Roles of the C-terminal Domain in Signal Transduction, Dimerisation, Desensitisation and Internalisation of the P2y1 Receptor

The human P2Y1 sequence of 373 amino acids was terminated at position 354 (construct P2Y1-T1) or at position 334 (P2Y1-T2), covering almost all of the tail. These proteins were N-terminally tagged with the Myc epitope, giving Myc-P2Y1, Myc-P2Y1-T1 and MycP2Y1-T2, and expressed separately in 293 cells: reaction with fluorescent anti-Myc antibody showed in confocal microscopy that all three are then localised to the cell membrane, with no obvious difference. The Ca transients evoked by the P2Y1 receptor agonist 2-methylthio-ADP (2-MeSADP) gave essentially identical EC50 values at 37C and maxima for Myc-P2Y1 (EC50: 2.8 nM + 4.5, n=3) and Myc-P2Y1-T1 (EC50: 3.7 nM + 3.6, n=3), but EC50 for Myc-P2Y1-T2 was 6.9-fold weaker (EC50: 25.3 nM + 16.2, n=3). Downstream signalling was tested at the level of extracellular-signal-regulated kinase (ERK): in phosphorylation of ERK2 in intact cells, EC50 for Myc-P2Y1-T1 was 38.2 nM + 25.6 (n=3) at 37C, not significantly different from Myc-P2Y1 (EC50: 46.7 nM + 28.6, n=3) value, and 2.9-fold weaker for Myc-P2Y1-T2 (EC50: 109.6 nM + 38.9, n=3). Also, the intact P2Y1 receptor is very sensitive to desensitisation at 37C: 10 min exposure to 100 nM 2MeSADP fully prevented a second response to up to 10 μM 2-MeSADP in both Ca and ERK transductions, a desensitisation reversed fully after 8 min wash. This behaviour was again fully retained after the T1 or T2 truncations.