Cell type‐dependent internalization of the ADP‐activated P2Y1 receptor

We have shown that phosphorylation of Ser352 and Ser354 in the C‐terminus of the P2Y 1 receptor is necessary for agonist‐promoted internalization in MDCK cells. To investigate internalization of the receptor in other model cell lines, we expressed the P2Y 1 receptor in HEK293 and 1321N1 cells; unexpectedly, agonist did not promote internalization in either cell line. Mutational analysis of Ser and Thr residues in the C‐tail of the P2Y 1 receptor revealed that a S336A mutation within a highly conserved SRAT motif restored agonist‐dependent internalization in HEK293 and 1321N1 cells, while it markedly increased the rate and extent of receptor internalization in MDCK cells. Phosphorylation studies showed that Ser336, Ser352 and Ser354 were phosphorylated in response to agonist addition in HEK293 and 1321N1 cells, while only Ser352 and Ser354 were phosphorylated in MDCK cells. The marked differences in P2Y 1 receptor internalization and phosphorylation in MDCK vs. HEK293/1321N1 cells suggest that an unidentified kinase exists in HEK293 and 1321N1 cells that phosphorylates Ser336 in an agonist‐dependent manner, thereby blocking internalization of the receptor. These data uncover a critical role for Ser336 in the regulation of agonist‐promoted internalization of the P2Y 1 receptor and define two disparate cell types with distinct internalization properties. ( Supported by NIH grant HL54889 ).