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 Vasopressin V2 is a promiscuous G protein-coupled receptor that is biased by its peptide ligands

2021 ◽  
Author(s):  
Franziska M. Heydenreich ◽  
Bianca Plouffe ◽  
Aurélien Rizk ◽  
Dalibor Milić ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Structural Changes ◽  
Peptide Ligands ◽  
Intrinsic Activity ◽  
Protein Activation ◽  
G Protein Activation ◽  
Added Benefit ◽  
V2 Receptor ◽  
G Protein Coupled

AbstractActivation of the G protein-coupled receptors by agonists may result in the activation of one or more G proteins, and in the recruitment of arrestins. The balance of activation of different pathways can be influenced by the ligand. Using BRET-based biosensors, we showed that the vasopressin V2 receptor activates or at least engages many different G proteins across all G protein subfamilies in response to its native agonist arginine vasopressin (AVP). This includes members of the Gi/o and G12/13 families that have not been previously reported. These signalling pathways are also activated by the synthetic peptide desmopressin and natural homologs of AVP, namely oxytocin and the non-mammalian hormone vasotocin. They demonstrated varying degrees of functional selectivity relative to AVP, as quantified using the operational model for quantifying ligand bias. Additionally, we modelled G protein activation as a Michaelis-Menten reaction. This approach provided a complementary way to quantify signalling bias, with an added benefit of clear separation of the effects of ligand affinity from the intrinsic activity of the receptor. These results showed that V2 receptor is not only promiscuous in its ability to engage several G proteins, but also that its signalling profile could be easily biased by small structural changes in the ligand.

scholarly journals Interaction of class A G protein-coupled receptors with G proteins.

2004 ◽  
Vol 51 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Rafał Slusarz ◽  
Jerzy Ciarkowski
Keyword(s):  
G Protein ◽  
G Proteins ◽  
G Protein Coupled Receptors ◽  
Alpha Subunit ◽  
Protein Activation ◽  
Class A ◽  
G Protein Activation ◽  
G Protein Coupled ◽  
Receptor Dimer

A model for interaction of class A G protein-coupled receptor with the G protein G(alpha) subunit is proposed using the rhodopsin-transducin (RD/Gt) prototype. The model combines the resolved interactions/distances, essential in the active RD*/Gt system, with the structure of Gt(alpha) C-terminal peptide bound to RD* while stabilizing it. Assuming the interactions involve conserved parts of the partners, the model specifies the conserved Helix 2 non-polar X- - -X, Helix 3 DRY and Helix 7/8 NP- -Y- - F RD* motifs interacting with the Gt(alpha) C-terminal peptide, in compliance with the structure of the latter. A concomitant role of Gt(alpha) and Gt(gamma) C-termini in stabilizing RD* could possibly be resolved assuming a receptor dimer as requisite for G protein activation.

scholarly journals Mutations in the ‘DRY’ motif of the CB1 cannabinoid receptor result in biased receptor variants

2014 ◽  
Vol 54 (1) ◽  
pp. 75-89 ◽  
Author(s):  
Pál Gyombolai ◽  
András D Tóth ◽  
Dániel Tímár ◽  
Gábor Turu ◽  
László Hunyady
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Double Mutant ◽  
Cannabinoid Receptor ◽  
Camp Accumulation ◽  
Protein Activation ◽  
Cb1 Cannabinoid Receptor ◽  
G Protein Activation ◽  
Dry Motif

The role of the highly conserved ‘DRY’ motif in the signaling of the CB1cannabinoid receptor (CB1R) was investigated by inducing single-, double-, and triple-alanine mutations into this site of the receptor. We found that the CB1R-R3.50A mutant displays a partial decrease in its ability to activate heterotrimeric Goproteins (∼80% of WT CB1R (CB1R-WT)). Moreover, this mutant showed an enhanced basal β-arrestin2 (β-arr2) recruitment. More strikingly, the double-mutant CB1R-D3.49A/R3.50A was biased toward β-arrs, as it gained a robustly increased β-arr1 and β-arr2 recruitment ability compared with the WT receptor, while its G-protein activation was decreased. In contrast, the double-mutant CB1R-R3.50A/Y3.51A proved to be G-protein-biased, as it was practically unable to recruit β-arrs in response to agonist stimulus, while still activating G-proteins, although at a reduced level (∼70% of CB1R-WT). Agonist-induced ERK1/2 activation of the CB1R mutants showed a good correlation with their β-arr recruitment ability but not with their G-protein activation or inhibition of cAMP accumulation. Our results suggest that G-protein activation and β-arr binding of the CB1R are mediated by distinct receptor conformations, and the conserved ‘DRY’ motif plays different roles in the stabilization of these conformations, thus mediating both G-protein- and β-arr-mediated functions of CB1R.

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling

Development ◽  
2001 ◽  
Vol 128 (5) ◽  
pp. 645-654
Author(s):  
X. Shi ◽  
S. Amindari ◽  
K. Paruchuru ◽  
D. Skalla ◽  
H. Burkin ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Cytoplasmic Domain ◽  
Protein Activation ◽  
Acrosomal Exocytosis ◽  
G Protein Activation ◽  
Galactosyltransferase I

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.

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.

Role of G proteins in stimulation of Na-H exchange by cell shrinkage

1992 ◽  
Vol 262 (2) ◽  
pp. C533-C536 ◽  
Author(s):  
B. A. Davis ◽  
E. M. Hogan ◽  
W. F. Boron
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Transport Processes ◽  
Cell Shrinkage ◽  
Protein Activation ◽  
Kinase C ◽  
G Protein Activation ◽  
Barnacle Muscle Fibers ◽  
Stimulation Of

Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.

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