Cytoskeletal protein 4.1G binds to the third intracellular loop of the A₁ adenosine receptor and inhibits receptor action

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. A ₁AR-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 A₁R-binding partner that can regulate adenosine action.