scholarly journals Heterotrimeric G proteins physically associated with the lipopolysaccharide receptor CD14 modulate both in vivo and in vitro responses to lipopolysaccharide.

1998 ◽  
Vol 102 (11) ◽  
pp. 2019-2027 ◽  
Author(s):  
K R Solomon ◽  
E A Kurt-Jones ◽  
R A Saladino ◽  
A M Stack ◽  
I F Dunn ◽  
...  
Keyword(s):  
G Proteins ◽  
Heterotrimeric G Proteins ◽  
Lipopolysaccharide Receptor

scholarly journals Interaction of Ras with phosphoinositide 3-kinase γ

1997 ◽  
Vol 326 (3) ◽  
pp. 891-895 ◽  
Author(s):  
Ignacio RUBIO ◽  
Pablo RODRIGUEZ-VICIANA ◽  
Julian DOWNWARD ◽  
Reinhard WETZKER
Keyword(s):  
Binding Site ◽  
G Proteins ◽  
Terminal Region ◽  
Regulatory Subunit ◽  
Heterotrimeric G Proteins ◽  
Modest Increase ◽  
Pi3k Activity ◽  
Phosphoinositide 3 Kinase ◽  
Stimulation Of

Phosphoinositide 3-kinase γ (PI3Kγ) can be activated in vitro by both α and βγ subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kα. Here we demonstrate the binding of Ras to PI3Kγ in vitro. An N-terminal region of PI3Kγ was identified as a binding site for Ras. After co-expression with PI3Kγ in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kα by Ras in the same cells.

scholarly journals Identification of Tetratricopeptide Repeat 1 as an Adaptor Protein That Interacts with Heterotrimeric G Proteins and the Small GTPase Ras

2003 ◽  
Vol 23 (11) ◽  
pp. 3847-3858 ◽  
Author(s):  
Caroline Marty ◽  
Darren D. Browning ◽  
Richard D. Ye
Keyword(s):  
G Proteins ◽  
Mammalian Cells ◽  
Active Form ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Tetratricopeptide Repeat ◽  
Heterotrimeric G Proteins ◽  
Interacting Protein

ABSTRACT The biological functions of heterotrimeric G proteins and small GTPases are modulated by both extracellular stimuli and intracellular regulatory proteins. Using Saccharomyces cerevisiae two-hybrid screening, we identified tetratricopeptide repeat 1 (TPR1), a 292-amino-acid protein with three TPR motifs, as a Gα16-binding protein. The interaction was confirmed both in vitro and in transfected mammalian cells, where TPR1 also binds to several other Gα proteins. TPR1 was found to interact with Ha-Ras preferentially in its active form. Overexpression of TPR1 promotes accumulation of active Ras. TPR1 was found to compete with the Ras-binding domain (RBD) of Raf-1 for binding to the active Ras, suggesting that it may also compete with Ras GTPase-activating protein, thus contributing to the accumulation of GTP-bound Ras. Expression of Gα16 strongly enhances the interaction between TPR1 and Ras. Removal of the TPR1 N-terminal 112 residues abolishes potentiation by Gα16 while maintaining the interaction with Gα16 and the ability to discriminate active Ras from wild-type Ras. We have also observed that LGN, a Gαi-interacting protein with seven TPR motifs, binds Ha-Ras. Thus, TPR1 is a novel adaptor protein for Ras and selected Gα proteins that may be involved in protein-protein interaction relating to G-protein signaling.

scholarly journals Complementary biosensors reveal different G-protein signaling modes triggered by GPCRs and non-receptor activators

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mikel Garcia-Marcos
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Cytoplasmic Proteins ◽  
Nucleotide Exchange Factor ◽  
In Cells

It has become evident that activation of heterotrimeric G-proteins by cytoplasmic proteins that are not G-protein-coupled receptors (GPCRs) plays a role in physiology and disease. Despite sharing the same biochemical guanine nucleotide exchange factor (GEF) activity as GPCRs in vitro, the mechanisms by which these cytoplasmic proteins trigger G-protein-dependent signaling in cells have not been elucidated. Heterotrimeric G-proteins can give rise to two active signaling species, Gα-GTP and dissociated Gβγ, with different downstream effectors, but how non-receptor GEFs affect the levels of these two species in cells is not known. Here, a systematic comparison of GPCRs and three unrelated non-receptor proteins with GEF activity in vitro (GIV/Girdin, AGS1/Dexras1, and Ric-8A) revealed high divergence in their contribution to generating Gα-GTP and free Gβγ in cells directly measured with live-cell biosensors. These findings demonstrate fundamental differences in how receptor and non-receptor G-protein activators promote signaling in cells despite sharing similar biochemical activities in vitro.

scholarly journals Latrophilins are essential for endothelial junctional fluid shear stress mechanotransduction

2020 ◽  
Author(s):  
Keiichiro Tanaka ◽  
Andrew Prendergast ◽  
Jared Hintzen ◽  
Abhishek Kumar ◽  
Minhwan Chung ◽  
...  
Keyword(s):  
Shear Stress ◽  
G Proteins ◽  
Fluid Shear Stress ◽  
Affinity Purification ◽  
Cell Junctions ◽  
Heterotrimeric G Proteins ◽  
Fluid Shear ◽  
Vascular Morphogenesis ◽  
Flow Activation

AbstractEndothelial cell (EC) responses to fluid shear stress (FSS) are crucial for vascular development, adult physiology and disease. PECAM1 is an important transducer but earlier events remain poorly understood. We therefore investigated heterotrimeric G proteins in FSS sensing. Knockdown (KD) in ECs of single Gα proteins had little effect but combined depletion of Gαi and Gαq/11 blocked all known PECAM1-dependent responses. Re-expression of Gαi2 and Gαq but not Gαi1 and Gαi3 rescued these effects. Sequence alignment and mutational studies identified that K307 in Gαi2 and Gq/11 (Q306 in Gαi1/3), determines participation in flow signaling. We developed pull-down assays for measuring Gα activation and found that this residue, localized to the GPCR interface, determines activation by FSS. We developed a protocol for affinity purification of GPCRs on activated Gα’s, which identified latrophilins (ADGRLs) as specific upstream interactors for Gαi2 and Gq/11. Depletion of latrophilin-2 blocked EC activation of Gαi2 and Gαq, downstream events in vitro, and flow-dependent vascular morphogenesis in zebrafish embryos. Surprisingly, latrophilin-2 depletion also blocked flow activation of two additional pathways activated at cell-cell junctions, Smad1/5 and Notch1, independently of Gα proteins. Latrophilins are thus central mediators of junctional shear stress mechanotransduction via Gα protein-dependent and -independent mechanisms.

Regulation of lipolysis by somatotropin: functional alteration of adrenergic and adenosine signaling in bovine adipose tissue

1997 ◽  
Vol 152 (3) ◽  
pp. 465-475 ◽  
Author(s):  
K L Houseknecht ◽  
D E Bauman
Keyword(s):  
Adipose Tissue ◽  
Signaling Proteins ◽  
Heterotrimeric G Proteins ◽  
Adrenergic Stimulation ◽  
Cellular Mechanisms ◽  
Lactating Cows ◽  
Adipose Tissue Lipolysis ◽  
Stimulation Of

To investigate the cellular mechanisms of somatotropin (ST) action on adipose tissue lipolysis, experiments were conducted using adipose tissue taken from lactating cows treated with excipient or ST (40 mg/day). Stimulation of lipolysis in vitro by the effectors isoproterenol with or without adenosine deaminase, dibutyryl cAMP with or without isobutylmethylxanthine, and forskolin was not altered by ST treatment. Conversely, the response to the antilipolytic effector, phenylisopropyladenosine (PIA), was significantly reduced in adipose tissue explants from ST or fasted cows. The different responses to adrenergic-stimulating agents (in vivo) and PIA (in vitro) were not due to differences in the abundance of α, β or γ subunits of the stimulatory (Gs) and inhibitory (Gi) subunits of the heterotrimeric G-proteins which bind to the β-adrenergic and adenosine receptors respectively. However, the functionality of Gi proteins, as assessed by their ability to be ADP-ribosylated by pertussis toxin, was significantly reduced in ST-treated but not fasted cows. These data highlight differential regulation of signaling proteins by ST and fasting, both of which result in enhanced in vivo response to adrenergic stimulation of lipolysis. Journal of Endocrinology (1997) 152, 465–475

scholarly journals Pharmacological characterization of repeated administration of the first generation abused synthetic cannabinoid CP47,497

2016 ◽  
Vol 27 (3) ◽  
Author(s):  
Travis W. Grim ◽  
Kimberly L. Samano ◽  
Bogna Ignatowska-Jankowska ◽  
Qing Tao ◽  
Laura J. Sim-Selly ◽  
...  
Keyword(s):  
G Proteins ◽  
First Generation ◽  
Repeated Administration ◽  
Synthetic Cannabinoid ◽  
In Vitro Assays ◽  
Pharmacological Effects ◽  
Pharmacological Characterization

AbstractA series of in vivo and in vitro assays were conducted to characterize the pharmacological effects of the first generation abused synthetic cannabinoid CP47,497, a racemic bicyclic cannabinoid that is similar in structure to the potent, high-efficacy synthetic cannabinoid CP55,940. CP47,497 was less efficacious than CP55,940 in activating G-proteins and dose-dependently produced common CB

scholarly journals A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins

2018 ◽  
Vol 293 (44) ◽  
pp. 16964-16983 ◽  
Author(s):  
Marcin Maziarz ◽  
Stefan Broselid ◽  
Vincent DiGiacomo ◽  
Jong-Chan Park ◽  
Alex Luebbers ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cell Function ◽  
Sequence Similarity ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Conserved Sequence ◽  
Discovery Pipeline

Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein–coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif–containing proteins.

scholarly journals In Vivo Metabolic Roles of G Proteins of the Gi Family Studied with Novel Mouse Models

Endocrinology ◽  
2021 ◽  
Author(s):  
Jürgen Wess
Keyword(s):  
G Proteins ◽  
Hepatic Glucose Production ◽  
Glucose Production ◽  
Plasma Free Fatty Acid ◽  
Genetically Engineered ◽  
Whole Body ◽  
Heterotrimeric G Proteins ◽  
Structural And Functional Properties ◽  
Genetically Engineered Mice

Abstract G protein-coupled receptors (GPCRs) are the target of ~30-35% of all FDA-approved drugs. The individual members of the GPCR superfamily couple to one or more functional classes of heterotrimeric G proteins. The physiological outcome of activating a particular GPCR in vivo depends on the pattern of receptor distribution and the type of G proteins activated by the receptor. Based on the structural and functional properties of their α-subunits, heterotrimeric G proteins are subclassified into four major families: Gs, Gi/o, Gq/11, and G12/13. Recent studies with genetically engineered mice have yielded important novel insights into the metabolic roles of Gi/o-type G proteins. For example, recent data indicate that Gi signaling in pancreatic α-cells plays a key role in regulating glucagon release and whole body glucose homeostasis. Receptor-mediated activation of hepatic Gi signaling stimulates hepatic glucose production, suggesting that inhibition of hepatic Gi signaling could prove clinically useful to reduce pathologically elevated blood glucose levels. Activation of adipocyte Gi signaling reduces plasma free fatty acid levels, thus leading to improved insulin sensitivity in obese, glucose-intolerant mice. These new data suggest that Gi-coupled receptors that are enriched in metabolically important cell types represent potential targets for the development of novel drugs useful for the treatment of type 2 diabetes and related metabolic disorders.

scholarly journals Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3710-3718 ◽  
Author(s):  
Ikuo Uchida ◽  
Ryoko Ishihara ◽  
Kiyoshi Tanaka ◽  
Eiji Hata ◽  
Sou-ichi Makino ◽  
...  
Keyword(s):  
G Proteins ◽  
Pertussis Toxin ◽  
Cho Cells ◽  
Salmonella Enterica ◽  
Phage Type ◽  
Chinese Hamster ◽  
Adp Ribosylation ◽  
Dependent Modification

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB – encoded by a prophage in S. Typhimurium DT104 – are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [32P]NAD and ArtA, and the label was released by HgCl2, which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA6Arg-Ala and ArtA115Glu-Ala, in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

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