PDZ ligand‐dependent phosphorylation of Ser336 in the C‐terminal tail of the P2Y1 receptor blocks agonist‐promoted internalization in HEK293 cells

Abstract

We have shown previously that phosphorylation of Ser352 and Ser354 in the C‐terminus of the ADP‐activated P2Y1 receptor is necessary for agonist‐promoted internalization in MDCK cells. Unexpectedly, ADP addition did not promote internalization of the P2Y1 receptor in HEK293 and 1321N1 cells. Mutational analysis of Ser and Thr residues in the C‐tail of the P2Y1 receptor revealed that a S336A mutation within a highly conserved SRAT motif conferred 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 in HEK293 and 1321N1 cells, while only Ser352 and Ser354 were phosphorylated in MDCK cells. Additionally, mutation of SRAT to DRAD to mimic Ser336 phosphorylation abolished agonist‐promoted internalization of the P2Y1 receptor in MDCK cells. Deletion of the PDZ ligand at the C‐terminus of the receptor conferred agonist‐promoted internalization and prevented phosphorylation of Ser336, suggesting recruitment of a PDZ protein‐dependent kinase that regulates internalization. This study reveals a critical role for Ser336 in the regulation of agonist‐promoted internalization of the P2Y1 receptor that depends on the cell type in which it is expressed. (Supported by NIH grant HL54889).