New Fluorescent Adenosine A₁-Receptor Agonists That Allow Quantification of Ligand−Receptor Interactions in Microdomains of Single Living Cells

Abstract

Fluorescence spectroscopy is becoming a valuable addition to the array of techniques available for scrutinizing ligand-receptor interactions in biological systems. In particular, scanning confocal microscopy and fluorescence correlation spectroscopy (FCS) allow the noninvasive imaging and quantification of these interactions in single living cells. To address the emerging need for fluorescently labeled ligands to support these technologies, we have developed a series of red-emitting agonists for the human adenosine A1-receptor that, collectively, are N6-aminoalkyl derivatives of adenosine or adenosine 5'-N-ethyl carboxamide. The agonists, which incorporate the commercially available fluorophore BODIPY [630/650], retain potent and efficacious agonist activity, as demonstrated by their ability to inhibit cAMP accumulation in chinese hamster ovary cells expressing the human adenosine A1-receptor. Visualization and confirmation of ligand-receptor interactions at the cell membrane were accomplished using confocal microscopy, and their suitability for use in FCS was demonstrated by quantification of agonist binding in small areas of cell membrane.

Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

“…The fast moving component, termed t D1 , is representative of free fluorescent ligand, whereas one or more slower moving components represent ligand-receptor complexes (termed t D2 and/or t D3 respectively). To date, FCS in combination with fluorescent ligands has been used to characterise the histamine-H 1 receptor (Rose et al, 2012), b 2 -adrenoceptor (Hegener et al, 2004;Prenner et al, 2007), galanin receptor (Pramanik et al, 2001), somatostatin receptor (Grant et al, 2004) and the adenosine-A 1 Middleton et al, 2007) and -A 3 (Cordeaux et al, 2008;Corriden et al, 2014) receptors. In all of these studies ligand-receptor complexes were shown to exist in two distinct diffusing components at the plasma membrane (designated t D2 and t D3 ), suggesting at least two states or populations of receptors exist.…”

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

“…It can be used at the single cell level and even at the level of single molecules (compare Table 1). This approach has already been used for the characterization of ligand binding to different GPCRs, including adenosine A 1 and A 3 (Middleton et al, 2007;Cordeaux et al, 2008;Corriden et al, 2014) and adrenergic receptors (Prenner et al, 2007).…”

The Effect of Allosteric Modulators on the Kinetics of Agonist-G Protein-Coupled Receptor Interactions in Single Living Cells

“…An advantage of this technique is that rapid removal of free ligand upon reservoir exchange allows agonist dissociation to be performed in the absence of a saturating concentration of competitive orthosteric ligand. Specifically, this study has directly quantified, at the single-cell level, the kinetics of the fluorescent adenosine derivative ABA-X-BY630 (Middleton et al, 2007) at the human A 1 AR and A 3 AR. Furthermore, the influence of the allosteric modulators PD81,723 and VUF5455 on the kinetics of ABA-X-BY630 binding and function has been investigated at the human A 1 AR and A 3 AR in live cells.…”