Hetero-oligomerization of the P2Y 11} and P2Y 1} receptor controls receptor internalization and ligand selectivity of the P2Y 11} receptor

Nucleotides signal through purinergic receptors. The nucleotide P2 receptors are subdivided into ionotropic P2X receptors and metabotropic P2Y receptors. The tissue-specificity of nucleotide signaling requires diversity of function within the family of the purinergic receptors. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. The P2Y 1} and P2Y 11} receptors were found to associate physically, when co-expressed in HEK293 cells. The association was detected by co-pulldown, immunoprecipitation and Fluorescence Resonance Energy Transfer (FRET) experiments. We found a striking functional consequence of the interaction between the P2Y 11} receptor and the P2Y 1} receptor. The interaction promotes agonist-induced internalization of the P2Y 11} receptor. This is remarkable because the P2Y 11} receptor by itself is completely endocytosis-reluctant. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells, co-expressing both P2Y 11} and P2Y 1} receptors, upon stimulation with ATP or the P2Y 1} receptor specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells the P2Y 11} receptor was found to associate functionally with the endogenous P2Y 1} receptors. Treatment of HEK293 cells with siRNA directed against the P2Y 1} receptor diminished the agonist-induced endocytosis of the heterologously expressed P2Y 11}GFP receptor. Pharmacological characteristics of the P2Y 11} receptor expressed in HEK293 cells were determined by recording Ca 2+} responses after nucleotide stimulation. This analysis revealed a ligand-specificity, which was different from the agonist profile established in cells expressing the P2Y 11} receptor as the only metabotropic nucleotide receptor. Thus, the hetero-oligomerization of the P2Y 1} and P2Y 11} receptors allows novel receptor functions of the P2Y 11} receptor in response to extracellular nucleotides.