Site-directed mutagenesis studies of human A(2A) adenosine receptors - Involvement of Glu(13) and His(278) in ligand binding and sodium modulation

To provide insights into interactions between ligands and A(2A) adenosine r eceptors, site-directed mutagenesis was used to test the roles of a glutami c acid residue in the first transmembrane domain (Glu13) and a histidine re sidue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, we re mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturati on experiments revealed that [H-3]ZM241385 (4-(2-[7-amino-2-(2-furyl)-1,2,4 -triazolo[1,5-a][1,3,5] triazin-5-yl-amino]ethyl)phenol) bound wild-type an d mutant receptors in membranes from COS-7 cells expressing human A(2A) ade nosine receptors with high affinity and low non-specific binding. It was fo und from the competition experiments that the affinity of the A(2A) adenosi ne receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13 Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3- fold) except in the case of theophylline, whose affinity was decreased appr oximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [H-3]ZM241385 were shifted to the right in both wild-type and m utant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mu tant receptors. Sodium ions also decreased [H-3]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former t han the latter. The results suggest that the two closely linked residues Gl u13 and His278 in A(2A) adenosine receptor are most important for agonist r ecognition and are partly responsible for the allosteric regulation by sodi um ions. BIOCHEM PHARMACOL 60;5:661-668, 2000. (C) 2000 Elsevier Science In c.