Comparative analysis of P2Y4 and P2Y6 receptor architecture in native and transfected neuronal systems

Although extensive studies provided molecular and pharmacological characterization of metabotropic P2Y receptors for extracellular nucleotides, little is still known about their quaternary structure. By the use of transfected cellular systems and SDS-PAGE, in our previous work we established the propensity of P2Y₄ receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y₄ and P2Y₆ subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y₄ and P2Y₆ receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y₄ or to the dimeric and monomeric P2Y₆ receptor. Moreover, dimeric P2Y₄ and monomeric P2Y₆ proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y₆ but not of P2Y₄ receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y₄ and P2Y₆ proteins homo-interact and posses the appropriate domains to associate with all P2Y_1,2,4,6,11 subtypes, in naive PC12 cells the endogenous P2Y₄ forms hetero-oligomers only with the P2Y₆ subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y₄ from P2Y₆ receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation.