scholarly journals Heterotrimeric G proteins promote FLS2 protein accumulation through inhibition of FLS2 autophagic degradation

2018 ◽  
Author(s):  
Jimi C. Miller ◽  
Stacey A. Lawrence ◽  
Nicole K. Clay
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Protein Complex ◽  
Heterotrimeric G Proteins ◽  
Immune Signaling ◽  
Heterotrimeric G Protein ◽  
Protein Levels ◽  
Autophagic Degradation ◽  
Protein Components

ABSTRACTFLAGELLIN-SENSITIVE 2 (FLS2) is a plant immune receptor that binds bacterial flagellin to activate immune signaling. This immune signal is transduced by a heterotrimeric G protein complex at the plasma membrane and activates downstream signaling. However, it is unknown whether the heterotrimeric G proteins have functions at other subcellular locations away from the plasma membrane. Here, we show that components of the heterotrimeric G protein complex stabilize FLS2 protein levels by inhibiting the autophagic degradation of FLS2. Using genetic analysis, we determined that mutations of G protein components resulted in reduced immune signaling in part due to decreased FLS2 protein levels. Furthermore, reduction of FLS2 protein levels was caused by elevated proteasomal and autophagic degradation of FLS2. Genetic inhibition of autophagy in G protein mutants rescued FLS2 levels and immunity. Our findings suggest that the heterotrimeric G protein components, in addition to being part of the heterotrimeric G protein complex that transduces signals at the plasma membrane, also function away from the plasma membrane to control FLS2 protein levels. These results expand the functional capacity of the heterotrimeric G protein complexes in plant immunity.

Anesthetics Inhibit Acetylcholine-promoted Guanine Nucleotide Exchange of Heterotrimeric G Proteins of Airway Smooth Muscle

2004 ◽  
Vol 101 (1) ◽  
pp. 120-126 ◽  
Author(s):  
Chie Sakihara ◽  
William J. Perkins ◽  
David O. Warner ◽  
Keith A. Jones
Keyword(s):  
Smooth Muscle ◽  
Muscle Contraction ◽  
G Protein ◽  
Muscarinic Receptor ◽  
G Proteins ◽  
Airway Smooth Muscle ◽  
Smooth Muscle Contraction ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Heterotrimeric G Protein

Background Anesthetics inhibit airway smooth muscle contraction in part by a direct effect on the smooth muscle cell. This study tested the hypothesis that the anesthetics halothane and hexanol, which both relax airway smooth muscle in vitro, inhibit acetylcholine-promoted nucleotide exchange at the alpha subunit of the Gq/11 heterotrimeric G protein (Galphaq/11; i.e., they inhibit muscarinic receptor-Galphaq/11 coupling). Methods The effect of halothane (0.38 +/- 0.02 mm) and hexanol (10 mm) on basal and acetylcholine-stimulated Galphaq/11 guanosine nucleotide exchange was determined in membranes prepared from porcine tracheal smooth muscle. The nonhydrolyzable, radioactive form of guanosine-5'-triphosphate, [S]GTPgammaS, was used as the reporter for Galphaq/11 subunit dissociation from the membrane to soluble fraction, which was immunoprecipitated with rabbit polyclonal anti-Galphaq/11 antiserum. Results Acetylcholine caused a significant time- and concentration-dependent increase in the magnitude of Galphaq/11 nucleotide exchange compared with basal values (i.e., without acetylcholine), reaching a maximal difference at 100 microm (35.9 +/-2.9 vs. 9.8 +/-1.2 fmol/mg protein, respectively). Whereas neither anesthetic had an effect on basal Galphaq/11 nucleotide exchange, both halothane and hexanol significantly inhibited the increase in Galphaq/11 nucleotide exchange produced by 30 microm acetylcholine (by 59% and 68%, respectively). Conclusions Halothane and hexanol interact with the receptor-heterotrimeric G-protein complex in a manner that prevents acetylcholine-promoted exchange of guanosine-5(')-triphosphate for guanosine-5'-diphosphate at Galphaq/11. These data are consistent with the ability of anesthetics to interfere with cellular processes mediated by heterotrimeric G proteins in many cells, including effects on muscarinic receptor-G-protein regulation of airway smooth muscle contraction.

scholarly journals Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions with GPCRs in solution

2015 ◽  
Vol 112 (11) ◽  
pp. E1181-E1190 ◽  
Author(s):  
Matthias Hillenbrand ◽  
Christian Schori ◽  
Jendrik Schöppe ◽  
Andreas Plückthun
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Conformational Changes ◽  
Protein Complex ◽  
Protein Complexes ◽  
Heterotrimeric G Proteins ◽  
Agonist Binding ◽  
Transient Nature ◽  
The Stability ◽  
Neurotensin Receptor 1

Agonist binding to G-protein–coupled receptors (GPCRs) triggers signal transduction cascades involving heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of conformational changes upon agonist binding, the details of interaction with the different G proteins, and the transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1or αsLand all possible βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1and γ11. Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.

scholarly journals Heterotrimeric G protein signaling in polycystic kidney disease

2016 ◽  
Vol 48 (7) ◽  
pp. 429-445 ◽  
Author(s):  
Taketsugu Hama ◽  
Frank Park
Keyword(s):  
Kidney Disease ◽  
G Protein ◽  
Polycystic Kidney Disease ◽  
G Proteins ◽  
G Protein Signaling ◽  
Protein Signaling ◽  
Heterotrimeric G Proteins ◽  
Polycystic Kidney ◽  
Heterotrimeric G Protein ◽  
Heterotrimeric G Protein Signaling

Autosomal dominant polycystic kidney disease (ADPKD) is a signalopathy of renal tubular epithelial cells caused by naturally occurring mutations in two distinct genes, polycystic kidney disease 1 ( PKD1) and 2 ( PKD2). Genetic variants in PKD1, which encodes the polycystin-1 (PC-1) protein, remain the predominant factor associated with the pathogenesis of nearly two-thirds of all patients diagnosed with PKD. Although the relationship between defective PC-1 with renal cystic disease initiation and progression remains to be fully elucidated, there are numerous clinical studies that have focused upon the control of effector systems involving heterotrimeric G protein regulation. A major regulator in the activation state of heterotrimeric G proteins are G protein-coupled receptors (GPCRs), which are defined by their seven transmembrane-spanning regions. PC-1 has been considered to function as an unconventional GPCR, but the mechanisms by which PC-1 controls signal processing, magnitude, or trafficking through heterotrimeric G proteins remains to be fully known. The diversity of heterotrimeric G protein signaling in PKD is further complicated by the presence of non-GPCR proteins in the membrane or cytoplasm that also modulate the functional state of heterotrimeric G proteins within the cell. Moreover, PC-1 abnormalities promote changes in hormonal systems that ultimately interact with distinct GPCRs in the kidney to potentially amplify or antagonize signaling output from PC-1. This review will focus upon the canonical and noncanonical signaling pathways that have been described in PKD with specific emphasis on which heterotrimeric G proteins are involved in the pathological reorganization of the tubular epithelial cell architecture to exacerbate renal cystogenic pathways.

Heterotrimeric G proteins in heart disease

2000 ◽  
Vol 78 (3) ◽  
pp. 187-198 ◽  
Author(s):  
Oliver Zolk ◽  
Ichiro Kouchi ◽  
Petra Schnabel ◽  
Michael Böhm
Keyword(s):  
Heart Failure ◽  
G Protein ◽  
G Proteins ◽  
Binding Proteins ◽  
Molecular Mechanisms ◽  
Heart Diseases ◽  
Membrane Receptors ◽  
Adrenergic System ◽  
Heterotrimeric G Proteins ◽  
Heterotrimeric G Protein

Guanine nucleotide binding proteins (G proteins) are largely grouped into three classes: heterotrimeric G proteins, ras-like or small molecular weight GTP binding proteins, and others like Gh. In the heart G proteins transduce signals from a variety of membrane receptors to generate diverse effects on contractility, heart rate, and myocyte growth. This central position of G proteins forming a switchboard between extracellular signals and intracellular effectors makes them candidates possibly involved in the pathogenesis of cardiac hypertrophy, heart failure, and arrhythmia. This review focuses primarily on discoveries of heterotrimeric G protein alterations in heart diseases that help us to understand the pathogenesis and pathophysiology. We also discuss the underlying molecular mechanisms of heterotrimeric G protein signalling.Key words: G proteins, signal transduction, adrenergic system, heart failure, hypertrophy.

scholarly journals High levels of caveolar cholesterol inhibit progesterone-induced genomic actions in human and guinea pig gallbladder muscle

2009 ◽  
Vol 296 (4) ◽  
pp. G948-G954 ◽  
Author(s):  
Ping Cong ◽  
Victor Pricolo ◽  
Piero Biancani ◽  
Jose Behar
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Gallbladder Disease ◽  
Scintillation Counter ◽  
Muscle Cells ◽  
Gallstone Formation ◽  
Protein Levels ◽  
Protein Activator ◽  
Bile Cholesterol

Gallbladder disease is prevalent during pregnancy. It has been suggested that this complication of pregnancy is attributable to increased bile cholesterol (Ch) induced by estrogens and to gallbladder hypomotility caused by increasing levels of progesterone (P4). Studies on nonpregnant gallbladders have shown that increased levels of bile Ch contribute to both gallstone formation and bile stasis. These studies investigated the effects of high levels of plasma membrane Ch on P4 on gallbladder muscle cells from human and guinea pigs. Contraction was studied in intact and permeabilized muscle cells. G proteins were determined by Western blot, and 3H-P4 incorporation by muscle cells was measured in the β-scintillation counter. High levels of caveolar Ch blocked the effects induced by P4 treatment for 6 h. They suppressed the expected P4 inhibition of GTP-γS (a G protein activator)-induced contraction and changes in G proteins by downregulating Gi3 and upregulating Gs protein levels. Ch inhibited these P4 actions at the caveolar 3 (CAV-3) level, since the P4 effects were antagonized by treatment with CAV-3 antibody, by reducing CAV-3 expression through CAV-3 siRNA. CAV-3 antibody and siRNA reduced caveolar Ch levels. High caveolar levels of Ch and CAV-3 antibody blocked the incorporation of 3H-P4 into caveolae. Treatment with GDP-βS (a G protein antagonist) had no effect on P4 actions. High caveolar Ch levels blocked the P4 effects on muscle contraction and G protein changes probably because both Ch and P4 require CAV-3 proteins for their transport across the plasma membrane.

scholarly journals Biased Signaling: Distinct Ligand-directed Plasma Membrane Signalosomes Using a Common RGS/G protein Core

2019 ◽  
Author(s):  
Timothy J. Ross-Elliott ◽  
Justin Watkins ◽  
Xiaoyi Shan ◽  
Fei Lou ◽  
Bernd Dreyer ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Lipid Rafts ◽  
Protein Complex ◽  
Heterotrimeric G Protein ◽  
Unknown Mechanism ◽  
Protein Core ◽  
Protein System ◽  
Biased Signaling ◽  
G Protein Coupled

Biased signaling occurs when different ligands that are directed at the same receptor launch different cellular outcomes. Because of their pharmacological importance, we know the most about biased ligands and little is known about other mechanisms to achieve signaling bias. In the canonical animal G protein system, endocytosis of a 7-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic signaling depending on the bias. In Arabidopsis, GPCRs are not required for G protein coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the prototype 7-transmembrane Regulator of G Signaling 1 protein AtRGS1 modulates G signaling and through ligand-dependent endocytosis, de-repression of signaling is initiated but canonical arrestins are not involved. Endocytosis initiates from two separate pools of plasma membrane: sterol-dependent domains, possibly lipid rafts, and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and newly-discovered arrestin-like adaptors. Different trafficking origins and trajectories lead to different cellular outcomes. Thus, compartmentation with its attendant signalosome architecture is a previously unknown mechanism to drive biased signaling.

Heterotrimeric G protein Giis involved in a signal transduction pathway for ATP release from erythrocytes

2004 ◽  
Vol 286 (3) ◽  
pp. H940-H945 ◽  
Author(s):  
Jeffrey J. Olearczyk ◽  
Alan H. Stephenson ◽  
Andrew J. Lonigro ◽  
Randy S. Sprague
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
G Proteins ◽  
Pertussis Toxin ◽  
Atp Release ◽  
Signal Transduction Pathway ◽  
Protein G ◽  
Transduction Pathway ◽  
Heterotrimeric G Proteins ◽  
Heterotrimeric G Protein

Erythrocytes are reported to release ATP in response to mechanical deformation and decreased oxygen tension. Previously we proposed that receptor-mediated activation of the heterotrimeric G protein Gsresulted in ATP release from erythrocytes. Here we investigate the hypothesis that activation of heterotrimeric G proteins of the Gisubtype are also involved in a signal transduction pathway for ATP release from rabbit erythrocytes. Heterotrimeric G proteins Gαi1, Gαi2, and Gαi3but not Gαowere identified in rabbit and human erythrocyte membranes. Pretreatment of rabbit erythrocytes with pertussis toxin (100 ng/ml, 2 h), which uncouples Gi/ofrom their effector proteins, inhibited deformation-induced ATP release. Incubation of rabbit and human erythrocytes with mastoparan (Mas, 10 μM) or Mas-7 (1 μM), which are compounds that directly activate Giproteins, resulted in ATP release. However, rabbit erythrocytes did not release ATP when incubated with Mas-17 (10 μM), which is an inactive Mas analog. In separate experiments, Mas (10 μM) but not Mas-17 (10 μM) increased intracellular concentrations of cAMP when incubated with rabbit erythrocytes. Importantly, Mas-induced ATP release from rabbit erythrocytes was inhibited after treatment with pertussis toxin (100 ng/ml, 2 h). These data are consistent with the hypothesis that the heterotrimeric G protein Giis a component of a signal transduction pathway for ATP release from erythrocytes.

scholarly journals The Role of Arabidopsis Heterotrimeric G-Protein Subunits in MLO2 Function and MAMP-Triggered Immunity

2013 ◽  
Vol 26 (9) ◽  
pp. 991-1003 ◽  
Author(s):  
Justine Lorek ◽  
Thomas Griebel ◽  
Alan M. Jones ◽  
Hannah Kuhn ◽  
Ralph Panstruga
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Protein Complex ◽  
Defense Responses ◽  
Resistance Locus ◽  
Callose Deposition ◽  
Heterotrimeric G Protein ◽  
Mildew Resistance ◽  
Genetic Ablation

Heterotrimeric G-proteins, composed of Gα, Gβ, and Gγ subunits, regulate many fundamental processes in plants. In animals, ligand binding to seven transmembrane (7TM) cell surface receptors designated G-protein coupled receptors (GPCR) leads to heterotrimeric G-protein activation. Because the plant G-protein complex is constitutively active, the exact role of plant 7TM proteins in this process is unclear. Members of the mildew resistance locus O (MLO) family represent the best-characterized 7TM plant proteins. Although genetic ablation of either MLO2 or G-proteins alters susceptibility to pathogens in Arabidopsis thaliana, it is unknown whether G-proteins directly couple signaling through MLO2. Here, we exploited two well-documented phenotypes of mlo2 mutants, broad-spectrum powdery mildew resistance and spontaneous callose deposition in leaf mesophyll cells, to assess the relationship of MLO2 proteins to the G-protein complex. Although our data reveal modulation of antifungal defense responses by Gβ and Gγ subunits and a role for the Gγ1 subunit in mlo2-conditioned callose deposition, our findings overall are inconsistent with a role of MLO2 as a canonical GPCR. We discovered that mutants lacking the Gβ subunit show delayed accumulation of a subset of defense-associated genes following exposure to the microbe-associated molecular pattern flg22. Moreover, Gβ mutants were found to be hypersusceptible to spray inoculation with the bacterial pathogen Pseudomonas syringae.

scholarly journals Gγ and Gα Identity Dictate a G-Protein Heterotrimer Plasma Membrane Targeting

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1246 ◽  
Author(s):  
Paweł Mystek ◽  
Beata Rysiewicz ◽  
Jan Gregrowicz ◽  
Marta Dziedzicka-Wasylewska ◽  
Agnieszka Polit
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
G Proteins ◽  
Protein Localization ◽  
Present Report ◽  
Lateral Diffusion ◽  
Resonance Energy ◽  
Fluorescence Lifetime Imaging ◽  
Heterotrimeric G Proteins ◽  
Protein Subunit

Heterotrimeric G-proteins along with G-protein-coupled receptors (GPCRs) regulate many biochemical functions by relaying the information from the plasma membrane to the inside of the cell. The lipid modifications of Gα and Gγ subunits, together with the charged regions on the membrane interaction surface, provide a peculiar pattern for various heterotrimeric complexes. In a previous study, we found that Gαs and Gαi3 prefer different types of membrane-anchor and subclass-specific lipid domains. In the present report, we examine the role of distinct Gγ subunits in the membrane localization and spatiotemporal dynamics of Gαs and Gαi3 heterotrimers. We characterized lateral diffusion and G-protein subunit interactions in living cells using fluorescence recovery after photobleaching (FRAP) microscopy and fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM), respectively. The interaction of Gγ subunits with specific lipids was confirmed, and thus the modulation of heterotrimeric G-protein localization. However, the Gα subunit also modulates trimer localization, and so the membrane distribution of heterotrimeric G-proteins is not dependent on Gγ only.

scholarly journals Characterization of Heterotrimeric G Protein γ4 Subunit in Rice

2018 ◽  
Vol 19 (11) ◽  
pp. 3596 ◽  
Author(s):  
Sakura Matsuta ◽  
Aki Nishiyama ◽  
Genki Chaya ◽  
Takafumi Itoh ◽  
Kotaro Miura ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Higher Plants ◽  
Yeast Cells ◽  
Mass Spectrometry Analysis ◽  
Heterotrimeric G Proteins ◽  
Subunit Gene ◽  
Heterotrimeric G Protein ◽  
Α Subunit ◽  
Γ Subunit

Heterotrimeric G proteins are the molecule switch that transmits information from external signals to intracellular target proteins in mammals and yeast cells. In higher plants, heterotrimeric G proteins regulate plant architecture. Rice harbors one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β-subunit gene (RGB1), and five γ-subunit genes (tentatively designated RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/qPE9-1/OsGGC3, and RGG5/OsGGC2) as components of the heterotrimeric G protein complex. Among the five γ-subunit genes, RGG1 encodes the canonical γ-subunit, RGG2 encodes a plant-specific type of γ-subunit with additional amino acid residues at the N-terminus, and the remaining three γ-subunit genes encode atypical γ-subunits with cysteine-rich C-termini. We characterized the RGG4/DEP1/DN1/qPE9-1/OsGGC3 gene product Gγ4 in the wild type (WT) and truncated protein Gγ4∆Cys in the RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutant, Dn1-1, as littele information regarding the native Gγ4 and Gγ4∆Cys proteins is currently available. Based on liquid chromatography-tandem mass spectrometry analysis, immunoprecipitated Gγ4 candidates were confirmed as actual Gγ4. Similar to α-(Gα) and β-subunits (Gβ), Gγ4 was enriched in the plasma membrane fraction and accumulated in the developing leaf sheath. As RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants exhibited dwarfism, tissues that accumulated Gγ4 corresponded to the abnormal tissues observed in RGG4/DEP1/DN1/qPE9-1/OsGGC3 mutants.

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