scholarly journals The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling

2004 ◽  
Vol 379 (3) ◽  
pp. 627-632 ◽  
Author(s):  
Sabine TRAVER ◽  
Anne SPLINGARD ◽  
Georges GAUDRIAULT ◽  
Jean de GUNZBURG
Keyword(s):  
G Protein ◽  
Muscarinic Acetylcholine Receptor ◽  
Maximal Activity ◽  
Stable Cell Line ◽  
Gtpase Activity ◽  
G Protein Signalling ◽  
Rgs Domain ◽  
Gdp Dissociation Inhibitor ◽  
G Protein Coupled

RGS (regulator of G-protein signalling) proteins stimulate the intrinsic GTPase activity of the α subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 has been shown previously to stimulate the GTPase activities of Gαo and Gαi subunits through its N-terminal RGS domain, and to down-modulate signalling from receptors coupled to Gi. It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Gαi. In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate Gi signalling, we generated RGS14 mutants invalidated in each of its domains, as well as truncated molecules, and assessed their effects on Gi signalling via the βγ pathway in a stable cell line ectopically expressing the Gi-coupled M2 muscarinic acetylcholine receptor (HEK-m2). We show that the RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of Gi. Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on Gi signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. We therefore propose that RGS14 combines the inhibition of Gi activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Gαi–GTP via its RGS domain to negatively regulate signalling downstream of Gi.

scholarly journals RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of Gαo

2000 ◽  
Vol 350 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Sabine TRAVER ◽  
Carole BIDOT ◽  
Nathalie SPASSKY ◽  
Tania BALTAUSS ◽  
Marie-France DE TAND ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
G Protein ◽  
Rgs Proteins ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Adult Mice ◽  
G Protein Signalling ◽  
Marked Preference

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS (‘regulators of G-protein signalling’) proteins that stimulate the GTPase activity of the GTP-binding α subunit of heterotrimeric G-proteins (Gα). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Gαo over other Gα subunits: RGS14 binds preferentially to Gαo in isolated brain membranes, and also interacts preferentially with Gαo (as compared with Gαi1) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Gαo, as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Gαo and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the Gi/o family and those involving the Ras-related GTPase Rap2.

scholarly journals RGS3 interacts with 14-3-3 via the N-terminal region distinct from the RGS (regulator of G-protein signalling) domain

2002 ◽  
Vol 365 (3) ◽  
pp. 677-684 ◽  
Author(s):  
Jiaxin NIU ◽  
Astrid SCHESCHONKA ◽  
Kirk M. DRUEY ◽  
Amanda DAVIS ◽  
Eleanor REED ◽  
...  
Keyword(s):  
Binding Site ◽  
G Protein ◽  
G Proteins ◽  
Rgs Proteins ◽  
Heterotrimeric G Proteins ◽  
G Protein Signalling ◽  
Vitro Binding ◽  
Rgs Domain ◽  
Hybrid Screening

RGS3 belongs to a family of the regulators of G-protein signalling (RGS), which bind and inhibit the Gα subunits of heterotrimeric G-proteins via a homologous RGS domain. Increasing evidence suggests that RGS proteins can also interact with targets other than G-proteins. Employing yeast two-hybrid screening of a cDNA library, we identified an interaction between RGS3 and the phosphoserine-binding protein 14-3-3. This interaction was confirmed by in vitro binding and co-immunoprecipitation experiments. RGS3-deletion analysis revealed the presence of a single 14-3-3-binding site located outside of the RGS domain. Ser264 was then identified as the 14-3-3-binding site of RGS3. The S264A mutation resulted in the loss of RGS3 binding to 14-3-3, without affecting its ability to bind Gαq. Signalling studies showed that the S264A mutant was more potent than the wild-type RGS3 in inhibition of G-protein-mediated signalling. Binding experiments revealed that RGS3 exists in two separate pools, either 14-3-3-bound or G-protein-bound, and that the 14-3-3-bound RGS3 is unable to interact with G-proteins. These data are consistent with the model wherein 14-3-3 serves as a scavenger of RGS3, regulating the amounts of RGS3 available for binding G-proteins. This study describes a new level in the regulation of G-protein signalling, in which the inhibitors of G-proteins, RGS proteins, can themselves be regulated by phosphorylation and binding 14-3-3.

scholarly journals Mapping physiological G protein-coupled receptor signaling pathways reveals a role for receptor phosphorylation in airway contraction

2016 ◽  
Vol 113 (16) ◽  
pp. 4524-4529 ◽  
Author(s):  
Sophie J. Bradley ◽  
Coen H. Wiegman ◽  
Max Maza Iglesias ◽  
Kok Choi Kong ◽  
Adrian J. Butcher ◽  
...  
Keyword(s):  
Lung Function ◽  
Signaling Pathways ◽  
G Protein ◽  
Physiological Responses ◽  
Muscarinic Acetylcholine Receptor ◽  
Smooth Muscle Contraction ◽  
Genetically Engineered ◽  
Receptor Phosphorylation ◽  
G Protein Coupled

G protein-coupled receptors (GPCRs) are known to initiate a plethora of signaling pathways in vitro. However, it is unclear which of these pathways are engaged to mediate physiological responses. Here, we examine the distinct roles of Gq/11-dependent signaling and receptor phosphorylation-dependent signaling in bronchial airway contraction and lung function regulated through the M3-muscarinic acetylcholine receptor (M3-mAChR). By using a genetically engineered mouse expressing a G protein-biased M3-mAChR mutant, we reveal the first evidence, to our knowledge, of a role for M3-mAChR phosphorylation in bronchial smooth muscle contraction in health and in a disease state with relevance to human asthma. Furthermore, this mouse model can be used to distinguish the physiological responses that are regulated by M3-mAChR phosphorylation (which include control of lung function) from those responses that are downstream of G protein signaling. In this way, we present an approach by which to predict the physiological/therapeutic outcome of M3-mAChR–biased ligands with important implications for drug discovery.

scholarly journals Adhesion G Protein–Coupled Receptors: From In Vitro Pharmacology to In Vivo Mechanisms

2015 ◽  
Vol 88 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Kelly R. Monk ◽  
Jörg Hamann ◽  
Tobias Langenhan ◽  
Saskia Nijmeijer ◽  
Torsten Schöneberg ◽  
...  
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
In Vitro Pharmacology ◽  
G Protein Coupled

scholarly journals The treatment effects of flaxseed-derived secoisolariciresinol diglycoside and its metabolite enterolactone on benign prostatic hyperplasia involve the G protein-coupled estrogen receptor 1

2016 ◽  
Vol 41 (12) ◽  
pp. 1303-1310 ◽  
Author(s):  
Guan-Yu Ren ◽  
Chun-Yang Chen ◽  
Wei-Guo Chen ◽  
Ya Huang ◽  
Li-Qiang Qin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Estrogen Receptor ◽  
Benign Prostatic Hyperplasia ◽  
G Protein ◽  
Therapeutic Potential ◽  
Prostatic Hyperplasia ◽  
Docking Simulations ◽  
G Protein Coupled

Secoisolariciresinol diglucoside (SDG), a lignan extracted from flaxseed, has been shown to suppress benign prostatic hyperplasia (BPH). However, little is known about the mechanistic basis for its anti-BPH activity. The present study showed that enterolactone (ENL), the mammalian metabolite of SDG, shared the similar binding site of G1 on a new type of membranous estrogen receptor, G-protein-coupled estrogen eceptor 1 (GPER), by docking simulations method. ENL and G1 (the specific agonist of GPER) inhibited the proliferation of human prostate stromal cell line WPMY-1 as shown by MTT assay and arrested cell cycle at the G0/G1 phase, which was displayed by propidium iodide staining following flow cytometer examination. Silencing GPER by short interfering RNA attenuated the inhibitory effect of ENL on WPMY-1 cells. The therapeutic potential of SDG in the treatment of BPH was confirmed in a testosterone propionate-induced BPH rat model. SDG significantly reduced the enlargement of the rat prostate and the number of papillary projections of prostatic alveolus and thickness of the pseudostratified epithelial and stromal cells when comparing with the model group. Mechanistic studies showed that SDG and ENL increased the expression of GPER both in vitro and in vivo. Furthermore, ENL-induced cell cycle arrest may be mediated by the activation of GPER/ERK pathway and subsequent upregulation of p53 and p21 and downregulation of cyclin D1. This work, in tandem with previous studies, will enhance our knowledge regarding the mechanism(s) of dietary phytochemicals on BPH prevention and ultimately expand the scope of adopting alternative approaches in BPH treatment.

Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

2000 ◽  
Vol 113 (13) ◽  
pp. 2463-2470 ◽  
Author(s):  
F. Santini ◽  
R.B. Penn ◽  
A.W. Gagnon ◽  
J.L. Benovic ◽  
J.H. Keen
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Coated Pits ◽  
Over Expression ◽  
Physiological Conditions ◽  
A Cell ◽  
G Protein Coupled ◽  
Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

N-ethyl-N-nitrosourea-based generation of mouse models for mutant G protein-coupled receptors

2006 ◽  
Vol 26 (3) ◽  
pp. 209-217 ◽  
Author(s):  
Johannes Grosse ◽  
Patrick Tarnow ◽  
Holger Römpler ◽  
Boris Schneider ◽  
Reinhard Sedlmeier ◽  
...  
Keyword(s):  
G Protein ◽  
Mouse Models ◽  
Germ Line ◽  
Random Mutagenesis ◽  
G Protein Coupled Receptors ◽  
In Vitro Assays ◽  
Vitro Fertilization ◽  
Type 3 ◽  
G Protein Coupled

Chemical random mutagenesis techniques with the germ line supermutagen N-ethyl- N-nitrosourea (ENU) have been established to provide comprehensive collections of mouse models, which were then mined and analyzed in phenotype-driven studies. Here, we applied ENU mutagenesis in a high-throughput fashion for a gene-driven identification of new mutations. Selected members of the large superfamily of G protein-coupled receptors (GPCR), melanocortin type 3 (Mc3r) and type 4 (Mc4r) receptors, and the orphan chemoattractant receptor GPR33, were used as model targets to prove the feasibility of this approach. Parallel archives of DNA and sperm from mice mutagenized with ENU were screened for mutations in these GPCR, and in vitro assays served as a preselection step before in vitro fertilization was performed to generate the appropriate mouse model. For example, mouse models for inherited obesity were established by selecting fully or partially inactivating mutations in Mc4r. Our technology described herein has the potential to provide mouse models for a GPCR dysfunction of choice within <4 mo and can be extended to other gene classes of interest.

scholarly journals Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

2015 ◽  
Vol 112 (27) ◽  
pp. 8427-8432 ◽  
Author(s):  
Katja Spiess ◽  
Mads G. Jeppesen ◽  
Mikkel Malmgaard-Clausen ◽  
Karen Krzywkowski ◽  
Kalpana Dulal ◽  
...  
Keyword(s):  
G Protein ◽  
Pseudomonas Exotoxin ◽  
Viral Pathogen ◽  
Virus Origin ◽  
Ligand Interactions ◽  
Toxin Protein ◽  
Novel Strategy ◽  
G Protein Coupled

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

Sign in / Sign up

Export Citation Format

Share Document