Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis

The objective of this study was to create constitutively active mutant huma n A, adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors, A, ARs mutated in trans membrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R10 8A and R108K in the DRY motif of TM3, were found to be constitutively activ e in both functional assays. The potency of the A, agonist CI-IB-MECA (1-ch loro-N-6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) in PLC activation wa s enhanced by at least an order of magnitude over wild type (EC50 951 nM) i n R108A and A229E mutant receptors. CI-IB-MECA was much less potent (>10-fo ld) in C88F, Y109F, and Y282F and mutants or inactive following double muta tion of the DRY motif. The degree of constitutive activation was more prono unced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cy tosolic region were responsible for constraining the receptor in a G protei n-uncoupled conformation. (C) 2001 Academic Press.