To identify binding partners of the A1AR (A1 adenosine receptor), yeast two-hybrid screening of a rat embryonic cDNA library was performed. This procedure led to the identification of erythrocyte membrane cytoskeletal protein (represented as 4.1G) as an A1AR-binding partner. Truncation studies revealed that the C-terminal domain of 4.1G was essential for binding to A1ARs and that the C-terminal domain of 4.1G and the third intracellular loop of A1ARs interacted. A1AR–4.1G interaction was also confirmed in studies using brain tissue. Studies in HEK-293 (human embryonic kidney 293) cells and Chinese-hamster ovary cells showed that 4.1G interfered with A1AR signal transduction, as 4.1G reduced A1AR-mediated inhibition of cAMP accumulation and intracellular calcium release. 4.1G also altered cell-surface A1AR expression. These observations identify 4.1G as a novel A1AR-binding partner that can regulate adenosine action.
Abbreviations used: AM, acetoxymethyl ester; AR, adenosine receptor; CHO, Chinese-hamster ovary; A1AR-CHO, CHO cells stably expressing A1AR; A1AR-ir, A1AR-immunoreactivity; [Ca2+]i, intracellular calcium; CPA, N6-cyclopentyladenosine; CTD, C-terminal domain; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; HA, haemagglutinin; HEK-293 cells, human embryonic kidney 293 cells; Y2H, yeast two-hybrid.