Comparative Analysis of the Efficacy of A1Adenosine Receptor Activation of Gi/oα G Proteins following Coexpression of Receptor and G Protein and Expression of A1Adenosine Receptor−Gi/oα Fusion Proteins†

Biochemistry ◽  
1999 ◽  
Vol 38 (8) ◽  
pp. 2272-2278 ◽  
Author(s):  
Alan Wise ◽  
Michael Sheehan ◽  
Stephen Rees ◽  
Melanie Lee ◽  
Graeme Milligan
Keyword(s):  
Comparative Analysis ◽  
G Protein ◽  
G Proteins ◽  
Fusion Proteins ◽  
Receptor Activation

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 Heterotrimeric G-protein shuttling via Gip1 extends the dynamic range of eukaryotic chemotaxis

2016 ◽  
Vol 113 (16) ◽  
pp. 4356-4361 ◽  
Author(s):  
Yoichiro Kamimura ◽  
Yukihiro Miyanaga ◽  
Masahiro Ueda
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Dynamic Range ◽  
Protein Translocation ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Heterotrimeric G Protein ◽  
Range Sensing ◽  
Interacting Protein ◽  
Wide Range

Chemotactic eukaryote cells can sense chemical gradients over a wide range of concentrations via heterotrimeric G-protein signaling; however, the underlying wide-range sensing mechanisms are only partially understood. Here we report that a novel regulator of G proteins, G protein-interacting protein 1 (Gip1), is essential for extending the chemotactic range of Dictyostelium cells. Genetic disruption of Gip1 caused severe defects in gradient sensing and directed cell migration at high but not low concentrations of chemoattractant. Also, Gip1 was found to bind and sequester G proteins in cytosolic pools. Receptor activation induced G-protein translocation to the plasma membrane from the cytosol in a Gip1-dependent manner, causing a biased redistribution of G protein on the membrane along a chemoattractant gradient. These findings suggest that Gip1 regulates G-protein shuttling between the cytosol and the membrane to ensure the availability and biased redistribution of G protein on the membrane for receptor-mediated chemotactic signaling. This mechanism offers an explanation for the wide-range sensing seen in eukaryotic chemotaxis.

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 Heparin and other polyanions uncouple α1-adrenoceptors from G-proteins

1991 ◽  
Vol 280 (3) ◽  
pp. 791-795 ◽  
Author(s):  
L L T Dasso ◽  
C W Taylor
Keyword(s):  
Adenylate Cyclase ◽  
G Protein ◽  
G Proteins ◽  
Alkaline Treatment ◽  
Receptor Activation ◽  
Maximal Effect ◽  
Intact Cells ◽  
High Affinity ◽  
Antagonist Binding ◽  
Coupling Mechanisms

Several polyanionic compounds antagonize the interaction between receptors and the G-proteins that regulate adenylate cyclase or K+ channels, possibly by binding to a basic stretch of the receptor that is proposed to mediate its interaction with the G-proteins. We have studied the effects of polyanions on the interaction between the liver alpha 1-adrenoceptor and the G-protein through which it stimulates polyphosphoinositide turnover. Heparin [concn. causing 50% of maximal effect (EC50) = 0.5 microM], Trypan Blue (EC50 7.1 microM) or suramin (EC50 2.1 microM) prevented formation of the high-affinity adrenaline-receptor-G-protein complex without affecting antagonist binding. After alkaline treatment of the membranes, previously reported to cause G-protein removal, binding of agonists was insensitive to both guanine nucleotides and heparin. We conclude that these polyanions uncouple the alpha 1-adrenoceptor from its G-protein, suggesting that similar coupling mechanisms may underlie receptor activation of the G-proteins that activate polyphosphoinositide hydrolysis and those that regulate adenylate cyclase. This action of heparin severely limits its utility as a selective antagonist of the Ins(1,4,5)P3 receptor in intact cells.

scholarly journals Implication of Gβγ proteins and c-SRC tyrosine kinase in parathyroid hormone-induced signal transduction in rat enterocytes

2006 ◽  
Vol 188 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Claudia Gentili ◽  
Ricardo Boland ◽  
Ana Russo de Boland
Keyword(s):  
Parathyroid Hormone ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
G Protein ◽  
G Proteins ◽  
Receptor Activation ◽  
Intestinal Cells ◽  
Second Phase ◽  
Acetoxymethyl Ester ◽  
Protein Tyrosine

Parathyroid hormone (PTH) interacts in target tissues with a G protein-coupled receptor (GPCR) localized in the plasma membrane. Although activation of GPCR can elicit rapid stimulation of cellular protein tyrosine phosphorylation, the mechanism by which G proteins activate protein-tyrosine kinases is not completely understood. In the present work, we demonstrate that PTH rapidly increases the activity of non-receptor tyrosine kinase c-Src in rat intestinal cells (enterocytes). The response is biphasic, the early phase is fast and transient, peaking at 30 s (+120%), while the second phase progressively increases up to 5 min (+220%). The hormone activates c-Src in intestinal cells through fast changes in tyrosine phosphorylation of the enzyme. The first event in the activation of c-Src is the dephosphorylation of Tyr527 (which happens after a few seconds of PTH treatment), followed by a second event of activation with phosphorylation at Tyr416 (+twofold, 5 min). Removal of external Ca2+ (EGTA, 0.5 mM) and chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid acetoxymethyl ester (BAPTA) (5 μM) suppressed Tyr527 dephosphorylation and Tyr416 phosphorylation, indicating that Ca2+ is an upstream activator of c-Src in enterocytes stimulated with PTH. The G protein subunits, Gαs and Gβ, are associated with c-Src in basal conditions and this association increases two- to threefold in cells treated with PTH. Blocking of Gβ subunits by preincubation of cells with a Gβ antibody abolished hormone-dependent c-Src Tyr416 phosphorylation and ERK1/ERK2 activation. The results of this work indicate that PTH activates c-Src in intestinal cells through conformational changes via G proteins and calcium-dependent modulation of tyrosine phosphorylation of the enzyme, and that PTH receptor activation leads via Gβγ–c-Src to the phosphorylation of the MAP kinases, ERK1 and ERK2.

scholarly journals Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling

2008 ◽  
Vol 19 (11) ◽  
pp. 4640-4650 ◽  
Author(s):  
Marilyne Labasque ◽  
Eric Reiter ◽  
Carine Becamel ◽  
Joël Bockaert ◽  
Philippe Marin
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cortical Neurons ◽  
Receptor Activation ◽  
Proximal Region ◽  
Dominant Negative ◽  
Binding Motif ◽  
Physical Interaction ◽  
Receptor Signaling ◽  
C Terminus

The serotonin (5-hydroxytryptamine; 5-HT)2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca2+-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT2C receptor agonists and their antidepressant-like activity.

G protein coupling profile of mGluR6 and expression of Gα proteins in retinal ON bipolar cells

2006 ◽  
Vol 23 (6) ◽  
pp. 909-916 ◽  
Author(s):  
LIANTIAN TIAN ◽  
PAUL J. KAMMERMEIER
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Glutamate Receptor ◽  
Intracellular Signaling ◽  
Metabotropic Glutamate Receptor ◽  
Sympathetic Neurons ◽  
Photoreceptor Cells ◽  
Receptor Activation ◽  
Bipolar Cells

Metabotropic glutamate receptor 6 (mGluR6) is a group III, pertussis toxin (PTX)-sensitive G protein coupled mGluR that plays a specialized role in the retina. Retinal ON bipolar cells, which receive direct glutamatergic input from photoreceptor cells, express mGluR6 as their primary postsynaptic glutamate receptor. Activation of mGluR6 in these cells initiates an intracellular signaling cascade ultimately leading to inhibition of a cation channel and cell hyperpolarization. The primary mediator of this pathway in vivo is Gαo, but the potential roles of other G proteins from the Gαi/o family in the regulation of this or other signaling pathways in ON bipolar cells are unclear. To determine which specific G proteins from the Gαi/o family are able to couple to mGluR6, a Gα reconstitution system was employed using PTX-insensitive Gα mutants expressed with mGluR6 in PTX-treated sympathetic neurons from the rat superior cervical ganglion (SCG). The efficiency of coupling to mGluR6 was Goa > Gob, Gi1 > Gi2, Gi3, whereas no coupling was observed with Gαz, nor with the retinal Gα proteins, rod (GNAT2) or cone (GNAT1) transducin (GαTr-R, GαTr-C). Finally, the expression of Gα proteins determined to couple with mGluR6 was examined in rat ON bipolar cells using single cell RT-PCR. Co-expression of mGluR6 message was used to distinguish ON from OFF bipolar cells. Expression of Gαo was detected in every ON bipolar cell examined. Message for Gαi1, which coupled moderately to mGluR6, was not detected in ON bipolar cells, nor was Gαi3, which coupled to mGluR6 in only a few cells but on average did not exhibit statistically significant coupling. Finally, though Gαi2 was detectable in ON bipolar cells, its coupling to mGluR6 in the SCG system was not significant. Together, these data indicate that signaling through mGluR6 in mammalian ON bipolar cells is highly focused, apparently acting through a single Gα protein subtype.

scholarly journals Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins

1999 ◽  
Vol 342 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Chee Wai FONG ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
G Proteins ◽  
Fusion Proteins ◽  
Hek293 Cells ◽  
Gtpase Activity ◽  
Prostanoid Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Stimulation Of

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with Gsα. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein Gs, Gi1, a G-protein which it fails to activate in co-expression studies, and a chimaeric Gi1-Gs6 (a form of Gi1 in which the C-terminal six amino acids were replaced with the equivalent sequence of Gs) were stably expressed in HEK293 cells. These were detected by [3H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-Gsα fusion protein, and both constructs were shown to interact with and activate endogenously expressed Gsα. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-Gi1α fusion. However, the fusion proteins containing either Gsα or Gi1-Gs6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-Gi1-Gs6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the Gsα-containing fusion protein and a 9-fold improvement when using the fusion protein containing Gi1-Gs6α to detect G-protein activation compared with expression of the isolated receptor.

scholarly journals Heterogeneity and dynamics of ERK and Akt activation by G protein-coupled receptors depend on the activated heterotrimeric G proteins

2021 ◽  
Author(s):  
Sergei Chavez-Abiega ◽  
Max Gronloh ◽  
Theodorus W.J. Gadella ◽  
Frank J. Bruggeman ◽  
Joachim Goedhart
Keyword(s):  
Single Cell ◽  
G Protein ◽  
G Proteins ◽  
Receptor Activation ◽  
Cellular Heterogeneity ◽  
Similar Response ◽  
Heterotrimeric G Proteins ◽  
Clustering Methods ◽  
Response Dynamics ◽  
Akt Activation

Kinases are fundamental regulators of cellular functions and play key roles in GPCR-mediated signaling pathways. Kinase activities are generally inferred from cell lysates, hiding the heterogeneity of the individual cellular responses to extracellular stimuli. Here, we study the dynamics and heterogeneity of ERK and Akt in genetically identical cells in response to activation of endogenously expressed GPCRs. We use kinase translocation reporters, high-content imaging, automated segmentation and clustering methods to assess cell-to-cell signaling heterogeneity. We observed ligand-concentration dependent response kinetics to histamine, α2-adrenergic, and S1P receptor stimulation that varied between cells. By using G protein inhibitors, we observed that Gq mediated the ERK and Akt responses to histamine. In contrast, Gi was necessary for ERK and Akt activation in response to α2-adrenergic receptor activation. ERK and Akt were also strongly activated by S1P, showing high heterogeneity at the single cell level, especially for ERK. In all cases, the cellular heterogeneity was not explained by distinct pre-stimulation levels or saturation of the measured response. Cluster analysis of time-series derived from 68,000 cells obtained under the different conditions revealed several distinct populations of cells that display similar response dynamics. The single-cell ERK responses to histamine and UK showed remarkably similar dynamics, despite the activation of different heterotrimeric G proteins. In contrast, the ERK response dynamics to S1P showed high heterogeneity, which was reduced by the inhibition of Gi. To conclude, we have set up an imaging and analysis strategy that reveals substantial cell-to-cell heterogeneity in kinase activity driven by GPCRs.

Sign in / Sign up

Export Citation Format

Share Document