Abstract

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 K_D-high (0.33 μM) and K_D-low (∼10 nM). Enhancers have little effect on K_D-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 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 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.