Despite previous work done in the rat hippocampal neuron (Ikeuchi et al., 1996) and in rat portal vein (Guibert et al., 1998), details of the physiological functions of the purinergic receptor still remain unclear. In this study, further insights into the significance of P1/P2 receptor coexistence may be obtained by studying the receptor regulation and signaling between them. Donor photobleaching FRET was used to show homodimerization of the purinergic P2Y₁ (P2Y1R) and the adenosine A₁ receptors (A1R) in living cells. FRET is dependent on the molecular proximity and energy transfer between the energy donor FITC-anti-Myc, and acceptor Cy3-anti-Myc, making it possible to study the interactions between the epitope-tagged P2Y1 and A1 receptors. In the presence of a G_i-linked A1R and a G_q-li...[ Read more ]

Despite previous work done in the rat hippocampal neuron (Ikeuchi et al., 1996) and in rat portal vein (Guibert et al., 1998), details of the physiological functions of the purinergic receptor still remain unclear. In this study, further insights into the significance of P1/P2 receptor coexistence may be obtained by studying the receptor regulation and signaling between them. Donor photobleaching FRET was used to show homodimerization of the purinergic P2Y₁ (P2Y1R) and the adenosine A₁ receptors (A1R) in living cells. FRET is dependent on the molecular proximity and energy transfer between the energy donor FITC-anti-Myc, and acceptor Cy3-anti-Myc, making it possible to study the interactions between the epitope-tagged P2Y1 and A1 receptors. In the presence of a G_i-linked A1R and a G_q-linked P2Y1R, the rate of ERK phosphorylation was brought on earlier in a time-course study when co-treated with both agonists as compared to treated with 2-MeSADP alone. Cross talk between A1R and P2Y1R must have contributed to the augmentation in signals which is a common occurrence between G_i- and G_q-linked receptors where synergism often occurs. A1R and P2Y1R transfected cells did not show synergistic activation of JNK and p38.

GPCR down-regulation includes agonist induced desensitization and receptor internalization. These regulations were examined using the epitope-tag Myc-P2Y1R construct. Transiently transfected HEK293 cells when pretreated with 2-MeSADP can induce desensitization, thereby diminished the subsequent 2-MeSADP stimulation of Ca²⁺ mobilization. Myc-P2Y1R when transiently expressed in HEK293 cells and visualized with FITC-anti-Myc showed membrane distribution but upon 2-MeSADP treatment, the receptor can internalize into the cytosol of the cells when tracked against time. Overall, the complexity of GPCRs regulation was studied by looking at the homodimerization of P2Y1R and A1R and since they were previously shown to heterodimerize, their signaling responses and their synergistic activation of ERK was studied.