation ◽  
G Protein Activation ◽  
G Protein Coupled

Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity.

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