Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1993 Dec 1;90(23):11009–11013. doi: 10.1073/pnas.90.23.11009

Purification and characterization of angiotensin II AT2 receptors from neonatal rat kidney.

G M Ciuffo 1, F M Heemskerk 1, J M Saavedra 1
PMCID: PMC47911  PMID: 8248203

Abstract

Angiotensin II (Ang II) AT2 receptors were purified 40,000-fold to a nearly homogeneous state after solubilization from neonatal rat kidney membranes with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonic acid. Comparable IC50 values for the soluble extract (0.32 nM) and membranes (0.31 nM) were obtained by competition curves with 125I-labeled CGP42112, a selective AT2 ligand. Binding to AT2 receptors in the soluble extract was not sensitive to dithiothreitol. AT2 receptors were further purified by gel filtration and a CGP42112 Sepharose affinity column. Ang II AT2 receptors were selectively eluted with 5 microM CGP42112 at 4 degrees C, and a single band with an apparent molecular mass of 71 kDa was obtained after SDS/PAGE. Two-dimensional electrophoresis confirmed the purity of the protein and an isoelectric point of 5.3-5.5 was obtained. A highly selective elution of the AT2 receptors from the affinity column was performed with 5 nM 125I-labeled CGP42112 at room temperature after the column was treated with 1 microM losartan in the presence of high salt. After cross-linking, a major labeled protein with similar molecular mass and isoelectric point was obtained. Dissociation of the radiolabeled protein was insensitive to losartan but was enhanced by CGP42112, PD123177, Ang II, and [Sar1]Ang II. In summary, Ang II AT2 receptors were purified by CGP42112 affinity chromatography and selective elution and retain the pharmacological specificity of particulate receptors.

Full text

PDF
11009

Images in this article

11012Fig. 4
on p.11012
11012Fig. 5
on p.11012

Selected References

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

  1. Bottari S. P., de Gasparo M., Steckelings U. M., Levens N. R. Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications. Front Neuroendocrinol. 1993 Apr;14(2):123–171. doi: 10.1006/frne.1993.1005. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bruns R. F., Lawson-Wendling K., Pugsley T. A. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983 Jul 1;132(1):74–81. doi: 10.1016/0003-2697(83)90427-x. [DOI] [PubMed] [Google Scholar]
  4. Bumpus F. M., Catt K. J., Chiu A. T., DeGasparo M., Goodfriend T., Husain A., Peach M. J., Taylor D. G., Jr, Timmermans P. B. Nomenclature for angiotensin receptors. A report of the Nomenclature Committee of the Council for High Blood Pressure Research. Hypertension. 1991 May;17(5):720–721. doi: 10.1161/01.hyp.17.5.720. [DOI] [PubMed] [Google Scholar]
  5. Carson M. C., Harper C. M., Baukal A. J., Aguilera G., Catt K. J. Physicochemical characterization of photoaffinity-labeled angiotensin II receptors. Mol Endocrinol. 1987 Feb;1(2):147–153. doi: 10.1210/mend-1-2-147. [DOI] [PubMed] [Google Scholar]
  6. Chiu A. T., Duncia J. V., McCall D. E., Wong P. C., Price W. A., Jr, Thoolen M. J., Carini D. J., Johnson A. L., Timmermans P. B. Nonpeptide angiotensin II receptor antagonists. III. Structure-function studies. J Pharmacol Exp Ther. 1989 Sep;250(3):867–874. [PubMed] [Google Scholar]
  7. Chiu A. T., Herblin W. F., McCall D. E., Ardecky R. J., Carini D. J., Duncia J. V., Pease L. J., Wong P. C., Wexler R. R., Johnson A. L. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989 Nov 30;165(1):196–203. doi: 10.1016/0006-291x(89)91054-1. [DOI] [PubMed] [Google Scholar]
  8. Ciuffo G. M., Viswanathan M., Seltzer A. M., Tsutsumi K., Saavedra J. M. Glomerular angiotensin II receptor subtypes during development of rat kidney. Am J Physiol. 1993 Aug;265(2 Pt 2):F264–F271. doi: 10.1152/ajprenal.1993.265.2.F264. [DOI] [PubMed] [Google Scholar]
  9. Desarnaud F., Marie J., Lombard C., Larguier R., Seyer R., Lorca T., Jard S., Bonnafous J. C. Deglycosylation and fragmentation of purified rat liver angiotensin II receptor: application to the mapping of hormone-binding domains. Biochem J. 1993 Jan 1;289(Pt 1):289–297. doi: 10.1042/bj2890289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dudley D. T., Summerfelt R. M. Regulated expression of angiotensin II (AT2) binding sites in R3T3 cells. Regul Pept. 1993 Mar 19;44(2):199–206. doi: 10.1016/0167-0115(93)90243-2. [DOI] [PubMed] [Google Scholar]
  11. Heemskerk F. M., Zorad S., Seltzer A., Saavedra J. M. Characterization of brain angiotensin II AT2 receptor subtype using [125I] CGP 42112A. Neuroreport. 1993 Jan;4(1):103–105. doi: 10.1097/00001756-199301000-00027. [DOI] [PubMed] [Google Scholar]
  12. Kiron M. A., Soffer R. L. Purification and properties of a soluble angiotensin II-binding protein from rabbit liver. J Biol Chem. 1989 Mar 5;264(7):4138–4142. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Marie J., Seyer R., Lombard C., Desarnaud F., Aumelas A., Jard S., Bonnafous J. C. Affinity chromatography purification of angiotensin II receptor using photoactivable biotinylated probes. Biochemistry. 1990 Sep 25;29(38):8943–8950. doi: 10.1021/bi00490a009. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Peach M. J. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev. 1977 Apr;57(2):313–370. doi: 10.1152/physrev.1977.57.2.313. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Saavedra J. M. Brain and pituitary angiotensin. Endocr Rev. 1992 May;13(2):329–380. doi: 10.1210/edrv-13-2-329. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Sen I., Bull H. G., Soffer R. L. Isolation of an angiotensin II-binding protein from liver. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1679–1683. doi: 10.1073/pnas.81.6.1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Servant G., Boulay G., Bossé R., Escher E., Guillemette G. Photoaffinity labeling of subtype 2 angiotensin receptor of human myometrium. Mol Pharmacol. 1993 May;43(5):677–683. [PubMed] [Google Scholar]
  23. Siemens I. R., Adler H. J., Addya K., Mah S. J., Fluharty S. J. Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification. Mol Pharmacol. 1991 Nov;40(5):717–726. [PubMed] [Google Scholar]
  24. Speth R. C., Rowe B. P., Grove K. L., Carter M. R., Saylor D. Sulfhydryl reducing agents distinguish two subtypes of angiotensin II receptors in the rat brain. Brain Res. 1991 May 10;548(1-2):1–8. doi: 10.1016/0006-8993(91)91098-l. [DOI] [PubMed] [Google Scholar]
  25. Speth R. C. [125I]CGP 42112 binding reveals differences between rat brain and adrenal AT2 receptor binding sites. Regul Pept. 1993 Mar 19;44(2):189–197. doi: 10.1016/0167-0115(93)90242-z. [DOI] [PubMed] [Google Scholar]
  26. Strömberg C., Näveri L., Saavedra J. M. Nonpeptide angiotensin AT1 and AT2 receptor ligands modulate the upper limit of cerebral blood flow autoregulation in rats. J Cereb Blood Flow Metab. 1993 Mar;13(2):298–303. doi: 10.1038/jcbfm.1993.37. [DOI] [PubMed] [Google Scholar]
  27. Sumners C., Richards E. M., Tang W., Raizada M. K. Angiotensin II type 2 receptor expression in neuronal cultures from spontaneously hypertensive rat brain. Regul Pept. 1993 Mar 19;44(2):181–188. doi: 10.1016/0167-0115(93)90241-y. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Tang S. S., Rogg H., Schumacher R., Dzau V. J. Characterization of nuclear angiotensin-II-binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology. 1992 Jul;131(1):374–380. doi: 10.1210/endo.131.1.1612017. [DOI] [PubMed] [Google Scholar]
  30. Tsutsumi K., Saavedra J. M. Characterization of AT2 angiotensin II receptors in rat anterior cerebral arteries. Am J Physiol. 1991 Sep;261(3 Pt 2):H667–H670. doi: 10.1152/ajpheart.1991.261.3.H667. [DOI] [PubMed] [Google Scholar]
  31. Tsutsumi K., Saavedra J. M. Increased dithiothreitol-insensitive, type 2 angiotensin II receptors in selected brain areas of young rats. Cell Mol Neurobiol. 1991 Apr;11(2):295–299. doi: 10.1007/BF00769042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tsutsumi K., Strömberg C., Viswanathan M., Saavedra J. M. Angiotensin-II receptor subtypes in fetal tissue of the rat: autoradiography, guanine nucleotide sensitivity, and association with phosphoinositide hydrolysis. Endocrinology. 1991 Aug;129(2):1075–1082. doi: 10.1210/endo-129-2-1075. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Whitebread S. E., Taylor V., Bottari S. P., Kamber B., de Gasparo M. Radioiodinated CGP 42112A: a novel high affinity and highly selective ligand for the characterization of angiotensin AT2 receptors. Biochem Biophys Res Commun. 1991 Dec 31;181(3):1365–1371. doi: 10.1016/0006-291x(91)92089-3. [DOI] [PubMed] [Google Scholar]
  35. 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 Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES