Activation of P2Y(2) receptors by extracellular nucleotides has been shown to induce phenotypic differentiation of human promonocytic U937 cells that is associated with the inflammatory response. The P2Y(2) receptor agonist, UTP, induced the phosphorylation of the MAP kinases MEK1/2 and ERK1/2 in a sequential manner, since ERK1/2 phosphorylation was abolished by the MEK1/2 inhibitor PD 098059. Other results indicated that P2Y(2) receptors can couple to MAP kinases via phosphatidylinositol 3-kinase (PI3K) and c-src. Accordingly, ERK1/2 phosphorylation induced by UTP was inhibited by the PI3K inhibitors, wortmannin and LY294002, and the c-src inhibitors, radicicol and PP2, but not by inhibitors of protein kinase C (PKC). The phosphorylation of ERK1/2 was independent of the ability of P2Y(2) receptors to increase the concentration of intracellular free calcium, since chelation of intracellular calcium by BAPTA did not diminish the phosphorylation of ERK1/2 induced by UTP. A 5-minute treatment with UTP reduced U937 cell responsiveness to a subsequent UTP challenge. UTP-induced desensitization was characterized by an increase in the EC(50) for receptor activation (from 0.44 to 9.3 microM) and a dramatic ( approximately 75%) decrease in the maximal calcium mobilization induced by a supramaximal dose of UTP. Phorbol ester treatment also caused P2Y(2) receptor desensitization (EC(50) = 12.3 microM UTP and maximal calcium mobilization reduced by approximately 33%). The protein kinase C inhibitor GF 109203X failed to significantly inhibit the UTP-induced desensitization of the P2Y(2) receptor, whereas the protein phosphatase inhibitor okadaic acid blocked receptor resensitization. Recovery of receptor activity after UTP-induced desensitization was evident in cells treated with agonist for 5 or 30 min. However, P2Y(2) receptor activity remained partially desensitized 30 min after pretreatment of cells with UTP for 1 h or longer. This sustained desensitized state correlated with a decrease in P2Y(2) receptor mRNA levels. Desensitization of ERK1/2 phosphorylation was induced by a 5-minute pretreatment with UTP, and cell responsiveness did not return even after a 30-minute incubation of cells in the absence of an agonist. Results suggest that desensitization of the P2Y(2) receptor may involve covalent modifications (i.e., receptor phosphorylation) that functionally uncouple the receptor from the calcium signaling pathway, and that transcriptional regulation may play a role in long-term desensitization. Our results indicate that calcium mobilization and ERK1/2 phosphorylation induced by P2Y(2) receptor activation are independent events in U937 monocytes.

SummaryPurification of HA-tagged P2Y 2 receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57-76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonistinduced desensitized state, suggesting a role for P2Y 2 receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y 2 nucleotide receptors from metabolically [ 32 P]-labeled cells indicated a 3.8 ± 0.2-fold increase in the [ 32 P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ~40% of the surface receptors were internalized after a 15 min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y 2 receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y 2 receptor signaling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y 2 receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbolinsensitive protein kinases in agonist-induced desensitization of the P2Y 2 nucleotide receptor.

Extracellular nucleotides exert a large number of physiological effects through activation of P2Y receptors. We expressed rat P2Y2 (rP2Y2) receptor, tagged with green fluorescent protein (GFP) in HEK-293 cells and visualized receptor translocation in live cells by confocal microscopy. Functional receptor expression was confirmed by determining [Ca2+]i responses. Agonist stimulation caused a time-dependent translocation of the receptor from the plasma membrane to the cytoplasm. Rearrangement of the actin cytoskeleton was observed during agonist-mediated rP2Y2-GFP receptor internalization. Colocalization of the internalized receptor with early endosomes, clathrin and lysosomes was detected by confocal microscopy. The inhibition of receptor endocytosis by either high-density medium or chlorpromazine in the presence of UTP indicates that the receptor was internalized by the clathrin-mediated pathway. The caveolin-mediated pathway was not involved. Targeting of the receptor from endosomes to lysosomes seems to involve the proteasome pathway, because proteasomal inhibition increased receptor recycling back to the plasma membrane.

G protein‐coupled P2Y nucleotide receptors have been described in cells of the immune system, including neutrophils, monocytes, macrophages, B‐ and T‐lymphocytes, granulocytes, and myeloblasts. In the monocyte/macrophage lineage, a P2Y2 receptor subtype activated equipotently by adenosine 5′‐triphosphate (ATP) and uridine 5′‐triphosphate (UTP) is coupled to phospholipase C and regulates low density lipoprotein uptake, superoxide production, gating of calcium channels, and phagocytosis. In U937 monocytes, P2Y2 receptor activation leads to phosphorylation of MKK3 and p38, mitogen‐activated protein kinases. P2Y2 receptors in U937 monocytes undergo agonist‐induced desensitization that decreases the potency and efficacy of subsequent doses of agonist. Cells recover rapidly from desensitization after short‐term (<30 minutes) agonist treatments, whereas long‐term (>1‐hour) treatments produced sustained desensitization correlating with a decrease in P2Y2 receptor mRNA levels. To investigate the molecular determinants of desensitization, a recombinant P2Y2 receptor was expressed in human astrocytoma cells in which it exhibited agonist‐induced desensitization and sequestration. P2Y2 receptors containing C‐terminal deletions of potential phosphorylation sites for protein kinases were resistant to desensitization and sequestration. Other results indicate that an integrin‐binding domain, arginine‐glycine‐aspartate (RGD), in the first extracellular loop of the P2Y2 receptor binds specifically to αvβ3 and αvβ5 integrins (vitronectin receptors), an intriguing finding considering the wide distribution of these receptors among immune cells. The RGD domain was necessary for localizing the receptor to focal adhesion complexes to promote efficient receptor signaling. Finally, positively charged amino acids were identified in the ligand binding site of the P2Y2 receptor, information that could promote the design of compounds for selective modulation of immune function. Drug Dev. Res. 45:222–228, 1998. © 1998 Wiley‐Liss, Inc.

A fluorescence spectroscopy experiment is described where students integrated biochemistry and instrumental analysis, while characterizing the green fluorescent protein excitation and emission spectra in terms of its phenolic and phenolate chromophores. Students studied the combined effect of pH and temperature on the protein's fluorescence, which led them to conclude that its fluorescence, denaturation, and renaturation was pH dependent. The importance of temperature control in fluorescence spectroscopy, and of control samples in experiments, was also stressed. This experiment is suitable for an upper-level biochemistry laboratory, as an extension of experiences involving bacterial transformation and hydrophobic interaction chromatography to purify the protein. It is also appropriate for an instrumental analysis laboratory using a provided protein sample.