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 Gi/oprotein-coupled A1adenosine receptor (A1R) and Gqprotein-coupled P2Y1receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1Rand P2Y2receptor, but not A1Rand dopamine D2receptor, also were found to coimmunoprecipitate in cotransfected cells. A1Ragonist and antagonist binding to cell membranes were reduced by coexpression of A1Rand P2Y1R, whereas a potent P2Y1Ragonist adenosine 5′-O-(2-thiotriphosphate) (ADPβS) revealed a significant potency to A1Rbinding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1Rcoexpressed cells showed an ADPβS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1Rantagonist, indicating that ADPβS binds A1Rand inhibits adenylyl cyclase activity via Gi/oproteins. Also, a high degree of A1Rand P2Y1Rcolocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1Rwith P2Y1Rgenerates A1Rwith P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.