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

Endocrinology ◽  
2007 ◽  
Vol 148 (7) ◽  
pp. 3236-3245 ◽  
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.

scholarly journals Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface

2004 ◽  
Vol 378 (3) ◽  
pp. 1015-1021 ◽  
Author(s):  
Joanne C. CHEUNG ◽  
Reinhart A. F. REITHMEIER
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Palmitic Acid ◽  
Anion Exchanger ◽  
Cell Surface Expression ◽  
Co2 Removal ◽  
Anion Exchanger 1 ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane.

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.

Activation of ERK, JNK, Akt, and G-protein coupled signaling by hybrid angiotensin II AT1/bradykinin B2 receptors expressed in HEK-293 cells

2007 ◽  
Vol 101 (1) ◽  
pp. 192-204 ◽  
Author(s):  
Jun Yu ◽  
David Lubinsky ◽  
Natia Tsomaia ◽  
Zhenhua Huang ◽  
Linda Taylor ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
G Protein ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
B2 Receptors ◽  
G Protein Coupled

scholarly journals Increased expression of G-protein-coupled receptor kinases 3 and 4 in hyperfunctioning thyroid nodules

2004 ◽  
Vol 182 (1) ◽  
pp. 173-182 ◽  
Author(s):  
C Voigt ◽  
HP Holzapfel ◽  
S Meyer ◽  
R Paschke
Keyword(s):  
G Protein ◽  
Thyroid Nodules ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
G Protein Coupled Receptor ◽  
Hek 293 ◽  
293 Cells ◽  
Receptor Kinases ◽  
G Protein Coupled

G-protein-coupled receptor kinases (GRKs) are implicated in the pathophysiology of human diseases such as arterial hypertension, heart failure and rheumatoid arthritis. While G-protein-coupled receptor kinases 2 and 5 have been shown to be involved in the desensitization of the rat thyrotropin receptor (TSHR), their role in the pathophysiology of hyperfunctioning thyroid nodules (HTNs) is unknown. Therefore, we analyzed the expression pattern of the known GRKs in human thyroid tissue and investigated their function in the pathology of HTNs. The expression of different GRKs in human thyroid and HTNs was measured by Western blotting. The influence of GRK expression on TSHR function was analyzed by coexpression experiments in HEK 293 cells. We demonstrate that in addition to GRKs 2, 5 and 6, GRKs 3 and 4 are also expressed in the human thyroid. GRKs 2, 3, 5 and 6 are able to desensitize the TSHR in vitro. This GRK-induced desensitization is amplified by the additional over-expression of beta-arrestin 1 or 2. We did not find any mutations in the GRKs 2, 3 and 5 from 14 HTNs without TSHR mutations and Gsalpha mutations. The expression of GRKs 3 and 4 was increased in HTNs independently from the existence of TSHR mutations or Gsalpha mutations. In conclusion, the increased expression of GRK 3 in HTNs and the ability of GRK 3 to desensitize the TSHR in vitro, suggest a potential role for GRK 3 as a negative feedback regulator for the constitutively activated cAMP pathway in HTNs.

scholarly journals Polycystin-1 Surface Localization Is Stimulated by Polycystin-2 and Cleavage at the G Protein-coupled Receptor Proteolytic Site

2010 ◽  
Vol 21 (24) ◽  
pp. 4338-4348 ◽  
Author(s):  
Hannah C. Chapin ◽  
Vanathy Rajendran ◽  
Michael J. Caplan
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Secretory Pathway ◽  
Channel Activity ◽  
G Protein Coupled Receptor ◽  
293 Cells ◽  
Proteolytic Site ◽  
Surface Localization ◽  
Autosomal Dominant Polycystic Kidney ◽  
G Protein Coupled

Polycystin (PC)1 and PC2 are membrane proteins implicated in autosomal dominant polycystic kidney disease. A physiologically relevant cleavage at PC1's G protein-coupled receptor proteolytic site (GPS) occurs early in the secretory pathway. Our results suggest that PC2 increases both PC1 GPS cleavage and PC1's appearance at the plasma membrane. Mutations that prevent PC1's GPS cleavage prevent its plasma membrane localization. PC2 is a member of the trp family of cation channels and is an important PC1 binding partner. The effect of PC2 on PC1 localization is independent of PC2 channel activity, as tested using channel-inhibiting PC2 mutations. PC1 and PC2 can interact through their C-terminal tails, but removing the C-terminal tail of either protein has no effect on PC1 surface localization in human embryonic kidney 293 cells. Experiments in polarized LLC-PK cells show that apical and ciliary PC1 localization requires PC2 and that this delivery is sensitive to PC2 truncation. In sum, our work shows that PC2 expression is required for the movement of PC1 to the plasma and ciliary membranes. In fibroblast cells this localization effect is independent of PC2's channel activity or PC1 binding ability but involves a stimulation of PC1's GPS cleavage before the PC1 protein's surface delivery.

scholarly journals Transport activity of chimaeric AE2-AE3 chloride/bicarbonate anion exchange proteins

2003 ◽  
Vol 371 (3) ◽  
pp. 687-696 ◽  
Author(s):  
Jocelyne FUJINAGA ◽  
Frederick B. LOISELLE ◽  
Joseph R. CASEY
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Cytoplasmic Domain ◽  
Anion Transport ◽  
Transport Activity ◽  
Surface Processing ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Anion Transport Activity

Chloride/bicarbonate anion exchangers (AEs), found in the plasma membrane of most mammalian cells, are involved in pH regulation and bicarbonate metabolism. Although AE2 and AE3 are highly similar in sequence, AE2-transport activity was 10-fold higher than AE3 (41 versus 4 mM · min−1 respectively), when expressed by transient transfection of HEK-293 cells. AE2–AE3 chimaeras were constructed to define the region responsible for differences in transport activity. The level of AE2 expression was approx. 30% higher than that of AE3. Processing to the cell surface, studied by chemical labelling and confocal microscopy, showed that AE2 is processed to the cell surface approx. 8-fold more efficiently than AE3. The efficiency of cell-surface processing was dependent on the cytoplasmic domain, since the AE2 domain conferred efficient processing upon the AE3 membrane domain, with a predominant role for amino acids 322–677 of AE2. AE2 that was expressed in HEK-293 cells was glycosylated, but little of AE3 was. However, AE2 expressed in the presence of the glycosylation inhibitor, tunicamycin, was not glycosylated, yet retained 85 ± 8% of anion-transport activity. Therefore glycosylation has little, if any, role in the cell-surface processing or activity of AE2 or AE3. We conclude that the low anion-transport activity of AE3 in HEK-293 cells is due to low level processing to the plasma membrane, possibly owing to protein interactions with the AE3 cytoplasmic domain.

scholarly journals Adenine nucleotides inhibit recombinant N-type calcium channels via G protein-coupled mechanisms in HEK 293 cells; involvement of the P2Y13 receptor-type

2004 ◽  
Vol 141 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Kerstin Wirkner ◽  
Joana Schweigel ◽  
Zoltan Gerevich ◽  
Heike Franke ◽  
Clemens Allgaier ◽  
...  
Keyword(s):  
Calcium Channels ◽  
G Protein ◽  
Adenine Nucleotides ◽  
Receptor Type ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
G Protein Coupled

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan™ High-Content Screening System

1999 ◽  
Vol 4 (2) ◽  
pp. 75-86 ◽  
Author(s):  
Bruce R. Conway ◽  
Lisa K. Minor ◽  
Jun Z. Xu ◽  
Joseph W. Gunnet ◽  
Robbin DeBiasio ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
G Protein ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Hek 293 ◽  
Whole Cells ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Green Fluorescent ◽  
G Protein Coupled

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan™ (Cellomics™, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a β2-adrenergic receptor (β2 AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate "spots" within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

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