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

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.

Download Full-text

GPR3 IS A CONSTITUTIVELY ACTIVE CELL SURFACE G PROTEIN-COUPLED RECEPTOR THAT MAINTAINS MEIOTIC ARREST OF Xenopus laevis OOCYTES

Biology of Reproduction ◽

10.1093/biolreprod/77.s1.201b ◽

2007 ◽

Vol 77 (Suppl_1) ◽

pp. 201-201

Author(s):

James Deng ◽

Stephen Hammes

Keyword(s):

Cell Surface ◽

Xenopus Laevis ◽

G Protein ◽

Active Cell ◽

Xenopus Laevis Oocytes ◽

G Protein Coupled Receptor ◽

Meiotic Arrest ◽

G Protein Coupled ◽

Constitutively Active

Download Full-text

A role for GPRx, a novel GPR3/6/12-related G-protein coupled receptor, in the maintenance of meiotic arrest in Xenopus laevis oocytes

Developmental Biology ◽

10.1016/j.ydbio.2008.02.047 ◽

2008 ◽

Vol 317 (1) ◽

pp. 380-388 ◽

Cited By ~ 20

Author(s):

Diana Ríos-Cardona ◽

Roberto R. Ricardo-González ◽

Ajay Chawla ◽

James E. Ferrell

Keyword(s):

Xenopus Laevis ◽

G Protein ◽

Xenopus Laevis Oocytes ◽

G Protein Coupled Receptor ◽

Meiotic Arrest ◽

G Protein Coupled

Download Full-text

Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane

Endocrinology ◽

10.1210/en.2006-1605 ◽

2007 ◽

Vol 148 (7) ◽

pp. 3236-3245 ◽

Cited By ~ 290

Author(s):

E. Filardo ◽

J. Quinn ◽

Y. Pang ◽

C. Graeber ◽

S. Shaw ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

G Protein ◽

Intracellular Camp ◽

Calcium Stores ◽

G Protein Coupled Receptor ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

G Protein Coupled

G protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor (7TMR), is associated with rapid estrogen-dependent, G protein signaling and specific estrogen binding. At present, the subcellular site of GPR30 action is unclear. Previous studies using antibodies and fluorochrome-labeled estradiol (E2) have failed to detect GPR30 on the cell surface, suggesting that GPR30 may function uniquely among 7TMRs as an intracellular receptor. Here, we show that detectable expression of GPR30 on the surface of transfected HEK-293 cells can be selected by fluorescence-activated cell sorting. Expression of GPR30 on the cell surface was confirmed by confocal microscopy using the lectin concanavalin A as a plasma membrane marker. Stimulation of GPR30-expressing HEK-293 cells with 17β-E2 caused sequestration of GPR30 from the cell surface and resulted in its codistribution with clathrin and mobilization of intracellular calcium stores. Evidence that GPR30 signals from the cell surface was obtained from experiments demonstrating that the cell-impermeable E2-protein conjugates E2-BSA and E2-horseradish peroxidase promote GPR30-dependent elevation of intracellular cAMP concentrations. Subcellular fractionation studies further support the plasma membrane as a site of GPR30 action with specific [3H]17β-E2 binding and G protein activation associated with plasma membrane but not microsomal, or other fractions, prepared from HEK-293 or SKBR3 breast cancer cells. These results suggest that GPR30, like other 7TMRs, functions as a plasma membrane receptor.

Download Full-text

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

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.32.1672 ◽

2009 ◽

Vol 32 (10) ◽

pp. 1672-1677 ◽

Cited By ~ 15

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

Download Full-text

Lipid Raft-dependent Glucagon-like Peptide-2 Receptor Trafficking Occurs Independently of Agonist-induced Desensitization

Molecular Biology of the Cell ◽

10.1091/mbc.e03-11-0825 ◽

2004 ◽

Vol 15 (8) ◽

pp. 3673-3687 ◽

Cited By ~ 28

Author(s):

Jennifer L. Estall ◽

Bernardo Yusta ◽

Daniel J. Drucker

Keyword(s):

Cell Surface ◽

G Protein ◽

Lipid Raft ◽

Receptor Activation ◽

Receptor Internalization ◽

Cell Surface Receptor ◽

G Protein Coupled Receptor ◽

Endosomal Acidification ◽

Glucagon Like Peptide ◽

G Protein Coupled

The intestinotrophic and cytoprotective actions of glucagon-like peptide-2 (GLP-2) are mediated by the GLP-2 receptor (GLP-2R), a member of the class II glucagon-secretin G protein-coupled receptor superfamily. Although native GLP-2 exhibits a short circulating half-life, long-acting degradation-resistant GLP-2 analogues are being evaluated for therapeutic use in human subjects. Accordingly, we examined the mechanisms regulating signaling, internalization, and trafficking of the GLP-2R to identify determinants of receptor activation and desensitization. Heterologous cells expressing the transfected rat or human GLP-2R exhibited a rapid, dose-dependent, and prolonged desensitization of the GLP-2–stimulated cAMP response and a sustained GLP-2–induced decrease in levels of cell surface receptor. Surprisingly, inhibitors of clathrin-dependent endocytosis failed to significantly decrease GLP-2R internalization, whereas cholesterol sequestration inhibited ligand-induced receptor internalization and potentiated homologous desensitization. The hGLP-2R localized to both Triton X-100–soluble and –insoluble (lipid raft) cellular fractions and colocalized transiently with the lipid raft marker caveolin-1. Although GLP-2R endocytosis was dependent on lipid raft integrity, the receptor transiently associated with green fluorescent protein tagged-early endosome antigen 1–positive vesicles and inhibitors of endosomal acidification attenuated the reappearance of the GLP-2R on the cell surface. Our data demonstrate that GLP-2R desensitization and raft-dependent trafficking represent distinct and independent cellular mechanisms and provide new evidence implicating the importance of a clathrin- and dynamin-independent, lipid raft-dependent pathway for homologous G protein-coupled receptor internalization.

Download Full-text

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

Anesthesiology ◽

10.1097/00000542-200310000-00025 ◽

2003 ◽

Vol 99 (4) ◽

pp. 911-917 ◽

Cited By ~ 4

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.

Download Full-text

GAP-43 augments G protein-coupled receptor transduction in Xenopus laevis oocytes.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.90.11.5327 ◽

1993 ◽

Vol 90 (11) ◽

pp. 5327-5331 ◽

Cited By ~ 41

Author(s):

S. M. Strittmatter ◽

S. C. Cannon ◽

E. M. Ross ◽

T. Higashijima ◽

M. C. Fishman

Keyword(s):

Xenopus Laevis ◽

G Protein ◽

Xenopus Laevis Oocytes ◽

G Protein Coupled Receptor ◽

G Protein Coupled

Download Full-text

The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011952 ◽

2020 ◽

Vol 295 (15) ◽

pp. 5124-5135 ◽

Cited By ~ 4

Author(s):

Michelle E. Boursier ◽

Sergiy Levin ◽

Kris Zimmerman ◽

Thomas Machleidt ◽

Robin Hurst ◽

...

Keyword(s):

Energy Transfer ◽

Cell Surface ◽

G Protein ◽

Dynamic Range ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Receptor Internalization ◽

Cell Surface Receptor ◽

Cellular Interactions ◽

G Protein Coupled

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

Download Full-text