Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1995 Feb 15;306(Pt 1):87–92. doi: 10.1042/bj3060087

Angiotensin II AT2 receptors are functionally coupled to protein tyrosine dephosphorylation in N1E-115 neuroblastoma cells.

C Nahmias 1, S M Cazaubon 1, M M Briend-Sutren 1, D Lazard 1, P Villageois 1, A D Strosberg 1
PMCID: PMC1136485  PMID: 7532401

Abstract

Murine N1E-115 neuroblastoma cells are shown to express a single class of angiotensin II (Ang II) receptors that display all the pharmacological properties defining the Ang II receptor subtype 2 (AT2): high affinity for 125I-labelled AT2-selective agonist CGP 42112 (Kd 91 +/- 19 pM); expected rank order of potency (CGP 42112 = (Sar1,Ile8)Ang II > or = Ang II > PD 123319 >> DUP 753) for several Ang II analogues; increased binding in the presence of the reducing reagent dithiothreitol (DTT); and insensitivity to analogues of GTP. Molecular cloning of cDNA encoding AT2 receptors from N1E-115 cells reveals nucleotide sequence identity with the AT2 subtype expressed in fetal tissue. Murine AT2 receptors transiently expressed in COS cells display the same pharmacological profile as endogenous Ang II receptors of N1E-115 cells. Taken together, these data reveal the exclusive presence of the AT2 receptor subtype in N1E-115 cells. Incubation of N1E-115 cells with Ang II leads to a marked decrease in the level of tyrosine phosphorylation of several proteins with apparent molecular masses of 80, 97, 120, 150 and 180 kDa respectively. Tyrosine dephosphorylation of the same set of proteins is observed after treatment with the AT2-specific agonist CGP 42112. The response to both effectors is rapid and transient, showing a maximum between 5 and 10 min, and returning to basal levels after 20-30 min. In both cases, tyrosine dephosphorylation can be prevented by co-incubation with an excess of the antagonist Sarile. These data thus establish that AT2 receptor activation leads to protein tyrosine dephosphorylation in N1E-115 cells, and support a possible role for AT2 receptors in the negative regulation of cell proliferation.

Full text

PDF
87

Images in this article

90Figure 3
on p.90
90Figure 4
on p.90
91Figure 5
on p.91

Selected References

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

  1. Aruffo A., Seed B. Molecular cloning of a CD28 cDNA by a high-efficiency COS cell expression system. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8573–8577. doi: 10.1073/pnas.84.23.8573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bottari S. P., King I. N., Reichlin S., Dahlstroem I., Lydon N., de Gasparo M. The angiotensin AT2 receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylate cyclase. Biochem Biophys Res Commun. 1992 Feb 28;183(1):206–211. doi: 10.1016/0006-291x(92)91629-5. [DOI] [PubMed] [Google Scholar]
  3. Bottari S. P., Taylor V., King I. N., Bogdal Y., Whitebread S., de Gasparo M. Angiotensin II AT2 receptors do not interact with guanine nucleotide binding proteins. Eur J Pharmacol. 1991 Jun 19;207(2):157–163. doi: 10.1016/0922-4106(91)90091-u. [DOI] [PubMed] [Google Scholar]
  4. Brechler V., Jones P. W., Levens N. R., de Gasparo M., Bottari S. P. Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells. Regul Pept. 1993 Mar 19;44(2):207–213. doi: 10.1016/0167-0115(93)90244-3. [DOI] [PubMed] [Google Scholar]
  5. Brechler V., Reichlin S., De Gasparo M., Bottari S. P. Angiotensin II stimulates protein tyrosine phosphatase activity through a G-protein independent mechanism. Receptors Channels. 1994;2(2):89–98. [PubMed] [Google Scholar]
  6. Buisson B., Bottari S. P., de Gasparo M., Gallo-Payet N., Payet M. D. The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108-15 cells. FEBS Lett. 1992 Sep 7;309(2):161–164. doi: 10.1016/0014-5793(92)81086-2. [DOI] [PubMed] [Google Scholar]
  7. Butcher R. D., Schöllmann C., Marmé D. Angiotensin II mediates intracellular signalling in vascular smooth muscle cells by activation of tyrosine-specific protein kinases and c-raf-1. Biochem Biophys Res Commun. 1993 Nov 15;196(3):1280–1287. doi: 10.1006/bbrc.1993.2391. [DOI] [PubMed] [Google Scholar]
  8. Cazaubon S., Parker P. J., Strosberg A. D., Couraud P. O. Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes. Biochem J. 1993 Jul 15;293(Pt 2):381–386. doi: 10.1042/bj2930381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chiu A. T., McCall D. E., Nguyen T. T., Carini D. J., Duncia J. V., Herblin W. F., Uyeda R. T., Wong P. C., Wexler R. R., Johnson A. L. Discrimination of angiotensin II receptor subtypes by dithiothreitol. Eur J Pharmacol. 1989 Oct 24;170(1-2):117–118. doi: 10.1016/0014-2999(89)90145-3. [DOI] [PubMed] [Google Scholar]
  10. Dudley D. T., Hubbell S. E., Summerfelt R. M. Characterization of angiotensin II (AT2) binding sites in R3T3 cells. Mol Pharmacol. 1991 Sep;40(3):360–367. [PubMed] [Google Scholar]
  11. Florio T., Pan M. G., Newman B., Hershberger R. E., Civelli O., Stork P. J. Dopaminergic inhibition of DNA synthesis in pituitary tumor cells is associated with phosphotyrosine phosphatase activity. J Biol Chem. 1992 Dec 5;267(34):24169–24172. [PubMed] [Google Scholar]
  12. Force T., Kyriakis J. M., Avruch J., Bonventre J. V. Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. J Biol Chem. 1991 Apr 5;266(10):6650–6656. [PubMed] [Google Scholar]
  13. Grady E. F., Sechi L. A., Griffin C. A., Schambelan M., Kalinyak J. E. Expression of AT2 receptors in the developing rat fetus. J Clin Invest. 1991 Sep;88(3):921–933. doi: 10.1172/JCI115395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hordijk P. L., Verlaan I., van Corven E. J., Moolenaar W. H. Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway. J Biol Chem. 1994 Jan 7;269(1):645–651. [PubMed] [Google Scholar]
  15. Huckle W. R., Dy R. C., Earp H. S. Calcium-dependent increase in tyrosine kinase activity stimulated by angiotensin II. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8837–8841. doi: 10.1073/pnas.89.18.8837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jaiswal N., Tallant E. A., Diz D. I., Khosla M. C., Ferrario C. M. Subtype 2 angiotensin receptors mediate prostaglandin synthesis in human astrocytes. Hypertension. 1991 Jun;17(6 Pt 2):1115–1120. doi: 10.1161/01.hyp.17.6.1115. [DOI] [PubMed] [Google Scholar]
  17. Jalink K., Eichholtz T., Postma F. R., van Corven E. J., Moolenaar W. H. Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. Cell Growth Differ. 1993 Apr;4(4):247–255. [PubMed] [Google Scholar]
  18. Janiak P., Pillon A., Prost J. F., Vilaine J. P. Role of angiotensin subtype 2 receptor in neointima formation after vascular injury. Hypertension. 1992 Dec;20(6):737–745. doi: 10.1161/01.hyp.20.6.737. [DOI] [PubMed] [Google Scholar]
  19. Kambayashi Y., Bardhan S., Takahashi K., Tsuzuki S., Inui H., Hamakubo T., Inagami T. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993 Nov 25;268(33):24543–24546. [PubMed] [Google Scholar]
  20. Lazard D., Villageois P., Briend-Sutren M. M., Cavaillé F., Bottari S., Strosberg A. D., Nahmias C. Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium. Eur J Biochem. 1994 Mar 15;220(3):919–926. doi: 10.1111/j.1432-1033.1994.tb18695.x. [DOI] [PubMed] [Google Scholar]
  21. Leung K. H., Roscoe W. A., Smith R. D., Timmermans P. B., Chiu A. T. Characterization of biochemical responses of angiotensin II (AT2) binding sites in the rat pheochromocytoma PC12W cells. Eur J Pharmacol. 1992 Sep 1;227(1):63–70. doi: 10.1016/0922-4106(92)90143-j. [DOI] [PubMed] [Google Scholar]
  22. Millan M. A., Jacobowitz D. M., Aguilera G., Catt K. J. Differential distribution of AT1 and AT2 angiotensin II receptor subtypes in the rat brain during development. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11440–11444. doi: 10.1073/pnas.88.24.11440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Molloy C. J., Taylor D. S., Weber H. Angiotensin II stimulation of rapid protein tyrosine phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J Biol Chem. 1993 Apr 5;268(10):7338–7345. [PubMed] [Google Scholar]
  24. Mukoyama M., Nakajima M., Horiuchi M., Sasamura H., Pratt R. E., Dzau V. J. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem. 1993 Nov 25;268(33):24539–24542. [PubMed] [Google Scholar]
  25. 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]
  26. Murphy T. J., Alexander R. W., Griendling K. K., Runge M. S., Bernstein K. E. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991 May 16;351(6323):233–236. doi: 10.1038/351233a0. [DOI] [PubMed] [Google Scholar]
  27. Nakajima M., Mukoyama M., Pratt R. E., Horiuchi M., Dzau V. J. Cloning of cDNA and analysis of the gene for mouse angiotensin II type 2 receptor. Biochem Biophys Res Commun. 1993 Dec 15;197(2):393–399. doi: 10.1006/bbrc.1993.2492. [DOI] [PubMed] [Google Scholar]
  28. Ohnishi J., Ishido M., Shibata T., Inagami T., Murakami K., Miyazaki H. The rat angiotensin II AT1A receptor couples with three different signal transduction pathways. Biochem Biophys Res Commun. 1992 Jul 31;186(2):1094–1101. doi: 10.1016/0006-291x(92)90859-j. [DOI] [PubMed] [Google Scholar]
  29. Pan M. G., Florio T., Stork P. J. G protein activation of a hormone-stimulated phosphatase in human tumor cells. Science. 1992 May 22;256(5060):1215–1217. doi: 10.1126/science.256.5060.1215. [DOI] [PubMed] [Google Scholar]
  30. Pucell A. G., Hodges J. C., Sen I., Bumpus F. M., Husain A. Biochemical properties of the ovarian granulosa cell type 2-angiotensin II receptor. Endocrinology. 1991 Apr;128(4):1947–1959. doi: 10.1210/endo-128-4-1947. [DOI] [PubMed] [Google Scholar]
  31. Reagan L. P., Ye X. H., Mir R., DePalo L. R., Fluharty S. J. Up-regulation of angiotensin II receptors by in vitro differentiation of murine N1E-115 neuroblastoma cells. Mol Pharmacol. 1990 Dec;38(6):878–886. [PubMed] [Google Scholar]
  32. Reagan L. P., Ye X., Maretzski C. H., Fluharty S. J. Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells. J Neurochem. 1993 Jan;60(1):24–31. doi: 10.1111/j.1471-4159.1993.tb05818.x. [DOI] [PubMed] [Google Scholar]
  33. Salles J. P., Gayral-Taminh M., Fauvel J., Delobbe I., Mignon-Conté M., Conté J. J., Chap H. Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells. J Biol Chem. 1993 Jun 15;268(17):12805–12811. [PubMed] [Google Scholar]
  34. Sasaki K., Yamano Y., Bardhan S., Iwai N., Murray J. J., Hasegawa M., Matsuda Y., Inagami T. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991 May 16;351(6323):230–233. doi: 10.1038/351230a0. [DOI] [PubMed] [Google Scholar]
  35. Sumners C., Tang W., Zelezna B., Raizada M. K. Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7567–7571. doi: 10.1073/pnas.88.17.7567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Takahasi K., Bardhan S., Kambayashi Y., Shirai H., Inagami T. Protein tyrosine phosphatase inhibition by angiotensin II in rat pheochromocytoma cells through type 2 receptor, AT2. Biochem Biophys Res Commun. 1994 Jan 14;198(1):60–66. doi: 10.1006/bbrc.1994.1009. [DOI] [PubMed] [Google Scholar]
  37. Tsutsumi K., Saavedra J. M. Heterogeneity of angiotensin II AT2 receptors in the rat brain. Mol Pharmacol. 1992 Feb;41(2):290–297. [PubMed] [Google Scholar]
  38. Tsutsumi K., Zorad S., Saavedra J. M. The AT2 subtype of the angiotensin II receptors has differential sensitivity to dithiothreitol in specific brain nuclei of young rats. Eur J Pharmacol. 1992 Jun 5;226(2):169–173. doi: 10.1016/0922-4106(92)90179-y. [DOI] [PubMed] [Google Scholar]
  39. Viswanathan M., Tsutsumi K., Correa F. M., Saavedra J. M. Changes in expression of angiotensin receptor subtypes in the rat aorta during development. Biochem Biophys Res Commun. 1991 Sep 30;179(3):1361–1367. doi: 10.1016/0006-291x(91)91723-p. [DOI] [PubMed] [Google Scholar]
  40. Whitebread S., Mele M., Kamber B., de Gasparo M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989 Aug 30;163(1):284–291. doi: 10.1016/0006-291x(89)92133-5. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES