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J. Biol. Chem., Vol. 276, Issue 15, 11939-11948, April 13, 2001

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Differential Regulation of the Uridine Nucleotide-activated P2Y4 and P2Y6 Receptors
SER-333 AND SER-334 IN THE CARBOXYL TERMINUS ARE INVOLVED IN AGONIST-DEPENDENT PHOSPHORYLATION DESENSITIZATION AND INTERNALIZATION OF THE P2Y4 RECEPTOR^*

Amy E. Brinson and T. Kendall Harden

From the Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Agonist-promoted regulation of the uridine nucleotide-activated human P2Y4 receptor (P2Y4-R) and P2Y6 receptor (P2Y6-R) was studied. Incubation of P2Y4-R-expressing 1321N1 human astrocytoma cells with the cognate agonist UTP resulted in rapid desensitization of the inositol phosphate response and a 50% loss of cell surface receptors. In contrast, incubation of P2Y6-R-expressing cells with the cognate agonist UDP caused neither rapid desensitization nor rapid loss of cell surface receptors. Removal of UTP from the medium of UTP-pretreated cells resulted in rapid and complete recovery of surface P2Y4-R even after 12 h of agonist treatment. Although extended incubation with UDP also caused a loss of surface P2Y6-R, rapid recovery of surface P2Y6-R did not occur following removal of agonist. Pharmacological studies indicated that neither protein kinase C nor other Ca²⁺-activated kinases were involved in agonist-promoted desensitization or loss of surface P2Y4-R or P2Y6-R. Mutational analyses were carried out to identify domains involved in agonist-dependent regulation of P2Y4-R. Sequential truncation of the carboxyl-terminal domain revealed that sequence between amino acids 332 and 343 was necessary for UTP-promoted desensitization and internalization. Further mutational analyses of the three serines in this domain confirmed that Ser-333 and Ser-334 play a major role in these agonist-promoted changes in P2Y4-R. Experiments were carried out with [³²P]P_i-labeled cells to ascertain the role of phosphorylation in regulation of P2Y4-R. Incubation with UTP for 2 min caused a marked increase in phosphorylation of both the wild-type P2Y4-R and the P2Y4-343 truncation mutant. In contrast, no UTP-promoted phosphorylation of the P2Y4-332 truncation mutant was observed. Taken together, these results demonstrate differential regulation of uridine nucleotide-activated P2Y4-R and P2Y6-R and indicate that Ser-333 and Ser-334 in the carboxyl terminus of P2Y4-R are important for UTP-dependent phosphorylation, desensitization, and loss of surface receptors.

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