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.
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 Ca(2+)-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 [(32)P]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.
Brinson
A E
AE
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
Harden
T K
TK
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004705:Endocytosis; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology*
D017931:DNA Primers; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; C104749:purinoceptor P2Y4; C099845:purinoceptor P2Y6; D012694:Serine; D014544:Uridine Triphosphate