2-[2-[4-[2-[2-[1,3-Dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5?-N-ethylcarboxamidoadenosine (FITC-APEC): A fluorescent ligand for A2a-adenosine receptors

McCabe, R. Tyler; Skolnick, Phil; Jacobson, Kenneth A.

doi:10.1007/bf00865279

Cited by 22 publications

(17 citation statements)

References 18 publications

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“…Specifically, the k on values we obtain for micelle-solubilized A 2A R are within ~10 fold of those for FITC-APEC 22 and 5–10 fold of those determined from ZM 241,385 and NECA using [ 3 H]-ZM 241,385 4 for wild-type A 2A R in membrane preparations. The k on values for ZM 241,385 agonist-selected, truncated variants (thermostabilized A 2A -Star2 and rant21) found also using exponential fits were ~50–500 fold higher than we determined in this study 31, 32 .…”

Section: Resultssupporting

confidence: 72%

“…22 Measurements were conducted in Corning Costar 96-well half-area black polystyrene plates (catalog # 3875, Corning Incorporated, Acton, MA) using a Synergy H1 plate reader (BioTek, Winooski, VT) at an excitation wavelength of 480–485 nm and emission wavelength of 520–528 nm. All measurements were taken at a constant gain (75); for assays at low fluorescent ligand concentration (0.5–1 nM) readings were also taken at a higher gain (100).…”

Section: Methodsmentioning

confidence: 99%

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A2AR Binding Kinetics in the Ligand Depletion Regime

et al. 2017

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Ligand binding plays a fundamental role in stimulating the downstream signaling of membrane receptors. Here, ligand-binding kinetics of the full-length human adenosine receptor A2A (A2AR) reconstituted in detergent micelles were measured using a fluorescently labeled ligand via fluorescence anisotropy. Importantly, to optimize the signal to noise ratio, these experiments were conducted in the ligand depletion regime. In the ligand depletion regime, the assumptions used to determine analytical solutions for one-site binding models for either one or two ligands in competition are no longer valid. We therefore implemented a numerical solution approach in order to analyze kinetic binding data as experimental conditions approach the ligand depletion regime. By comparing the results from the numerical and the analytical solutions, we highlight the ligand-receptor ratios at which the analytical solution begins to lose predictive accuracy. Using the numerical solution approach, we determined the kinetic rate constants of the fluorescent ligand, FITC-APEC, and those for three unlabeled ligands using competitive association experiments. The association and dissociation rate constants of the unlabeled ligands determined from the competitive association experiments were then independently validated using competitive dissociation data. Based on this study, a numerical solution is recommended to determine kinetic ligand-binding parameters for experiments conducted in the ligand-depletion regime.

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Section: Resultssupporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

A2AR Binding Kinetics in the Ligand Depletion Regime

et al. 2017

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“…To determine if the variants were able to bind ligand, a high affinity (K D = 57 nM) fluorescent agonist, FITC-APEC [27], was used to visualize the active receptors at the cell surface. For these experiments we used HEK-293 cells expressing untagged A 2A R variants; Fig.…”

Section: Resultsmentioning

confidence: 99%

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition

Naranjo

Chevalier

Cousins

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.

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“…PTP derivatives of varying chain length 1 – 6 were prepared by coupling FITC to various chain-extended alkyl amino derivatives, in which the anchoring point was the exocyclic amine common to many structural classes of AR antagonists. FITC was used previously in a fluorescent agonist conjugate designed for binding to the A 2A AR [29]. Derivatives 2 – 4 contained homologous n -alkyl spacer chains, and 5 and 6 contained multiple ether linkages.…”

Section: Resultsmentioning

confidence: 99%

Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry

Kozma

Kumar

Federico

et al. 2012

Biochemical Pharmacology

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The physiological role of the A3 adenosine receptor (AR) was explored in cardiac ischaemia, inflammatory diseases and cancer. We report a new fluorophore-conjugated human (h) A3AR antagonist for application to cell-based assays in ligand discovery and for receptor imaging. Fluorescent pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-ylamine (pyrazolo-triazolo-pyrimidine, PTP) and triazolo[1,5-c]quinazolin-5-yl)amine (triazolo-quinazoline, TQ) AR antagonists were compared. A chain-extended and click-conjugated Alexa Fluor-488 TQ derivative (MRS5449) displayed a radioligand binding Ki value of 6.4 ± 2.5 nM in hA3AR-expressing CHO cell membranes. MRS5449 antagonized hA3AR agonist-induced inhibition of cyclic AMP accumulation in a concentration-dependent manner (KB 4.8 nM). Using flow cytometry (FCM), MRS5449 saturated hA3ARs with very high specific-to-nonspecific binding ratio with an equilibrium binding constant 5.15 nM, comparable to the Kd value of 6.65 nM calculated from kinetic experiments. Ki values of known AR antagonists in inhibition of MRS5449 binding in whole cell FCM were consistent with radioligand binding in membranes, but agonist binding was 5–20 fold weaker than obtained with agonist radioligand [125I]I-AB-MECA. Further binding analysis of MRS5549 suggested multiple agonist binding states of the A3AR. Molecular docking predicted binding modes of these fluorescent antagonists. Thus, MRS5449 is a useful tool for hA3AR characterization.

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2-[2-[4-[2-[2-[1,3-Dihydro-1,1-bis(4-hydroxyphenyl)-3-oxo-5-isobenzofuranthioureidyl]ethylaminocarbonyl]ethyl]phenyl] ethylamino]-5?-N-ethylcarboxamidoadenosine (FITC-APEC): A fluorescent ligand for A2a-adenosine receptors

Abstract: The fluorescein conjugate,

Cited by 22 publications

References 18 publications

A2AR Binding Kinetics in the Ligand Depletion Regime

A2AR Binding Kinetics in the Ligand Depletion Regime

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition

Novel fluorescent antagonist as a molecular probe in A3 adenosine receptor binding assays using flow cytometry

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