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 P2Y(4) receptor to form dimeric interactions. Here we focused on endogenously expressed P2Y(4) and P2Y(6) subtypes, comparing their oligomeric complexes under Blue Native (BN) gel electrophoresis. We provided evidence that P2Y(4) and P2Y(6) receptors form high order complexes in native neuronal phenotypes and that the oligomers can be disaggregated down to the dimeric P2Y(4) or to the dimeric and monomeric P2Y(6) receptor. Moreover, dimeric P2Y(4) and monomeric P2Y(6) proteins display selective microdomain partitioning in lipid rafts from specialized subcellular compartments such as synaptosomes. Ligand activation by UTP shifted the oligomerization of P2Y(6) but not of P2Y(4) receptor, as analysed by BN electrophoresis. Finally, whereas transfected P2Y(4) and P2Y(6) 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 P2Y(4) forms hetero-oligomers only with the P2Y(6) subunit. In conclusion, our results indicate that quaternary structure distinguishing P2Y(4) from P2Y(6) receptors might be crucial for specific ligand activation, membrane partitioning and consequent functional regulation.
D'Ambrosi
Nadia
N
Santa Lucia Foundation/CNR, Via Del Fosso di Fiorano 64, 00143 Roma, Italy. n.dambrosi@hsantalucia.it <n.dambrosi@hsantalucia.it>
Iafrate
Monia
M
Saba
Elena
E
Rosa
Patrizia
P
Volonté
Cinzia
C
D000818:Animals; D015153:Blotting, Western; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016716:PC12 Cells; D020836:Protein Structure, Quaternary; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics*
D009419:Nerve Tissue Proteins; D018048:Receptors, Purinergic P2; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6