RESUMO
BACKGROUND AND PURPOSE: The P2Y(1) receptor promotes chloride secretion in epithelial cells, a process critical for regulation of extracellular ion and fluid levels. Here we have examined the role of phosphorylation in agonist-induced internalization of P2Y(1) receptors. EXPERIMENTAL APPROACH: A high-affinity radiolabelled antagonist, MRS2500, was used to quantify cell surface-binding sites of P2Y(1) receptors in Madin-Darby canine kidney (MDCK) epithelial cells, following exposure to agonists. The regions in the carboxyl terminus involved in both agonist-induced internalization of the receptor and its phosphorylation were identified by mutational analysis. KEY RESULTS: Endogenous and stably expressed recombinant P2Y(1) receptors rapidly internalized with similar time courses in response to agonist in MDCK cells, ensuring that the levels of recombinant receptor achieved by retroviral infection did not adversely affect function of the internalization machinery. Four protein kinase C inhibitors of varying specificity did not affect internalization of recombinant receptors. Agonist-promoted internalization of a series of truncated P2Y(1) receptors identified a region between residues 349 and 359 in the carboxyl terminus as critical for regulation. Two amino acids within this region, Ser352 and Ser354, were shown to be both necessary and sufficient for agonist-promoted receptor phosphorylation and internalization. CONCLUSIONS AND IMPLICATIONS: Our results firmly establish Ser352 and Ser354 in the carboxyl terminus of P2Y(1) receptors as critical residues for agonist-induced receptor internalization in MDCK cells. As the mechanism mediating this regulation requires phosphorylation of these key residues, the relevant receptor-regulated protein kinase can now be identified.
