Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1995 Dec;86(4):620–628.

Independent down-regulation of EP2 and EP3 subtypes of the prostaglandin E2 receptors on U937 human monocytic cells.

L Zeng 1, S An 1, E J Goetzl 1
PMCID: PMC1384064  PMID: 8567030

Abstract

Co-expression of EP2 and EP3 subtypes of prostaglandin E2 (PGE2) receptors (R) by U937 human monocytic cells permitted comparative studies of desensitization of each subtype. Specific binding of [3H]PGE2 to membranes of U937 cells showed a Kd of 2.9 +/- 0.3 nM (mean +/- SEM) and a Bmax of 40.5 +/- 1.0 fmol/mg protein, and was competitively inhibited by PGE2 > or = PGE1 > PGF2 alpha > PGD2 > PGI2. EP2 R and EP3 R mRNA were detected by reverse transcription-polymerase chain reaction and Northern blots. EP3 R expression was demonstrated by inhibition of [3H]PGE2 binding with the EP1/EP3 agonist sulprostone [50% inhibitory concentration (IC50 = 3.3 +/- 0.6 nM)] and the EP3/EP2 agonist M&B 28767 (IC50 = 2.1 +/- 0.3 nM), but not with the EP1 antagonist SC-19220. EP2 R protein was identified by Western blot analysis using specific rabbit IgG antibodies to an amino-terminal peptide of the EP2 R. EP2 R transduced PGE2 stimulation of significant increases in cellular [cAMP]i [50% effective concentration (EC50 = 20 +/- 2.5 nM)], and EP3 R mediated sulprostone inhibition of forskolin elevation of [cAMP]i (IC50 = 1.3 +/- 0.4 nM). Pretreatment of U937 cells with phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), for 1 hr reduced the total number, but not the affinity, of PGE2 R by down-regulating principally EP2 R. In contrast, a 24-hr exposure to PMA, which is known to down-regulate PKC, suppressed both the total number and affinity of PGE2 R on U937 cells with concurrent reductions in EP2 R and EP3 R. The down-regulation of EP2 R by PMA at 1 hr was blocked by staurosporine, an inhibitor of PKC, whereas the down-regulation of EP3 R by PMA at 24 hr was blocked by indomethacin. Pretreatment of U937 cells with PGE2 for 1 and 24 hr reduced both the binding affinity and the total number of PGE2 R, by co-ordinate suppression of the EP2 R and EP3 R. Desensitization of EP2 R and EP3 R for 1 hr with PGE2 suppressed subsequent PGE2-evoked chemokinetic responses to PGE2, whereas selective down-regulation of EP2 R alone by PMA for 1 hr had no effects on U937 cell migration. Thus expression of each subtype of PGE2 R is regulated independently and EP3 R, but not EP2 R, transduces PGE2 effects on migration of mononuclear phagocytes.

Full text

PDF
620

Images in this article

623Figure 2
on p.623
623Figure 3
on p.623

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adam M., Boie Y., Rushmore T. H., Müller G., Bastien L., McKee K. T., Metters K. M., Abramovitz M. Cloning and expression of three isoforms of the human EP3 prostanoid receptor. FEBS Lett. 1994 Jan 31;338(2):170–174. doi: 10.1016/0014-5793(94)80358-7. [DOI] [PubMed] [Google Scholar]
  2. An S., Yang J., So S. W., Zeng L., Goetzl E. J. Isoforms of the EP3 subtype of human prostaglandin E2 receptor transduce both intracellular calcium and cAMP signals. Biochemistry. 1994 Dec 6;33(48):14496–14502. doi: 10.1021/bi00252a016. [DOI] [PubMed] [Google Scholar]
  3. An S., Yang J., Xia M., Goetzl E. J. Cloning and expression of the EP2 subtype of human receptors for prostaglandin E2. Biochem Biophys Res Commun. 1993 Nov 30;197(1):263–270. doi: 10.1006/bbrc.1993.2470. [DOI] [PubMed] [Google Scholar]
  4. Bell J. D., Buxton I. L., Brunton L. L. Enhancement of adenylate cyclase activity in S49 lymphoma cells by phorbol esters. Putative effect of C kinase on alpha s-GTP-catalytic subunit interaction. J Biol Chem. 1985 Mar 10;260(5):2625–2628. [PubMed] [Google Scholar]
  5. Boggs J. M., Koo C. H., Goetzl E. J. Down-regulation of receptor antigen in leukotriene B4-induced chemotactic deactivation of human polymorphonuclear leucocytes. Immunology. 1991 Jun;73(2):212–216. [PMC free article] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Collins M. K., Rozengurt E. Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps. J Cell Physiol. 1984 Feb;118(2):133–142. doi: 10.1002/jcp.1041180205. [DOI] [PubMed] [Google Scholar]
  8. Downward J., Waterfield M. D., Parker P. J. Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity. J Biol Chem. 1985 Nov 25;260(27):14538–14546. [PubMed] [Google Scholar]
  9. Findlay D. M., Michelangeli V. P., Robinson P. J. Protein kinase-C-induced down-regulation of calcitonin receptors and calcitonin-activated adenylate cyclase in T47D and BEN cells. Endocrinology. 1989 Nov;125(5):2656–2663. doi: 10.1210/endo-125-5-2656. [DOI] [PubMed] [Google Scholar]
  10. Freyaldenhoven A. M., Gutierrez G. E., Lifschitz M. D., Katz M. S. Protein kinase C differentially modulates PTH- and PGE2-sensitive adenylate cyclase in osteoblast-like cells. Am J Physiol. 1992 Jan;262(1 Pt 1):E87–E95. doi: 10.1152/ajpendo.1992.262.1.E87. [DOI] [PubMed] [Google Scholar]
  11. Goetzl E. J., An S., Smith W. L. Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases. FASEB J. 1995 Aug;9(11):1051–1058. doi: 10.1096/fasebj.9.11.7649404. [DOI] [PubMed] [Google Scholar]
  12. Goetzl E. J., An S., Zeng L. Specific suppression by prostaglandin E2 of activation-induced apoptosis of human CD4+CD8+ T lymphoblasts. J Immunol. 1995 Feb 1;154(3):1041–1047. [PubMed] [Google Scholar]
  13. Goetzl E. J., Hoe K. Y. Chemotactic factor receptors of human PMN leucocytes. I. Effects on migration of labelling plasma membrane determinants with impermeant covalent reagents and inhibition of labelling by chemotactic factors. Immunology. 1979 Jun;37(2):407–418. [PMC free article] [PubMed] [Google Scholar]
  14. Goldring S. R., Amento E. P., Roelke M. S., Krane S. M. The adenosine 3':5'-cyclic monophosphate response to prostaglandin E2 is altered in U937 cells in association with maturational events induced by activated T lymphocytes and 1,25-dihydroxyvitamin D3. J Immunol. 1986 May 1;136(9):3461–3466. [PubMed] [Google Scholar]
  15. Grinstein S., Mack E., Mills G. B. Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes. Biochem Biophys Res Commun. 1986 Jan 14;134(1):8–13. doi: 10.1016/0006-291x(86)90519-x. [DOI] [PubMed] [Google Scholar]
  16. Harris P., Ralph P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J Leukoc Biol. 1985 Apr;37(4):407–422. doi: 10.1002/jlb.37.4.407. [DOI] [PubMed] [Google Scholar]
  17. Honda A., Sugimoto Y., Namba T., Watabe A., Irie A., Negishi M., Narumiya S., Ichikawa A. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP2 subtype. J Biol Chem. 1993 Apr 15;268(11):7759–7762. [PubMed] [Google Scholar]
  18. Huang C. L., Cogan M. G., Cragoe E. J., Jr, Ives H. E. Thrombin activation of the Na+/H+ exchanger in vascular smooth muscle cells. Evidence for a kinase C-independent pathway which is Ca2+-dependent and pertussis toxin-sensitive. J Biol Chem. 1987 Oct 15;262(29):14134–14140. [PubMed] [Google Scholar]
  19. Jacobs S., Sahyoun N. E., Saltiel A. R., Cuatrecasas P. Phorbol esters stimulate the phosphorylation of receptors for insulin and somatomedin C. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6211–6213. doi: 10.1073/pnas.80.20.6211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Katada T., Gilman A. G., Watanabe Y., Bauer S., Jakobs K. H. Protein kinase C phosphorylates the inhibitory guanine-nucleotide-binding regulatory component and apparently suppresses its function in hormonal inhibition of adenylate cyclase. Eur J Biochem. 1985 Sep 2;151(2):431–437. doi: 10.1111/j.1432-1033.1985.tb09120.x. [DOI] [PubMed] [Google Scholar]
  21. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  22. Murayama T., Nomura Y., Ui M. Enhancement of adenosine A2 and prostaglandin E1 receptor-mediated cAMP generation by prior exposure of Swiss 3T3 fibroblasts to Ca2+-mobilizing receptor agonists or phorbol ester. Activation of protein kinase C triggers increases in the receptor density in cell membranes. J Biol Chem. 1989 Sep 15;264(26):15186–15191. [PubMed] [Google Scholar]
  23. Niedel J. E., Kuhn L. J., Vandenbark G. R. Phorbol diester receptor copurifies with protein kinase C. Proc Natl Acad Sci U S A. 1983 Jan;80(1):36–40. doi: 10.1073/pnas.80.1.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ralph P., Moore M. A., Nilsson K. Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. J Exp Med. 1976 Jun 1;143(6):1528–1533. doi: 10.1084/jem.143.6.1528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Regan J. W., Bailey T. J., Pepperl D. J., Pierce K. L., Bogardus A. M., Donello J. E., Fairbairn C. E., Kedzie K. M., Woodward D. F., Gil D. W. Cloning of a novel human prostaglandin receptor with characteristics of the pharmacologically defined EP2 subtype. Mol Pharmacol. 1994 Aug;46(2):213–220. [PubMed] [Google Scholar]
  26. Sibley D. R., Nambi P., Peters J. R., Lefkowitz R. J. Phorbol diesters promote beta-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes. Biochem Biophys Res Commun. 1984 Jun 29;121(3):973–979. doi: 10.1016/0006-291x(84)90772-1. [DOI] [PubMed] [Google Scholar]
  27. Sugimoto Y., Namba T., Honda A., Hayashi Y., Negishi M., Ichikawa A., Narumiya S. Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype. J Biol Chem. 1992 Apr 5;267(10):6463–6466. [PubMed] [Google Scholar]
  28. Sundström C., Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. 1976 May 15;17(5):565–577. doi: 10.1002/ijc.2910170504. [DOI] [PubMed] [Google Scholar]
  29. Yang J., Xia M., Goetzl E. J., An S. Cloning and expression of the EP3-subtype of human receptors for prostaglandin E2. Biochem Biophys Res Commun. 1994 Feb 15;198(3):999–1006. doi: 10.1006/bbrc.1994.1142. [DOI] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES