RT Journal Article SR Electronic T1 Allosteric Enhancers of A1 Adenosine Receptors Increase Receptor-G Protein Coupling and Counteract Guanine Nucleotide Effects on Agonist Binding JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 1557 OP 1564 DO 10.1124/mol.64.6.1557 VO 64 IS 6 A1 Heidi Figler A1 Ray A. Olsson A1 Joel Linden YR 2003 UL http://molpharm.aspetjournals.org/content/64/6/1557.abstract AB Endogenous ligands of G protein-coupled receptors bind to orthosteric sites that are topologically distinct from allosteric sites. Certain aminothiophenes such as (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl-4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A1 adenosine receptor (A1AR). In equilibrium binding assays, 125I-N6-aminobenzyladenosine (125I-ABA) binds to two affinity states of A1AR with KD-high (0.33 μM) and KD-low (∼10 nM). Enhancers have little effect on KD-high but convert all A1AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5′-O-(3-thio)triphosphate (GTPγS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of 125I-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA1 membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t1/2 values of 1.85 and 12.8 min. Allosteric enhancers selectively increase the number of slow binding sites, possibly by stabilizing newly formed receptor-G protein complexes. A new rapid assay method scores enhancer activity on a scale from 0 to 100 based on their ability to prevent the rapid dissociation of 125I-ABA from A1AR in response to GTPγS. Compared with PD81,723, ATL525 (100 μM) scores higher (27 versus 79) and has less antagonist activity. ATL525 functionally enhances A1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A1AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPγS.