scholarly journals Measuring G Protein Activation with Spectrally Resolved Fluorescence Fluctuation Spectroscopy

2021 ◽  
Author(s):  
Daniel J. Foust ◽  
David W. Piston
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Matrix Analysis ◽  
Coupling Mechanism ◽  
Protein Signaling ◽  
Protein Activation ◽  
Collision Coupling ◽  
Assembled Complexes ◽  
Spectrally Resolved

AbstractG protein-coupled receptor signaling has been posited to occur through either collision coupling or pre-assembled complexes with G protein transducers. To investigate the dynamics of G protein signaling, we introduce fluorescence covariance matrix analysis (FCMA), a novel implementation of fluorescence cumulant analysis applied to spectrally resolved fluorescence images. We labeled the GPCR, Gα, and Gβγ units with distinct fluorescent protein labels and we applied FCMA to measure directly the complex formation during stimulation of dopamine and adrenergic receptors. To determine the prevalence of hetero-oligomers, we compared the GPCR data to those from control samples expressing three fluorescent protein labels with known stoichiometries. Interactions between Gα and Gβγ subunits determined by FCMA were sensitive to stimulation with GPCR ligands. However, GPCR/G protein interactions were too weak to be distinguished from background. These findings support a collision coupling mechanism rather than pre-assembled complexes for the two GPCRs studied.

scholarly journals Regulator of G protein signaling 17 represents a novel target for treating cisplatin induced hearing loss

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Asmita Dhukhwa ◽  
Raheem F. H. Al Aameri ◽  
Sandeep Sheth ◽  
Debashree Mukherjea ◽  
Leonard Rybak ◽  
...  
Keyword(s):  
Hearing Loss ◽  
G Protein ◽  
Protein Interactions ◽  
Cannabinoid Receptor ◽  
Significant Proportion ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Drug Induced ◽  
Male Wistar Rats

AbstractRegulators of G protein signaling (RGS) accelerate the GTPase activity of G proteins to enable rapid termination of the signals triggered by G protein-coupled receptors (GPCRs). Activation of several GPCRs, including cannabinoid receptor 2 (CB2R) and adenosine A1 receptor (A1AR), protects against noise and drug-induced ototoxicity. One such drug, cisplatin, an anticancer agent used to treat various solid tumors, produces permanent hearing loss in experimental animals and in a high percentage of cancer patients who undergo treatments. In this study we show that cisplatin induces the expression of the RGS17 gene and increases the levels of RGS17 protein which contributes to a significant proportion of the hearing loss. Knockdown of RGS17 suppressed cisplatin-induced hearing loss in male Wistar rats, while overexpression of RGS17 alone produced hearing loss in vivo. Furthermore, RGS17 and CB2R negatively regulate the expression of each other. These data suggest that RGS17 mediates cisplatin ototoxicity by uncoupling cytoprotective GPCRs from their normal G protein interactions, thereby mitigating the otoprotective contributions of endogenous ligands of these receptors. Thus, RGS17 represents a novel mediator of cisplatin ototoxicity and a potential therapeutic target for treating hearing loss.

scholarly journals RGS9-2–controlled adaptations in the striatum determine the onset of action and efficacy of antidepressants in neuropathic pain states

2015 ◽  
Vol 112 (36) ◽  
pp. E5088-E5097 ◽  
Author(s):  
Vasiliki Mitsi ◽  
Dimitra Terzi ◽  
Immanuel Purushothaman ◽  
Lefteris Manouras ◽  
Sevasti Gaspari ◽  
...  
Keyword(s):  
Neuropathic Pain ◽  
G Protein ◽  
Protein Interactions ◽  
Antidepressant Drug ◽  
Sequencing Analysis ◽  
Protein Signaling ◽  
Cellular Mechanisms ◽  
Onset Of Action ◽  
Injury Model ◽  
Drug Actions

The striatal protein Regulator of G-protein signaling 9-2 (RGS9-2) plays a key modulatory role in opioid, monoamine, and other G-protein–coupled receptor responses. Here, we use the murine spared-nerve injury model of neuropathic pain to investigate the mechanism by which RGS9-2 in the nucleus accumbens (NAc), a brain region involved in mood, reward, and motivation, modulates the actions of tricyclic antidepressants (TCAs). Prevention of RGS9-2 action in the NAc increases the efficacy of the TCA desipramine and dramatically accelerates its onset of action. By controlling the activation of effector molecules by G protein α and βγ subunits, RGS9-2 affects several protein interactions, phosphoprotein levels, and the function of the epigenetic modifier histone deacetylase 5, which are important for TCA responsiveness. Furthermore, information from RNA-sequencing analysis reveals that RGS9-2 in the NAc affects the expression of many genes known to be involved in nociception, analgesia, and antidepressant drug actions. Our findings provide novel information on NAc-specific cellular mechanisms that mediate the actions of TCAs in neuropathic pain states.

Competition Between Internal AlF4 − and Receptor-Mediated Stimulation of Dorsal Raphe Neuron G-Proteins Coupled to Calcium Current Inhibition

2000 ◽  
Vol 83 (3) ◽  
pp. 1273-1282 ◽  
Author(s):  
Yuan Chen ◽  
Nicholas J. Penington
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Inhibitory Effect ◽  
Dorsal Raphe ◽  
Protein Activation ◽  
Bath Application ◽  
G Protein Activation ◽  
Rate Kinetics ◽  
Kinetics Of ◽  
Dorsal Raphé

Intracellular aluminum fluoride (AlF4 −), placed in a patch pipette, activated a G-protein, resulting in a “tonic” inhibition of the Ca2+ current of isolated serotonergic neurons of the rat dorsal raphe nucleus. Serotonin (5-HT) also inhibits the Ca2+ current of these cells. After external bath application and quick removal of 5-HT to an AlF4 −containing cell, there was a reversal or transient disinhibition (TD) of the inhibitory effect of AlF4 − on Ca2+current. A short predepolarization of the membrane potential to +70 mV, a condition that is known to reverse G-protein–mediated inhibition, reversed the inhibitory effect of AlF4 − on Ca2+ current and brought the Ca2+ current to the same level as that seen at the peak of the TD current. With AlF4 − in the pipette, the TD phenomenon could be eliminated by lowering pipette MgATP, or by totally chelating pipette Al3+. In the presence of AlF4 −, but with either lowered MgATP or extreme efforts to eliminate pipette Al3+, the rate of recovery from 5-HT on wash was slowed, a condition opposite to that where a TD occurred. The putative complex of AlF4 −-bound G-protein (Gα·GDP·AlF4 −) appeared to free G-βγ-subunits, mimicking the effect on Ca2+ channels of the G·GTP complex. The on-rate of the inhibition of Ca2+ current, after a depolarizing pulse, by βγ-subunits released by AlF4 − in the pipette was significantly slower than that of the agonist-activated G-protein. Theoff-rate of the AlF4 −-mediated inhibition in response to a depolarizing pulse, a measure of the affinity of the free G-βγ-subunit for the Ca2+ channel, was slightly slower than that of the agonist stimulated G-protein. In summary, AlF4 − modified the off-rate kinetics of G-protein activation by agonists, but had little effect on the kinetics of the interaction of the βγ-subunit with Ca2+channels. Agonist application temporarily reversed the effects of AlF4 −, making it a complementary tool to GTP-γ-S for the study of G-protein interactions.

Mechanical and chemical activation of GPR68 (OGR1) probed with a genetically-encoded fluorescent reporter

2021 ◽  
Author(s):  
Alper D. Ozkan ◽  
Tina Gettas ◽  
Audrey Sogata ◽  
Wynn Phaychanpheng ◽  
Miou Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
Cancer Progression ◽  
Fluid Shear Stress ◽  
Fluorescent Protein ◽  
Chemical Activation ◽  
G Protein Signaling ◽  
Mechanical Stimuli ◽  
Protein Signaling ◽  
Fluorescent Reporter ◽  
Membrane Stretch

G-protein coupled receptor (GPCR) 68 (GPR68, or OGR1) couples extracellular acidifications and mechanical stimuli to G-protein signaling and plays important roles in vascular physiology, neuroplasticity, and cancer progression. Inspired by previous GPCR-based reporters, here, we inserted a cyclic permuted fluorescent protein into the third intracellular loop of GPR68 to create a genetically-encoded fluorescent reporter of GPR68 activation we call "iGlow". iGlow responds to known physiological GPR68 activators such as fluid shear stress and extracellular acidifications. In addition, iGlow responds to Ogerin, a synthetic GPR68-selective agonist, but not to a non-active Ogerin analog, showing the specificity of iGlow-mediated fluorescence signals. Flow-induced iGlow activation is not eliminated by pharmacological modulation of downstream G-protein signaling, disruption of actin filaments, or application of GsMTx4, an inhibitor of certain mechanosensitive ion channels activated by membrane stretch. Deletion of the conserved Helix 8, proposed to mediate mechanosensitivity in certain GPCRs, does not eliminate flow-induced iGlow activation. iGlow could be useful to investigate the contribution of GPR68-dependent signaling in health and disease.

scholarly journals C5a-Stimulated Recruitment of β-Arrestin2 to the Nonsignaling 7-Transmembrane Decoy Receptor C5L2

2009 ◽  
Vol 14 (9) ◽  
pp. 1067-1075 ◽  
Author(s):  
Lambertus H.C. Van Lith ◽  
Julia Oosterom ◽  
Andrea Van Elsas ◽  
Guido J.R. Zaman
Keyword(s):  
G Protein ◽  
Fluorescent Protein ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Ligand Interaction ◽  
C5a Receptor ◽  
Decoy Receptor ◽  
Complementation Assay ◽  
Fragment Complementation

C5L2 (or GPR77) is a high-affinity receptor for the complement fragment C5a and its desarginated product, C5a-desArg. Unlike the classical C5a receptor CD88, C5L2 does not couple to intracellular G-protein-signaling pathways but is thought to function as a decoy receptor. The authors show that stimulation of C5L2 with C5a and C5a-desArg induces redistribution of green fluorescent protein—labeled β-arrestin2 to cytoplasmic vesicles. C3a and C3a-desArg were inactive in the β-arrestin translocation assay. Direct interaction of ligand-stimulated C5L2 with β-arrestin was confirmed using a novel β-galactosidase fragment complementation assay. In this assay, C5L2 was labeled with a mutationally altered peptide fragment of β-galactosidase, whereas β-arrestin2 was labeled with a corresponding deletion mutant of the enzyme. Stable transfection of the modified C5L2 and subsequent stimulation with C5a or C5a-desArg restored β-galactosidase activity in a dose-dependent manner. The subnanomolar potency of β-arrestin coupling in the β-galactosidase fragment complementation assay is in agreement with the affinity of the receptor-ligand interaction. C5L2 is the first example of a 7-transmembrane decoy receptor that couples to β-arrestin in a ligand-dependent manner. This observation supports the notion that G-protein-signaling and β-arrestin coupling can be 2 separate activities of 7-transmembrane receptors. Furthermore, the β-arrestin assays described in this article provide methods of screening for selective C5L2 modulators. ( Journal of Biomolecular Screening 2009:1067-1075)

Structural basis of function in heterotrimeric G proteins

2006 ◽  
Vol 39 (2) ◽  
pp. 117-166 ◽  
Author(s):  
William M. Oldham ◽  
Heidi E. Hamm
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Complex ◽  
Effector Proteins ◽  
Protein Signaling ◽  
Structural Determinants ◽  
Α Subunit ◽  
Protein Activation ◽  
Downstream Effector ◽  
G Protein Activation

1. Introduction 22. Heterotrimeric G-protein structure 32.1. G-protein α subunit 32.2. G-protein βγ dimer 82.3. Unique role of Gβ5 in complexes with RGS proteins 92.4. Heterotrimer structure 102.5. Lipid modifications direct membrane association 113. Receptor–G protein complex 113.1. Low affinity interactions between inactive receptors (R) and G proteins 113.2. Receptor activation exposes the high-affinity G-protein binding site 123.3. Receptor–G protein interface 143.4. Structural determinants of receptor–G protein specificity 153.5. Models of the receptor–G protein complex 173.6. Sequential interactions may form the receptor–G protein complex 194. Molecular basis for G-protein activation 194.1. Potential mechanisms of receptor-catalyzed GDP release 204.2. GTP-mediated alteration of the receptor–G protein complex 235. Activation of downstream effector proteins 245.1. Gα interactions with effectors 245.2. Gβγ interactions with effectors and regulatory proteins 266. G-protein inactivation 286.1. Intrinsic GTPase-activity of Gα 286.2. GTPase-activating proteins 307. Novel regulation of G-protein signaling 318. New approaches to study G-protein dynamics 328.1. Nuclear magnetic resonance spectroscopy 328.2. Site-directed labeling techniques 338.3. Mapping allosteric connectivity with computational approaches 348.4. Studies of G-protein function in living cells 369. Conclusions 3710. References 38Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein α subunit (Gα) binds GDP and Gβγ. Receptors activate G proteins by catalyzing GTP for GDP exchange on Gα, leading to a structural change in the Gα(GTP) and Gβγ subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Gα subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Gα, how these changes may lead to subunit dissociation and allow Gα and Gβγ to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor–G protein complex and how this interaction leads to GDP release from Gα. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor–G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

scholarly journals A Dynamic, Split-Luciferase-Based Mini-G Protein Sensor to Functionally Characterize Ligands at All Four Histamine Receptor Subtypes

2020 ◽  
Vol 21 (22) ◽  
pp. 8440
Author(s):  
Carina Höring ◽  
Ulla Seibel ◽  
Katharina Tropmann ◽  
Lukas Grätz ◽  
Denise Mönnich ◽  
...  
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Signal Amplitude ◽  
Histamine Receptor ◽  
Receptor Activation ◽  
Functional Assay ◽  
Receptor Subtypes ◽  
Protein Activation ◽  
Drug Candidates ◽  
Specific Effects

In drug discovery, assays with proximal readout are of great importance to study target-specific effects of potential drug candidates. In the field of G protein-coupled receptors (GPCRs), the determination of GPCR-G protein interactions and G protein activation by means of radiolabeled GTP analogs ([35S]GTPγS, [γ-32P]GTP) has widely been used for this purpose. Since we were repeatedly faced with insufficient quality of radiolabeled nucleotides, there was a requirement to implement a novel proximal functional assay for the routine characterization of putative histamine receptor ligands. We applied the split-NanoLuc to the four histamine receptor subtypes (H1R, H2R, H3R, H4R) and recently engineered minimal G (mini-G) proteins. Using this method, the functional response upon receptor activation was monitored in real-time and the four mini-G sensors were evaluated by investigating selected standard (inverse) agonists and antagonists. All potencies and efficacies of the studied ligands were in concordance with literature data. Further, we demonstrated a significant positive correlation of the signal amplitude and the mini-G protein expression level in the case of the H2R, but not for the H1R or the H3R. The pEC50 values of histamine obtained under different mini-G expression levels were consistent. Moreover, we obtained excellent dynamic ranges (Z’ factor) and the signal spans were improved for all receptor subtypes in comparison to the previously performed [35S]GTPγS binding assay.

Sign in / Sign up

Export Citation Format

Share Document