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. 1996 Sep 17;93(19):10040–10045. doi: 10.1073/pnas.93.19.10040

Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.

L Hunyady 1, M Zhang 1, G Jagadeesh 1, M Bor 1, T Balla 1, K J Catt 1
PMCID: PMC38332  PMID: 8816747

Abstract

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.

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Selected References

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

  1. Anderson K. M., Murahashi T., Dostal D. E., Peach M. J. Morphological and biochemical analysis of angiotensin II internalization in cultured rat aortic smooth muscle cells. Am J Physiol. 1993 Jan;264(1 Pt 1):C179–C188. doi: 10.1152/ajpcell.1993.264.1.C179. [DOI] [PubMed] [Google Scholar]
  2. Baldwin J. M. The probable arrangement of the helices in G protein-coupled receptors. EMBO J. 1993 Apr;12(4):1693–1703. doi: 10.1002/j.1460-2075.1993.tb05814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balla T., Sim S. S., Iida T., Choi K. Y., Catt K. J., Rhee S. G. Agonist-induced calcium signaling is impaired in fibroblasts overproducing inositol 1,3,4,5-tetrakisphosphate. J Biol Chem. 1991 Dec 25;266(36):24719–24726. [PubMed] [Google Scholar]
  4. Barrett P. Q., Bollag W. B., Isales C. M., McCarthy R. T., Rasmussen H. Role of calcium in angiotensin II-mediated aldosterone secretion. Endocr Rev. 1989 Nov;10(4):496–518. doi: 10.1210/edrv-10-4-496. [DOI] [PubMed] [Google Scholar]
  5. Bernstein K. E., Berk B. C. The biology of angiotensin II receptors. Am J Kidney Dis. 1993 Nov;22(5):745–754. doi: 10.1016/s0272-6386(12)80441-0. [DOI] [PubMed] [Google Scholar]
  6. Bianchi C., Gutkowska J., De Léan A., Ballak M., Anand-Srivastava M. B., Genest J., Cantin M. Fate of [125I]angiotensin II in adrenal zona glomerulosa cells. Endocrinology. 1986 Jun;118(6):2605–2607. doi: 10.1210/endo-118-6-2605. [DOI] [PubMed] [Google Scholar]
  7. Bihoreau C., Monnot C., Davies E., Teutsch B., Bernstein K. E., Corvol P., Clauser E. Mutation of Asp74 of the rat angiotensin II receptor confers changes in antagonist affinities and abolishes G-protein coupling. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5133–5137. doi: 10.1073/pnas.90.11.5133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blüml K., Mutschler E., Wess J. Functional role of a cytoplasmic aromatic amino acid in muscarinic receptor-mediated activation of phospholipase C. J Biol Chem. 1994 Apr 15;269(15):11537–11541. [PubMed] [Google Scholar]
  9. Blüml K., Mutschler E., Wess J. Identification of an intracellular tyrosine residue critical for muscarinic receptor-mediated stimulation of phosphatidylinositol hydrolysis. J Biol Chem. 1994 Jan 7;269(1):402–405. [PubMed] [Google Scholar]
  10. Campbell P. T., Hnatowich M., O'Dowd B. F., Caron M. G., Lefkowitz R. J., Hausdorff W. P. Mutations of the human beta 2-adrenergic receptor that impair coupling to Gs interfere with receptor down-regulation but not sequestration. Mol Pharmacol. 1991 Feb;39(2):192–198. [PubMed] [Google Scholar]
  11. Catt K. J., Hunyady L., Balla T. Second messengers derived from inositol lipids. J Bioenerg Biomembr. 1991 Feb;23(1):7–27. doi: 10.1007/BF00768836. [DOI] [PubMed] [Google Scholar]
  12. Chaki S., Guo D. F., Yamano Y., Ohyama K., Tani M., Mizukoshi M., Shirai H., Inagami T. Role of carboxyl tail of the rat angiotensin II type 1A receptor in agonist-induced internalization of the receptor. Kidney Int. 1994 Dec;46(6):1492–1495. doi: 10.1038/ki.1994.427. [DOI] [PubMed] [Google Scholar]
  13. Cheung A. H., Dixon R. A., Hill W. S., Sigal I. S., Strader C. D. Separation of the structural requirements for agonist-promoted activation and sequestration of the beta-adrenergic receptor. Mol Pharmacol. 1990 Jun;37(6):775–779. [PubMed] [Google Scholar]
  14. Cheung A. H., Huang R. R., Strader C. D. Involvement of specific hydrophobic, but not hydrophilic, amino acids in the third intracellular loop of the beta-adrenergic receptor in the activation of Gs. Mol Pharmacol. 1992 Jun;41(6):1061–1065. [PubMed] [Google Scholar]
  15. Clark C. D., Palzkill T., Botstein D. Systematic mutagenesis of the yeast mating pheromone receptor third intracellular loop. J Biol Chem. 1994 Mar 25;269(12):8831–8841. [PubMed] [Google Scholar]
  16. Collins S., Caron M. G., Lefkowitz R. J. From ligand binding to gene expression: new insights into the regulation of G-protein-coupled receptors. Trends Biochem Sci. 1992 Jan;17(1):37–39. doi: 10.1016/0968-0004(92)90425-9. [DOI] [PubMed] [Google Scholar]
  17. Conchon S., Monnot C., Teutsch B., Corvol P., Clauser E. Internalization of the rat AT1a and AT1b receptors: pharmacological and functional requirements. FEBS Lett. 1994 Aug 8;349(3):365–370. doi: 10.1016/0014-5793(94)00703-9. [DOI] [PubMed] [Google Scholar]
  18. Crozat A., Penhoat A., Saez J. M. Processing of angiotensin II (A-II) and (Sar1,Ala8)A-II by cultured bovine adrenocortical cells. Endocrinology. 1986 Jun;118(6):2312–2318. doi: 10.1210/endo-118-6-2312. [DOI] [PubMed] [Google Scholar]
  19. Ganguly A., Davis J. S. Role of calcium and other mediators in aldosterone secretion from the adrenal glomerulosa cells. Pharmacol Rev. 1994 Dec;46(4):417–447. [PubMed] [Google Scholar]
  20. Griendling K. K., Delafontaine P., Rittenhouse S. E., Gimbrone M. A., Jr, Alexander R. W. Correlation of receptor sequestration with sustained diacylglycerol accumulation in angiotensin II-stimulated cultured vascular smooth muscle cells. J Biol Chem. 1987 Oct 25;262(30):14555–14562. [PubMed] [Google Scholar]
  21. Hedin K. E., Duerson K., Clapham D. E. Specificity of receptor-G protein interactions: searching for the structure behind the signal. Cell Signal. 1993 Sep;5(5):505–518. doi: 10.1016/0898-6568(93)90046-o. [DOI] [PubMed] [Google Scholar]
  22. Hunyady L., Baukal A. J., Balla T., Catt K. J. Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction. J Biol Chem. 1994 Oct 7;269(40):24798–24804. [PubMed] [Google Scholar]
  23. Hunyady L., Bor M., Balla T., Catt K. J. Critical role of a conserved intramembrane tyrosine residue in angiotensin II receptor activation. J Biol Chem. 1995 Apr 28;270(17):9702–9705. doi: 10.1074/jbc.270.17.9702. [DOI] [PubMed] [Google Scholar]
  24. Hunyady L., Bor M., Balla T., Catt K. J. Identification of a cytoplasmic Ser-Thr-Leu motif that determines agonist-induced internalization of the AT1 angiotensin receptor. J Biol Chem. 1994 Dec 16;269(50):31378–31382. [PubMed] [Google Scholar]
  25. Hunyady L., Bor M., Baukal A. J., Balla T., Catt K. J. A conserved NPLFY sequence contributes to agonist binding and signal transduction but is not an internalization signal for the type 1 angiotensin II receptor. J Biol Chem. 1995 Jul 14;270(28):16602–16609. doi: 10.1074/jbc.270.28.16602. [DOI] [PubMed] [Google Scholar]
  26. Hunyady L., Merelli F., Baukal A. J., Balla T., Catt K. J. Agonist-induced endocytosis and signal generation in adrenal glomerulosa cells. A potential mechanism for receptor-operated calcium entry. J Biol Chem. 1991 Feb 15;266(5):2783–2788. [PubMed] [Google Scholar]
  27. Hunyady L., Tian Y., Sandberg K., Balla T., Catt K. J. Divergent conformational requirements for angiotensin II receptor internalization and signaling. Kidney Int. 1994 Dec;46(6):1496–1498. doi: 10.1038/ki.1994.428. [DOI] [PubMed] [Google Scholar]
  28. Högger P., Shockley M. S., Lameh J., Sadée W. Activating and inactivating mutations in N- and C-terminal i3 loop junctions of muscarinic acetylcholine Hm1 receptors. J Biol Chem. 1995 Mar 31;270(13):7405–7410. doi: 10.1074/jbc.270.13.7405. [DOI] [PubMed] [Google Scholar]
  29. Kapas S., Hinson J. P., Puddefoot J. R., Ho M. M., Vinson G. P. Internalization of the type I angiotensin II receptor (AT1) is required for protein kinase C activation but not for inositol trisphosphate release in the angiotensin II stimulated rat adrenal zona glomerulosa cell. Biochem Biophys Res Commun. 1994 Nov 15;204(3):1292–1298. doi: 10.1006/bbrc.1994.2603. [DOI] [PubMed] [Google Scholar]
  30. Kjelsberg M. A., Cotecchia S., Ostrowski J., Caron M. G., Lefkowitz R. J. Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem. 1992 Jan 25;267(3):1430–1433. [PubMed] [Google Scholar]
  31. Kolakowski L. F., Jr GCRDb: a G-protein-coupled receptor database. Receptors Channels. 1994;2(1):1–7. [PubMed] [Google Scholar]
  32. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  33. Lund K. A., Opresko L. K., Starbuck C., Walsh B. J., Wiley H. S. Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization. J Biol Chem. 1990 Sep 15;265(26):15713–15723. [PubMed] [Google Scholar]
  34. Marie J., Maigret B., Joseph M. P., Larguier R., Nouet S., Lombard C., Bonnafous J. C. Tyr292 in the seventh transmembrane domain of the AT1A angiotensin II receptor is essential for its coupling to phospholipase C. J Biol Chem. 1994 Aug 19;269(33):20815–20818. [PubMed] [Google Scholar]
  35. Moro O., Shockley M. S., Lameh J., Sadée W. Overlapping multi-site domains of the muscarinic cholinergic Hm1 receptor involved in signal transduction and sequestration. J Biol Chem. 1994 Mar 4;269(9):6651–6655. [PubMed] [Google Scholar]
  36. 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]
  37. Ohyama K., Yamano Y., Chaki S., Kondo T., Inagami T. Domains for G-protein coupling in angiotensin II receptor type I: studies by site-directed mutagenesis. Biochem Biophys Res Commun. 1992 Dec 15;189(2):677–683. doi: 10.1016/0006-291x(92)92254-u. [DOI] [PubMed] [Google Scholar]
  38. Probst W. C., Snyder L. A., Schuster D. I., Brosius J., Sealfon S. C. Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell Biol. 1992 Jan-Feb;11(1):1–20. doi: 10.1089/dna.1992.11.1. [DOI] [PubMed] [Google Scholar]
  39. Prossnitz E. R., Quehenberger O., Cochrane C. G., Ye R. D. The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling. Biochem J. 1993 Sep 1;294(Pt 2):581–587. doi: 10.1042/bj2940581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ren Q., Kurose H., Lefkowitz R. J., Cotecchia S. Constitutively active mutants of the alpha 2-adrenergic receptor. J Biol Chem. 1993 Aug 5;268(22):16483–16487. [PubMed] [Google Scholar]
  41. Samama P., Cotecchia S., Costa T., Lefkowitz R. J. A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem. 1993 Mar 5;268(7):4625–4636. [PubMed] [Google Scholar]
  42. Savarese T. M., Fraser C. M. In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors. Biochem J. 1992 Apr 1;283(Pt 1):1–19. doi: 10.1042/bj2830001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Spät A., Enyedi P., Hajnóczky G., Hunyady L. Generation and role of calcium signal in adrenal glomerulosa cells. Exp Physiol. 1991 Nov;76(6):859–885. doi: 10.1113/expphysiol.1991.sp003550. [DOI] [PubMed] [Google Scholar]
  44. Strader C. D., Fong T. M., Tota M. R., Underwood D., Dixon R. A. Structure and function of G protein-coupled receptors. Annu Rev Biochem. 1994;63:101–132. doi: 10.1146/annurev.bi.63.070194.000533. [DOI] [PubMed] [Google Scholar]
  45. Thomas W. G., Baker K. M., Motel T. J., Thekkumkara T. J. Angiotensin II receptor endocytosis involves two distinct regions of the cytoplasmic tail. A role for residues on the hydrophobic face of a putative amphipathic helix. J Biol Chem. 1995 Sep 22;270(38):22153–22159. doi: 10.1074/jbc.270.38.22153. [DOI] [PubMed] [Google Scholar]
  46. Thomas W. G., Thekkumkara T. J., Motel T. J., Baker K. M. Stable expression of a truncated AT1A receptor in CHO-K1 cells. The carboxyl-terminal region directs agonist-induced internalization but not receptor signaling or desensitization. J Biol Chem. 1995 Jan 6;270(1):207–213. doi: 10.1074/jbc.270.1.207. [DOI] [PubMed] [Google Scholar]
  47. Ullian M. E., Linas S. L. Role of receptor cycling in the regulation of angiotensin II surface receptor number and angiotensin II uptake in rat vascular smooth muscle cells. J Clin Invest. 1989 Sep;84(3):840–846. doi: 10.1172/JCI114244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vinson G. P., Ho M. M., Puddefoot J. R., Teja R., Barker S., Kapas S., Hinson J. P. Internalisation of the type I angiotensin II receptor (AT1) and angiotensin II function in the rat adrenal zona glomerulosa cell. Endocr Res. 1995 Feb-May;21(1-2):211–217. doi: 10.3109/07435809509030437. [DOI] [PubMed] [Google Scholar]
  49. Wang C., Jayadev S., Escobedo J. A. Identification of a domain in the angiotensin II type 1 receptor determining Gq coupling by the use of receptor chimeras. J Biol Chem. 1995 Jul 14;270(28):16677–16682. doi: 10.1074/jbc.270.28.16677. [DOI] [PubMed] [Google Scholar]
  50. Weiner J. L., Guttierez-Steil C., Blumer K. J. Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway. J Biol Chem. 1993 Apr 15;268(11):8070–8077. [PubMed] [Google Scholar]
  51. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  52. Yu S. S., Lefkowitz R. J., Hausdorff W. P. Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization. J Biol Chem. 1993 Jan 5;268(1):337–341. [PubMed] [Google Scholar]

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