Allosteric enhancers of A1 adenosine receptors increase receptor-G protein coupling and counteract guanine nucleotide effects on agonist binding

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-dimethyl3-thienyl)-[3-(trifluromethyl)-phenyl]-methanone (PD81,723) and 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-biphenyl4-yl-methanone (ATL525) are positive allosteric regulators, or enhancers, of the human A 1 adenosine receptor (A(1)AR). In equilibrium binding assays, I-125-N-6-aminobenzyladenosine (I-125-ABA) binds to two affinity states of A(1)AR with K-D-high (0.33 muM) and K-D-low (similar to10 nM). Enhancers have little effect on K-D-high but convert all A(1)AR binding sites to the high-affinity state. Enhancers decrease the potency of guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) as an inhibitor of agonist binding by 100-fold and increase agonist-stimulated guanine nucleotide exchange. The association of I-125-ABA to high-affinity receptors on Chinese hamster ovary (CHO)-hA(1) membranes does not follow theoretical single-site association kinetics but is approximated by a bi-exponential equation with t(1/2) 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 I-125-ABA from A(1)AR in response to GTPgammaS. Compared with PD81,723, ATL525 (100 muM) scores higher ( 27 versus 79) and has less antagonist activity. ATL525 functionally enhances A 1 signaling to inhibit cAMP accumulation in CHO-hA1 cells. These data suggest that simultaneously binding orthosteric and allosteric enhancer ligands convert the A(1)AR from partly to fully coupled to G proteins and prevents rapid uncoupling upon binding of GTPgammaS.