scholarly journals Identification of G protein-coupled receptors by RNase H-mediated hybrid depletion using Xenopus laevis oocytes as expression system

FEBS Letters ◽  
1990 ◽  
Vol 266 (1-2) ◽  
pp. 192-194 ◽  
Author(s):  
Wolfgang Meyerhof ◽  
Dietmar Richter
Keyword(s):  
Xenopus Laevis ◽  
G Protein ◽  
Expression System ◽  
Rnase H ◽  
G Protein Coupled Receptors ◽  
Xenopus Laevis Oocytes ◽  
G Protein Coupled

scholarly journals Melatonin promotes sleep by activating the BK channel in C. elegans

2020 ◽  
Vol 117 (40) ◽  
pp. 25128-25137
Author(s):  
Longgang Niu ◽  
Yan Li ◽  
Pengyu Zong ◽  
Ping Liu ◽  
Yuan Shui ◽  
...  
Keyword(s):  
G Protein ◽  
Neurotransmitter Release ◽  
Expression System ◽  
Molecular Target ◽  
Bk Channel ◽  
G Protein Coupled Receptors ◽  
Wild Type ◽  
Heterologous Expression System ◽  
C Elegans ◽  
G Protein Coupled

Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2. A peptide acting to release free Gβγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gβλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gβλ.

scholarly journals The Xenopus laevis Isoform of G Protein-Coupled Receptor 3 (GPR3) Is a Constitutively Active Cell Surface Receptor that Participates in Maintaining Meiotic Arrest in X. laevis Oocytes

2008 ◽  
Vol 22 (8) ◽  
pp. 1853-1865 ◽  
Author(s):  
James Deng ◽  
Stephanie Lang ◽  
Christopher Wylie ◽  
Stephen R. Hammes
Keyword(s):  
Cell Surface ◽  
Xenopus Laevis ◽  
G Protein ◽  
Oocyte Maturation ◽  
Cell Surface Receptor ◽  
Xenopus Laevis Oocytes ◽  
Intracellular Camp ◽  
G Protein Coupled Receptor ◽  
Meiotic Arrest ◽  
G Protein Coupled

Abstract Oocytes are held in meiotic arrest in prophase I until ovulation, when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed “maturation.” Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Gαs-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here we characterized the signaling and functional properties of GPR3 using the more amenable model system of Xenopus laevis oocytes. We cloned the X. laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intraoocyte cAMP, in large part via Gβγ signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides reduced intracellular cAMP levels and enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, human chorionic gonadotropin-treatment of follicle-enclosed oocytes triggered metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Together, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may play a partial role in sensitizing oocytes for maturation by inactivating constitutive GPR3 signaling.

scholarly journals Identification of a Novel Planarian G-Protein-Coupled Receptor That Responds to Serotonin in Xenopus laevis Oocytes

2009 ◽  
Vol 32 (10) ◽  
pp. 1672-1677 ◽  
Author(s):  
Kaneyasu Nishimura ◽  
Kazuhiro Unemura ◽  
Jun Tsushima ◽  
Yosuke Yamauchi ◽  
Jun Otomo ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
G Protein ◽  
Xenopus Laevis Oocytes ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled

High-Level Expression of G Protein-Coupled Receptors with the Aid of the Semliki Forest Virus Expression System

1995 ◽  
Vol 15 (1-4) ◽  
pp. 23-32 ◽  
Author(s):  
Kenneth Lundstrom ◽  
Ann Mills ◽  
Elisabeth Allet ◽  
Karin Ceszkowski ◽  
Georges Agudo ◽  
...  
Keyword(s):  
G Protein ◽  
Semliki Forest Virus ◽  
Expression System ◽  
G Protein Coupled Receptors ◽  
High Level Expression ◽  
Virus Expression ◽  
High Level ◽  
G Protein Coupled

Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors

2008 ◽  
Vol 389 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Ankita Roy ◽  
Arun Kumar Shukla ◽  
Winfried Haase ◽  
Hartmut Michel
Keyword(s):  
G Protein ◽  
Rhodobacter Sphaeroides ◽  
Structural Information ◽  
Membrane Surface ◽  
Expression System ◽  
Gene Promoter ◽  
Photosynthetic Bacterium ◽  
G Protein Coupled Receptors ◽  
Recombinant Receptors ◽  
G Protein Coupled

AbstractG protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacteriumRhodobacter sphaeroidesfor the production of human recombinant GPCRs. The advantage of employingR. sphaeroidesas a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoterpufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host –R. sphaeroides– to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.

Effects of Antidepressants on G Protein–coupled Receptor Signaling and Viability in Xenopus laevis  Oocytes

2003 ◽  
Vol 99 (4) ◽  
pp. 911-917 ◽  
Author(s):  
Danja Strümper ◽  
Marcel E. Durieux ◽  
Barbara Tröster ◽  
Klaus Hahnenkamp ◽  
Cristina Vitan ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
G Protein ◽  
Lysophosphatidic Acid ◽  
Local Anesthetics ◽  
Xenopus Laevis Oocytes ◽  
Receptor Signaling ◽  
G Protein Coupled Receptor ◽  
Prolonged Incubation ◽  
Low Concentrations ◽  
G Protein Coupled

Background Tricyclic antidepressants are structurally related to local anesthetics, suggesting that part of their analgesic action may result from properties shared with local anesthetics. Because local anesthetics block G protein-coupled receptor signaling (which explains, in part, their inflammatory modulating properties), the authors studied whether antidepressants have similar effects. Methods Peak Ca-activated Cl currents induced in Xenopus laevis oocytes by lysophosphatidic acid (10(-4) m) were measured using a voltage clamp. The effects of a 30-, 120-, or 240-min incubation in amitriptyline, nortriptyline, imipramine, or fluoxetine were determined. Results After a 30-min incubation, low concentrations (10(-7)-10(-5) m) of antidepressants had no effect on lysophosphatidic acid-induced currents. After prolonged incubation, only amitriptyline or nortriptyline inhibited lysophosphatidic acid signaling (each to 58% of the control response at 10(-7) m after 240 min). At low concentrations, none of the compounds induced membrane damage (defined as a holding current of > 1 microA, 2% in control cells). Imipramine at 10(-3) m induced damage in 100% of oocytes, and fluoxetine at 10(-4) m induced damage in 71% of oocytes (P < 0.05 vs. control). Amitriptyline and nortriptyline had no effect. Conclusions These findings are in part different from those obtained with local anesthetics and suggest that interference with G protein-coupled signaling might explain, in part, the analgesic properties of some antidepressants. However, use of antidepressants in high concentrations may be associated with cellular toxicity.

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