scholarly journals Deficiency of GABARAP but Not Its Paralogs Causes Enhanced EGF-Induced EGFR Degradation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1296 ◽  
Author(s):  
Jochen Dobner ◽  
Indra M. Simons ◽  
Kerstin Rufinatscha ◽  
Sebastian Hänsch ◽  
Melanie Schwarten ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Target Gene ◽  
Egf Receptor ◽  
Mapk Signaling ◽  
Living Cells ◽  
Aminobutyric Acid ◽  
Receptor Associated Protein ◽  
Acid Type ◽  
Membrane Tethers

The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.

Identification of Clathrin Heavy Chain as a Direct Interaction Partner for the γ-Aminobutyric Acid Type A Receptor Associated Protein†

Biochemistry ◽  
2007 ◽  
Vol 46 (50) ◽  
pp. 14537-14543 ◽  
Author(s):  
Jeannine Mohrlüder ◽  
Yvonne Hoffmann ◽  
Thomas Stangler ◽  
Karen Hänel ◽  
Dieter Willbold
Keyword(s):  
Heavy Chain ◽  
Direct Interaction ◽  
Type A ◽  
Aminobutyric Acid ◽  
Interaction Partner ◽  
Receptor Associated Protein ◽  
Clathrin Heavy Chain ◽  
Acid Type

scholarly journals Propofol at Clinically Relevant Concentrations Increases Neuronal Differentiation But Is Not Toxic to Hippocampal Neural Precursor Cells In Vitro 

2012 ◽  
Vol 117 (5) ◽  
pp. 1080-1090 ◽  
Author(s):  
Jeffrey W. Sall ◽  
Greg Stratmann ◽  
Jason Leong ◽  
Elliott Woodward ◽  
Philip E. Bickler
Keyword(s):  
Cell Death ◽  
Lactate Dehydrogenase ◽  
Neuronal Differentiation ◽  
Cell Function ◽  
Type A ◽  
Receptor Activation ◽  
Aminobutyric Acid ◽  
Neural Precursor ◽  
Acid Type

Background Propofol in the early postnatal period has been shown to cause brain cell death. One proposed mechanism for cognitive dysfunction after anesthesia is alteration of neural stem cell function and neurogenesis. We examined the effect of propofol on neural precursor or stem cells (NPCs) grown in vitro. Methods Hippocampal-derived NPCs from postnatal day 2 rats were exposed to propofol or Diprivan. NPCs were then analyzed for bromodeoxyuridine incorporation to measure proliferation. Cell death was measured by lactate dehydrogenase release. Immunocytochemistry was used to evaluate the expression of neuronal and glial markers in differentiating NPCs exposed to propofol. Results Propofol dose dependently increases the release of lactate dehydrogenase from NPCs under both proliferating and differentiating conditions at supraclinical concentrations (more than 7.1 µM). Both Diprivan and propofol had the same effect on NPCs. Propofol-mediated release of lactate dehydrogenase is not inhibited by blocking the γ-aminobutyric acid type A receptor or extracellular calcium influx and is not mediated by caspase-3/7. Direct γ-aminobutyric acid type A receptor activation did not have the same effect. In differentiating NPCs, 6 h of propofol at 2.1 µM increased the number neurons but not glial cells 4 days later. Increased neuronal differentiation was not blocked by bicuculline. Conclusions Only supraclinical concentrations of propofol or Diprivan kill NPCs in culture by a non-γ-aminobutyric acid type A, noncaspase-3 mechanism. Clinically relevant doses of propofol increase neuronal fate choice by a non-γ-aminobutyric acid type A mechanism.

scholarly journals Methoxycarbonyl-etomidate

2009 ◽  
Vol 111 (2) ◽  
pp. 240-249 ◽  
Author(s):  
Joseph F. Cotten ◽  
S Shaukat Husain ◽  
Stuart A. Forman ◽  
Keith W. Miller ◽  
Elizabeth W. Kelly ◽  
...  
Keyword(s):  
Half Life ◽  
Human Liver ◽  
Type A ◽  
Aminobutyric Acid ◽  
Gamma Aminobutyric Acid ◽  
Loss Of Righting Reflex ◽  
Acid Type ◽  
Righting Reflex ◽  
S9 Fraction

Background Etomidate is a rapidly acting sedative-hypnotic that provides hemodynamic stability. It causes prolonged suppression of adrenocortical steroid synthesis; therefore, its clinical utility and safety are limited. The authors describe the results of studies to define the pharmacology of (R)-3-methoxy-3-oxopropyl1-(1-phenylethyl)-1H-imidazole-5-carboxylate (MOC-etomidate), the first etomidate analogue designed to be susceptible to ultra-rapid metabolism. Methods The gamma-aminobutyric acid type A receptor activities of MOC-etomidate and etomidate were compared by using electrophysiological techniques in human alpha1beta2gamma2l receptors. MOC-etomidate's hypnotic concentration was determined in tadpoles by using a loss of righting reflex assay. Its in vitro metabolic half-life was measured in human liver S9 fraction, and the resulting metabolite was provisionally identified by using high-performance liquid chromatography/mass spectrometry techniques. The hypnotic and hemodynamic actions of MOC-etomidate, etomidate, and propofol were defined in rats. The abilities of MOC-etomidate and etomidate to inhibit corticosterone production were assessed in rats. Results MOC-etomidate potently enhanced gamma-aminobutyric acid type A receptor function and produced loss of righting reflex in tadpoles. Metabolism in human liver S9 fraction was first-order, with an in vitro half-life of 4.4 min versus more than 40 min for etomidate. MOC-etomidate's only detectable metabolite was a carboxylic acid. In rats, MOC-etomidate produced rapid loss of righting reflex that was extremely brief and caused minimal hemodynamic changes. Unlike etomidate, MOC-etomidate produced no adrenocortical suppression 30 min after administration. Conclusions MOC-etomidate is an etomidate analogue that retains etomidate's important favorable pharmacological properties. However, it is rapidly metabolized, ultra-short-acting, and does not produce prolonged adrenocortical suppression after bolus administration.

scholarly journals Analogues of Etomidate

2014 ◽  
Vol 121 (2) ◽  
pp. 290-301 ◽  
Author(s):  
Ervin Pejo ◽  
Peter Santer ◽  
Spencer Jeffrey ◽  
Hilary Gallin ◽  
S. Shaukat Husain ◽  
...  
Keyword(s):  
Adrenocorticotropic Hormone ◽  
Type A ◽  
Chiral Center ◽  
Aminobutyric Acid ◽  
Adrenocortical Function ◽  
Receptor Subtypes ◽  
Structure Activity ◽  
Acid Type ◽  
Righting Reflex

Abstract Background: R-etomidate possesses unique desirable properties but potently suppresses adrenocortical function. Consequently, efforts are being made to define structure–activity relationships with the goal of designing analogues with reduced adrenocortical toxicity. The authors explored the pharmacological impact of modifying etomidate’s chiral center using R-etomidate, S-etomidate, and two achiral etomidate analogues (cyclopropyl etomidate and dihydrogen etomidate). Methods: The γ-aminobutyric acid type A receptor modulatory potencies of drugs were assessed in oocyte-expressed α1(L264T)β3γ2L and α1(L264T)β1γ2L γ-aminobutyric acid type A receptors (for each drug, n = 6 oocytes per subtype). In rats, hypnotic potencies and durations of action were measured using a righting reflex assay (n = 26 to 30 doses per drug), and adrenocortical potencies were quantified by using an adrenocorticotropic hormone stimulation test (n = 20 experiments per drug). Results: All four drugs activated both γ-aminobutyric acid type A receptor subtypes in vitro and produced hypnosis and suppressed adrenocortical function in rats. However, drug potencies in each model ranged by 1 to 2 orders of magnitude. R-etomidate had the highest γ-aminobutyric acid type A receptor modulatory, hypnotic, and adrenocortical inhibitory potencies. Respectively, R-etomidate, S-etomidate, and cyclopropyl etomidate were 27.4-, 18.9-, and 23.5-fold more potent activators of receptors containing β3 subunits than β1 subunits; however, dihydrogen etomidate’s subunit selectivity was only 2.48-fold and similar to that of propofol (2.08-fold). S-etomidate was 1/23rd as potent an adrenocortical inhibitor as R-etomidate. Conclusion: The linkage between the structure of etomidate’s chiral center and its pharmacology suggests that altering etomidate’s chiral center may be used as part of a strategy to design analogues with more desirable adrenocortical activities and/or subunit selectivities.

Doxorubicin-induced glomerulosclerosis with proteinuria in GFP-GABARAP transgenic mice

2012 ◽  
Vol 302 (3) ◽  
pp. F380-F389 ◽  
Author(s):  
Miyuki Takagi-Akiba ◽  
Katsuhiko Asanuma ◽  
Isei Tanida ◽  
Norihiro Tada ◽  
Juan Alejandro Oliva Trejo ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Single Injection ◽  
Aminobutyric Acid ◽  
Wild Type ◽  
Glomerular Function ◽  
Receptor Associated Protein ◽  
Green Fluorescent ◽  
Precise Role

Autophagy is a process of cellular degradation, and its dysfunction elicits many pathological symptoms. However, the contribution of autophagy to kidney glomerular function has not been fully clarified. We previously reported that LC3, a promising executor of autophagy, played an important role in recovery from podocyte damage in an experimental nephrosis model (Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y. FASEB J 17: 1165–1167, 2003). γ-Aminobutyric acid A receptor-associated protein (GABARAP), has recently been characterized as another homolog of LC3, although its precise role in autophagy remains unclear. We recently generated green fluorescent protein (GFP)-GABARAP transgenic mice, in which GFP-GABARAP is abundantly expressed in glomerular podocytes. We found that the transgenic mice showed no obvious phenotype, and podocytes isolated from these mice manifested autophagic activity almost equivalent to that of wild-type mice when measured in vitro. Surprisingly, a single injection of doxorubicin caused a greater increase in proteinuria and sclerotic glomeruli in transgenic mice compared with wild-type mice. Under these conditions, neither GFP-GABARAP nor endogenous GABARAP appeared to be recruited to autophagosomes, and both remained in the cytosol. Moreover, the cytosolic GFP-GABARAP was significantly colocalized with p62 to form aggregates. These results indicate that the GFP-GABARAP/p62 complex is responsible for impairment of glomerular function and that it retards recovery from the effects of doxorubicin.

scholarly journals Effects of γ-Aminobutyric Acid Type A Receptor Modulation by Flumazenil on Emergence from General Anesthesia

2016 ◽  
Vol 125 (1) ◽  
pp. 147-158 ◽  
Author(s):  
Seyed A. Safavynia ◽  
Glenda Keating ◽  
Iris Speigel ◽  
Jonathan A. Fidler ◽  
Matthias Kreuzer ◽  
...  
Keyword(s):  
General Anesthesia ◽  
Expression System ◽  
Type A ◽  
Aminobutyric Acid ◽  
Clinical Effects ◽  
General Anesthetics ◽  
Isoflurane Anesthesia ◽  
Receptor Modulation ◽  
Acid Type

Abstract Background Transitions into conscious states are partially mediated by inactivation of sleep networks and activation of arousal networks. Pharmacologic hastening of emergence from general anesthesia has largely focused on activating subcortical monoaminergic networks, with little attention on antagonizing the γ-aminobutyric acid type A receptor (GABAAR). As the GABAAR mediates the clinical effects of many common general anesthetics, the authors hypothesized that negative GABAAR modulators would hasten emergence, possibly via cortical networks involved in sleep. Methods The authors investigated the capacity of the benzodiazepine rescue agent, flumazenil, which had been recently shown to promote wakefulness in hypersomnia patients, to alter emergence. Using an in vivo rodent model and an in vitro GABAAR heterologous expression system, they measured flumazenil’s effects on behavioral, neurophysiologic, and electrophysiologic correlates of emergence from isoflurane anesthesia. Results Animals administered intravenous flumazenil (0.4 mg/kg, n = 8) exhibited hastened emergence compared to saline-treated animals (n = 8) at cessation of isoflurane anesthesia. Wake-like electroencephalographic patterns occurred sooner and exhibited more high-frequency electroencephalography power after flumazenil administration (median latency ± median absolute deviation: 290 ± 34 s) compared to saline administration (473 ± 186 s; P = 0.042). Moreover, in flumazenil-treated animals, there was a decreased impact on postanesthesia sleep. In vitro experiments in human embryonic kidney-293T cells demonstrated that flumazenil inhibited isoflurane-mediated GABA current enhancement (n = 34 cells, 88.7 ± 2.42% potentiation at 3 μM). Moreover, flumazenil exhibited weak agonist activity on the GABAAR (n = 10 cells, 10.3 ± 3.96% peak GABA EC20 current at 1 μM). Conclusions Flumazenil can modulate emergence from isoflurane anesthesia. The authors highlight the complex role GABAARs play in mediating consciousness and provide mechanistic links between emergence from anesthesia and arousal.

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