Hetero-oligomerization of the P2Y₁₁ receptor with the P2Y₁ receptor controls the internalization and ligand selectivity of the P2Y₁₁ receptor

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y₁ and P2Y₁₁, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y₁₁ receptor and the P2Y₁ receptor where this interaction promotes agonist-induced internalization of the P2Y₁₁ receptor. This is remarkable because the P2Y₁₁ receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y₁₁ and P2Y₁ receptors, upon stimulation with ATP or the P2Y₁ receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y₁₁ receptor was found to functionally associate with endogenous P2Y₁ receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y₁ receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP–P2Y₁₁ receptor. Pharmacological characteristics of the P2Y₁₁ receptor expressed in HEK293 cells were determined by recording Ca²⁺ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y₁₁ receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y₁ and P2Y₁₁ receptors allows novel functions of the P2Y₁₁ receptor in response to extracellular nucleotides.