scholarly journals The Influence of Monovalent Cations on Trimeric G Protein Gi1α Activity in HEK293 Cells Stably Expressing DOR-Gi1α (Cys351-Ile351) Fusion Protein

2011 ◽  
pp. 541-547 ◽  
Author(s):  
M. VOŠAHLÍKOVÁ ◽  
P. SVOBODA
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Binding Assay ◽  
Significant Inhibition ◽  
Chloride Anion ◽  
Hek293 Cells ◽  
Monovalent Cations ◽  
High Affinity ◽  
Monovalent Ions ◽  
High Level

The effect of monovalent cations on trimeric G protein Gi1α was measured at equimolar concentration of chloride anion in pertussis-toxin (PTX)-treated HEK293 cells stably expressing PTX-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein by high-affinity [35S]GTPS binding assay. The high basal level of binding was detected in absence of DOR agonist and monovalent ions and this high level was inhibited with the order of: Na+ > K+ > Li+. The first significant inhibition was detected at 1 mM NaCl. The inhibition by monovalent ions was reversed by increasing concentrations of DOR agonist DADLE. The maximum DADLE response was also highest for sodium and decreased in the order of: Na+ > K+ ≈ Li+. Our data indicate i) an inherently high activity of trimeric G protein Gi1α when expressed within DOR-Gi1α fusion protein and determined in the absence of monovalent cations, ii) preferential sensitivity of DOR-Gi1α to sodium as far as maximum of agonist response is involved.

A Model to Study Platelet Integrin Variants Using Transfected Human Embryonic Kidney-293 Cells Expressing Fluorescent αIIbβ3-Fusion Proteins

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5364-5364
Author(s):  
Volker Rainer Stoldt ◽  
Abdelouahid El Khattouti ◽  
Rudiger E. Scharf
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Laser Scanning Microscopy ◽  
Hek293 Cells ◽  
Confocal Laser ◽  
Chimeric Antibody ◽  
Human Embryonic Kidney ◽  
G Protein Coupled

Abstract The HPA-1 polymorphism of platelet integrin αIIbβ3 (GPIIb-IIIa) arises from a thymidine to cytosine transition in position 1565 of the ITGB3 gene. This transition leads to an amino acid exchange at residue 33 of the mature β3 subunit. The resulting isoforms are HPA-1a (Leu33) or HPA-1b (Pro33). We have shown that the HPA-1b variant of αIIbβ3 is associated with premature manifestation of myocardial infarction in patients suffering from coronary artery disease (Zotz et al. J Thromb Haemost 2005). This observation has lead to the hypothesis that the HPA-1b variant of αIIbβ3 may increase platelet thrombogenicity. Recently, we have also demonstrated that HPA-1b/1b platelets adhering onto fibrinogen are more resistant than HPA-1a/1a platelets when exposed to arterial shear rates of 1000 to 1500 sec-1 (Loncar et al. Thromb J 2007). To explore the molecular nature of the postulated prothrombotic phenotype of HPA-1b in further detail, we have now overexpressed the yellow fluorescent protein (YFP) or the cyan fluorescent protein (CFP) fused to the cytoplasmic tails of the αIIb or β3 subunit of both αIIbβ3 variants in human embryonic kidney-293 (HEK293) cells. Clones were screened for their cyan and yellow fluorescence. Ten positive clones of each αIIbβ3 variant were detected and characterized by Western blotting identifying the 140 kD αIIb-CFP fusion protein and the 113 kD β3-YFP fusion protein with appropriate antibodies directed against the αIIb subunit, the β3 subunit or the fluorescent proteins. Stable HEK293 clones expressing the HPA-1 receptor isoforms on the cell surface were functionally analyzed by flow cytometry (Becton Dickinson), confocal laser scanning microscopy (Zeiss), and a fluorescence imager (Thermo). We used fluorophore (PE)-conjugated complex-specific (PM6/248) and activation-dependent (PAC-1) antibodies and fluorescently tagged fibrinogen (Alexa647- Fg) in combination with phorbol-12-myristate-13-acetate (PMA) or organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol, SAG). Corresponding controls were performed with the chimeric antibody abciximab (ReoPro) to block fibrinogen binding to αIIbβ3 or with pertussis toxin (PTX) to inhibit G-protein-coupled inside-out signal transduction. Functional integrity of the integrin variants (HPA-1a and HPA-1b) was demonstrated by intact activation through G-protein-coupled receptors with SAG and by specific binding of Alexa647 fibrinogen to αIIbβ3 indicating proper insertion of the receptor complex into the plasma membrane of transfected cells. In the presence of PTX or abciximab, activation and ligand binding function of αIIbβ3 were completely (>95%) inhibited in both isoforms of HPA-1. Cytoplasmic conformational changes upon integrin activation using either PMA or SAG were followed by fluorescence resonance energy transfer (FRET) and quantified by FRET signal disappearance due to allosteric changes of the cytoplasmic domains. Stimulation with PMA and SAG caused FRET signal disappearance to same extent in each HPA-1 variant. However, the dynamics of signal disappearance appeared to be faster in the HPA-1b than in the HPA-1a variant of the cell clones studied so far. This observation corresponds to the prothrombotic phenotype of HPA-1b. In conclusion, our results demonstrate that we have generated a cellular model which can be useful to study molecular properties of αIIbβ3 variants and to explore the nature of the prothrombotic HPA-1b phenotype in further detail.

scholarly journals Measurement of agonist-induced guanine nucleotide turnover by the G-protein Gi1α when constrained within an α2A-adrenoceptor-Gi1α fusion protein

1997 ◽  
Vol 325 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Alan WISE ◽  
I. Craig CARR ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Pertussis Toxin ◽  
Gtpase Activity ◽  
Α Subunit ◽  
Fusion Construct ◽  
High Affinity ◽  
C Terminus ◽  
Adrenoceptor Agonist ◽  
Stimulation Of

A fusion protein was generated between the porcine α2A-adrenoceptor and a pertussis-toxin-insensitive (Cys351 → Gly) variant of the α subunit of Gi1α by direct in-frame fusion of the N-terminus of the G-protein to the C-terminus of the receptor. The fusion protein could be transiently expressed to high levels in COS-7 cells. Addition of the α2-adrenoceptor agonist 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304) to membranes of pertussis-toxin-treated transfected cells resulted in a concentration-dependent stimulation of high-affinity GTPase activity. Vmax estimations for the GTPase activity demonstrated an induced catalytic-centre activity of 2.0±0.2 min-1 for Gi1α when the α2A-adrenoceptor was maximally stimulated by UK14304 with a Km for GTP of 0.37±0.07 μM. Co-expression of excess β1γ2 along with the α2A-adrenoceptor-Gi1α fusion protein resulted in greater maximal UK14304-induced stimulation of high-affinity GTPase activity (2.1±0.2-fold) without alteration in agonist EC50. These studies demonstrate the functionality of the fusion construct, its capacity to interact with βγ complex and its utility in measuring agonist regulation of the catalytic-centre activity of GTP by a receptor-associated G-protein.

scholarly journals Pharmacological Comparison of a Recombinant CB1 Cannabinoid Receptor with Its Ga16 Fusion Product

2002 ◽  
Vol 7 (3) ◽  
pp. 281-289
Author(s):  
Renée S. Martin ◽  
Paul H. Reynen ◽  
Joyce J. Calixto ◽  
Christian L. Reyes ◽  
Thomas K. Chang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
Cannabinoid Receptor ◽  
Rank Order ◽  
Hek293 Cells ◽  
Fusion Product ◽  
Protein Subunit ◽  
Binding Studies ◽  
Competition Binding ◽  
In Cells

The pharmacology of G protein-coupled receptors is widely accepted to depend on the G protein subunit to which the agonist-stimulated receptor couples. In order to investigate whether CB1 agonist-mediated signal transduction via an engineered Gaα16 system is different than that of the Gi/o coupling normally preferred by the CB1 receptor, we transfected the human recombinant CB1 receptor (hCB1) or a fusion protein comprising the hCB1 receptor and Gα16 (hCB1-Gα16) into HEK293 cells. From competition binding studies, the rank order of ligand affinities at the hCB1-Gaα16 fusion protein was found to be similar to that for hCB1: HU 210 > CP 55,940 ≥ SR 141716A > WIN 55212-2 > anandamide > JWH 015. Agonists increased [35S]GTPγS binding or inhibited forskolin-stimulated cAMP, presumably by coupling to Gi/o, in cells expressing hCB1 but not hCB1-Gα16. However, an analogous rank order of potencies was observed for these agonists in their ability to evoke increases in intracellular calcium concentration in cells expressing hCBq-Gaα16 but not hCB1. These data demonstrate that ligand affinities for the hCB1, receptor are not affected by fusion to the Gα16 subunit. Furthermore, there is essentially no difference in the function of the hCB1, receptor when coupled to Gi/o, or Gα16.

Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2624-2629 ◽  
Author(s):  
James R. Van Brocklyn ◽  
Markus H. Gräler ◽  
Günter Bernhardt ◽  
John P. Hobson ◽  
Martin Lipp ◽  
...  
Keyword(s):  
G Protein ◽  
Hek293 Cells ◽  
Hematopoietic Tissue ◽  
G Protein Coupled Receptor ◽  
High Affinity ◽  
Extracellular Signal ◽  
Sphingosine 1 Phosphate ◽  
High Affinity Receptor ◽  
High Concentrations ◽  
G Protein Coupled

Abstract EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [32P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [32P]SPP was saturable, demonstrating high affinity (KD = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a Gi/o protein, resulting in the activation of growth-related signaling pathways.

scholarly journals Analysis of agonist function at fusion proteins between the IP prostanoid receptor and cognate, unnatural and chimaeric G-proteins

1999 ◽  
Vol 342 (2) ◽  
pp. 457-463 ◽  
Author(s):  
Chee Wai FONG ◽  
Graeme MILLIGAN
Keyword(s):  
Fusion Protein ◽  
G Protein ◽  
G Proteins ◽  
Fusion Proteins ◽  
Hek293 Cells ◽  
Gtpase Activity ◽  
Prostanoid Receptor ◽  
Protein Activation ◽  
G Protein Activation ◽  
Stimulation Of

Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with Gsα. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor and fusion proteins generated between this form of the receptor and the α subunits of its cognate G-protein Gs, Gi1, a G-protein which it fails to activate in co-expression studies, and a chimaeric Gi1-Gs6 (a form of Gi1 in which the C-terminal six amino acids were replaced with the equivalent sequence of Gs) were stably expressed in HEK293 cells. These were detected by [3H]ligand-binding studies and by immunoblotting with both an anti-FLAG antibody and with appropriate antisera to the G-proteins. Each construct displayed similar affinity to bind the agonist iloprost. Iloprost stimulated adenylate cyclase activity in clones expressing both IP prostanoid receptor and the IP prostanoid receptor-Gsα fusion protein, and both constructs were shown to interact with and activate endogenously expressed Gsα. Addition of iloprost to membranes of cells expressing the isolated receptor resulted in a small stimulation of high-affinity GTPase activity. Iloprost produced no stimulation of GTPase activity which could be attributed to the IP prostanoid receptor-Gi1α fusion. However, the fusion proteins containing either Gsα or Gi1-Gs6α produced substantially greater stimulation of GTPase activity than the isolated IP prostanoid receptor. Treatment of cells expressing the IP prostanoid receptor-Gi1-Gs6α fusion protein with a combination of cholera and pertussis toxins allowed direct measurement of agonist activation of the receptor-linked G-protein. Normalization of such results for levels of expression of the IP prostanoid receptor constructs demonstrated a 5-fold higher stimulation of GTPase activity when using the Gsα-containing fusion protein and a 9-fold improvement when using the fusion protein containing Gi1-Gs6α to detect G-protein activation compared with expression of the isolated receptor.

Sphingosine-1-phosphate is a ligand for the G protein-coupled receptor EDG-6

Blood ◽  
2000 ◽  
Vol 95 (8) ◽  
pp. 2624-2629
Author(s):  
James R. Van Brocklyn ◽  
Markus H. Gräler ◽  
Günter Bernhardt ◽  
John P. Hobson ◽  
Martin Lipp ◽  
...  
Keyword(s):  
G Protein ◽  
Hek293 Cells ◽  
Hematopoietic Tissue ◽  
G Protein Coupled Receptor ◽  
High Affinity ◽  
Extracellular Signal ◽  
Sphingosine 1 Phosphate ◽  
High Affinity Receptor ◽  
High Concentrations ◽  
G Protein Coupled

EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known sphingosine-1-phosphate (SPP) receptors EDG-1, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [32P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [32P]SPP was saturable, demonstrating high affinity (KD = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-1-phosphate, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a Gi/o protein, resulting in the activation of growth-related signaling pathways.

scholarly journals Molecular Recognition: Perspective and a New Approach

Sensors ◽  
2021 ◽  
Vol 21 (8) ◽  
pp. 2757
Author(s):  
W. Rudolf Seitz ◽  
Casey J. Grenier ◽  
John R. Csoros ◽  
Rongfang Yang ◽  
Tianyu Ren
Keyword(s):  
Molecular Recognition ◽  
Molecularly Imprinted Polymers ◽  
Binding Sites ◽  
Binding Kinetics ◽  
Molecularly Imprinted ◽  
New Approach ◽  
Imprinted Polymers ◽  
High Affinity ◽  
Water Blocking ◽  
High Level

This perspective presents an overview of approaches to the preparation of molecular recognition agents for chemical sensing. These approaches include chemical synthesis, using catalysts from biological systems, partitioning, aptamers, antibodies and molecularly imprinted polymers. The latter three approaches are general in that they can be applied with a large number of analytes, both proteins and smaller molecules like drugs and hormones. Aptamers and antibodies bind analytes rapidly while molecularly imprinted polymers bind much more slowly. Most molecularly imprinted polymers, formed by polymerizing in the presence of a template, contain a high level of covalent crosslinker that causes the polymer to form a separate phase. This results in a material that is rigid with low affinity for analyte and slow binding kinetics. Our approach to templating is to use predominantly or exclusively noncovalent crosslinks. This results in soluble templated polymers that bind analyte rapidly with high affinity. The biggest challenge of this approach is that the chains are tangled when the templated polymer is dissolved in water, blocking access to binding sites.

scholarly journals High level expression of transfected G protein αi3subunit is required for plasma membrane targeting and adenylyl cyclase inhibition in NIH 3T3 fibroblasts

FEBS Letters ◽  
1992 ◽  
Vol 312 (2-3) ◽  
pp. 223-228 ◽  
Author(s):  
Sylvie Hermouet ◽  
Philippe de Mazancourt ◽  
Allen M. Spiegel ◽  
Marilyn Gist Farquhar ◽  
Bridget S. Wilson
Keyword(s):  
Plasma Membrane ◽  
Adenylyl Cyclase ◽  
G Protein ◽  
Membrane Targeting ◽  
High Level Expression ◽  
3T3 Fibroblasts ◽  
High Level ◽  
Nih 3T3
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