@article {Maymol.110.064493, author = {Lauren T May and Tim J Self and Stephen J Briddon and Stephen J Hill}, title = {THE EFFECT OF ALLOSTERIC MODULATORS ON THE KINETICS OF AGONIST-G PROTEIN-COUPLED RECEPTOR INTERACTIONS IN SINGLE LIVING CELLS.}, elocation-id = {mol.110.064493}, year = {2010}, doi = {10.1124/mol.110.064493}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, abstract = {Allosteric binding sites on adenosine -A1 and -A3 receptors represent attractive therapeutic targets for amplifying, in a spatially and temporally selective manner, the tissue protective actions of endogenous adenosine. This study has directly quantified the kinetics of agonist/GPCR interactions at the single cell level, reflecting the physiological situation where intracellular signalling proteins can exert major allosteric effects on agonist-receptor interactions. The association and dissociation rate constants at both A1 and A3 receptors and therefore the affinity of the fluorescent adenosine derivative, ABA-X-BY630, were concentration-independent. The equilibrium dissociation constant of ABA-X-BY630 at A1 and A3 receptors was approximately 50 nM and 10 nM respectively; suggesting that, even in live cells, low agonist concentrations predominantly detect high affinity receptor states. At A1 receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (koff=1.95{\textpm}0.09 min-1) as compared to the presence of the allosteric enhancer PD81,723 (10 μM; koff=0.80{\textpm}0.03 min-1) and allosteric inhibitor VUF5455 (1 μM; koff=1.48{\textpm}0.16 min-1). In contrast, ABA-X-BY630 dissociation from A3 receptors was significantly slower in the absence (koff=0.78{\textpm}0.18 min-1) as compared to the presence of the allosteric inhibitors, VUF 5455 (1 μM; koff=3.15{\textpm}0.12 min-1) and PD81,723 (10 μM; koff=2.46{\textpm}0.18 min-1). An allosteric mechanism of action has previously not been identified for PD81,723 at the A3 receptor or VUF5455 at the A1 receptor. Furthermore, the marked enhancement in fluorescent agonist dissociation by VUF5455 in living cells contrasts previous observations from broken cell preparations and emphasizes the need to study the allosteric regulation of agonist binding in living cells.The American Society for Pharmacology and Experimental Therapeutics}, issn = {0026-895X}, URL = {https://molpharm.aspetjournals.org/content/early/2010/06/28/mol.110.064493}, eprint = {https://molpharm.aspetjournals.org/content/early/2010/06/28/mol.110.064493.full.pdf}, journal = {Molecular Pharmacology} }