Heteromeric association creates a P2Y-like adenosine receptor

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G(i/o) protein-coupled A(1) adenosine receptor (A(1)R) and G(q) protein-coupled P2Y(1) receptor (P2Y(1)R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A(1)R and P2Y(2) receptor, but not A(1)R and dopamine D(2) receptor, also were found to coimmunoprecipitate in cotransfected cells. A(1)R agonist and antagonist binding to cell membranes were reduced by coexpression of A(1)R and P2Y(1)R, whereas a potent P2Y(1)R agonist adenosine 5′-O-(2-thiotriphosphate) (ADPβS) revealed a significant potency to A(1)R binding only in the cotransfected cell membranes. Moreover, the A(1)R/P2Y(1)R coexpressed cells showed an ADPβS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A(1)R antagonist, indicating that ADPβS binds A(1)R and inhibits adenylyl cyclase activity via G(i/o) proteins. Also, a high degree of A(1)R and P2Y(1)R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A(1)R with P2Y(1)R generates A(1)R with P2Y(1)R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.