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. 1997 Nov 17;16(22):6737–6747. doi: 10.1093/emboj/16.22.6737

Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.

U Gether 1, S Lin 1, P Ghanouni 1, J A Ballesteros 1, H Weinstein 1, B K Kobilka 1
PMCID: PMC1170278  PMID: 9362488

Abstract

Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of 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. Allen L. F., Lefkowitz R. J., Caron M. G., Cotecchia S. G-protein-coupled receptor genes as protooncogenes: constitutively activating mutation of the alpha 1B-adrenergic receptor enhances mitogenesis and tumorigenicity. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11354–11358. doi: 10.1073/pnas.88.24.11354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altenbach C., Yang K., Farrens D. L., Farahbakhsh Z. T., Khorana H. G., Hubbell W. L. Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study. Biochemistry. 1996 Sep 24;35(38):12470–12478. doi: 10.1021/bi960849l. [DOI] [PubMed] [Google Scholar]
  3. Arnis S., Fahmy K., Hofmann K. P., Sakmar T. P. A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin. J Biol Chem. 1994 Sep 30;269(39):23879–23881. [PubMed] [Google Scholar]
  4. 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]
  5. Benovic J. L., Shorr R. G., Caron M. G., Lefkowitz R. J. The mammalian beta 2-adrenergic receptor: purification and characterization. Biochemistry. 1984 Sep 25;23(20):4510–4518. doi: 10.1021/bi00315a002. [DOI] [PubMed] [Google Scholar]
  6. Bouvier M., Collins S., O'Dowd B. F., Campbell P. T., de Blasi A., Kobilka B. K., MacGregor C., Irons G. P., Caron M. G., Lefkowitz R. J. Two distinct pathways for cAMP-mediated down-regulation of the beta 2-adrenergic receptor. Phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem. 1989 Oct 5;264(28):16786–16792. [PubMed] [Google Scholar]
  7. Choe H., Farzan M., Sun Y., Sullivan N., Rollins B., Ponath P. D., Wu L., Mackay C. R., LaRosa G., Newman W. The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell. 1996 Jun 28;85(7):1135–1148. doi: 10.1016/s0092-8674(00)81313-6. [DOI] [PubMed] [Google Scholar]
  8. Choudhary M. S., Sachs N., Uluer A., Glennon R. A., Westkaemper R. B., Roth B. L. Differential ergoline and ergopeptine binding to 5-hydroxytryptamine2A receptors: ergolines require an aromatic residue at position 340 for high affinity binding. Mol Pharmacol. 1995 Mar;47(3):450–457. [PubMed] [Google Scholar]
  9. Cronet P., Sander C., Vriend G. Modeling of transmembrane seven helix bundles. Protein Eng. 1993 Jan;6(1):59–64. doi: 10.1093/protein/6.1.59. [DOI] [PubMed] [Google Scholar]
  10. Dohlman H. G., Caron M. G., DeBlasi A., Frielle T., Lefkowitz R. J. Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor. Biochemistry. 1990 Mar 6;29(9):2335–2342. doi: 10.1021/bi00461a018. [DOI] [PubMed] [Google Scholar]
  11. Elling C. E., Schwartz T. W. Connectivity and orientation of the seven helical bundle in the tachykinin NK-1 receptor probed by zinc site engineering. EMBO J. 1996 Nov 15;15(22):6213–6219. [PMC free article] [PubMed] [Google Scholar]
  12. Fanelli F., Menziani M. C., De Benedetti P. G. Molecular dynamics simulations of m3-muscarinic receptor activation and QSAR analysis. Bioorg Med Chem. 1995 Nov;3(11):1465–1477. doi: 10.1016/0968-0896(95)00131-y. [DOI] [PubMed] [Google Scholar]
  13. Farahbakhsh Z. T., Ridge K. D., Khorana H. G., Hubbell W. L. Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study. Biochemistry. 1995 Jul 11;34(27):8812–8819. doi: 10.1021/bi00027a033. [DOI] [PubMed] [Google Scholar]
  14. Farrens D. L., Altenbach C., Yang K., Hubbell W. L., Khorana H. G. Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin. Science. 1996 Nov 1;274(5288):768–770. doi: 10.1126/science.274.5288.768. [DOI] [PubMed] [Google Scholar]
  15. Feng Y., Broder C. C., Kennedy P. E., Berger E. A. HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science. 1996 May 10;272(5263):872–877. doi: 10.1126/science.272.5263.872. [DOI] [PubMed] [Google Scholar]
  16. Fraser C. M. Site-directed mutagenesis of beta-adrenergic receptors. Identification of conserved cysteine residues that independently affect ligand binding and receptor activation. J Biol Chem. 1989 Jun 5;264(16):9266–9270. [PubMed] [Google Scholar]
  17. Gether U., Ballesteros J. A., Seifert R., Sanders-Bush E., Weinstein H., Kobilka B. K. Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility. J Biol Chem. 1997 Jan 31;272(5):2587–2590. doi: 10.1074/jbc.272.5.2587. [DOI] [PubMed] [Google Scholar]
  18. Gether U., Lin S., Kobilka B. K. Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem. 1995 Nov 24;270(47):28268–28275. doi: 10.1074/jbc.270.47.28268. [DOI] [PubMed] [Google Scholar]
  19. Guan X. M., Kobilka T. S., Kobilka B. K. Enhancement of membrane insertion and function in a type IIIb membrane protein following introduction of a cleavable signal peptide. J Biol Chem. 1992 Nov 5;267(31):21995–21998. [PubMed] [Google Scholar]
  20. Javitch J. A., Fu D., Chen J., Karlin A. Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method. Neuron. 1995 Apr;14(4):825–831. doi: 10.1016/0896-6273(95)90226-0. [DOI] [PubMed] [Google Scholar]
  21. Kirkpatrick S., Gelatt C. D., Jr, Vecchi M. P. Optimization by simulated annealing. Science. 1983 May 13;220(4598):671–680. doi: 10.1126/science.220.4598.671. [DOI] [PubMed] [Google Scholar]
  22. Kobilka B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal Biochem. 1995 Oct 10;231(1):269–271. doi: 10.1006/abio.1995.1533. [DOI] [PubMed] [Google Scholar]
  23. Kobilka B. Adrenergic receptors as models for G protein-coupled receptors. Annu Rev Neurosci. 1992;15:87–114. doi: 10.1146/annurev.ne.15.030192.000511. [DOI] [PubMed] [Google Scholar]
  24. Lefkowitz R. J., Cotecchia S., Samama P., Costa T. Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci. 1993 Aug;14(8):303–307. doi: 10.1016/0165-6147(93)90048-O. [DOI] [PubMed] [Google Scholar]
  25. Liu J., Schöneberg T., van Rhee M., Wess J. Mutational analysis of the relative orientation of transmembrane helices I and VII in G protein-coupled receptors. J Biol Chem. 1995 Aug 18;270(33):19532–19539. doi: 10.1074/jbc.270.33.19532. [DOI] [PubMed] [Google Scholar]
  26. Luo X., Zhang D., Weinstein H. Ligand-induced domain motion in the activation mechanism of a G-protein-coupled receptor. Protein Eng. 1994 Dec;7(12):1441–1448. doi: 10.1093/protein/7.12.1441. [DOI] [PubMed] [Google Scholar]
  27. MaloneyHuss K., Lybrand T. P. Three-dimensional structure for the beta 2 adrenergic receptor protein based on computer modeling studies. J Mol Biol. 1992 Jun 5;225(3):859–871. doi: 10.1016/0022-2836(92)90406-a. [DOI] [PubMed] [Google Scholar]
  28. Mizobe T., Maze M., Lam V., Suryanarayana S., Kobilka B. K. Arrangement of transmembrane domains in adrenergic receptors. Similarity to bacteriorhodopsin. J Biol Chem. 1996 Feb 2;271(5):2387–2389. doi: 10.1074/jbc.271.5.2387. [DOI] [PubMed] [Google Scholar]
  29. Mouillac B., Caron M., Bonin H., Dennis M., Bouvier M. Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells. J Biol Chem. 1992 Oct 25;267(30):21733–21737. [PubMed] [Google Scholar]
  30. Méjean A., Guillaume J. L., Strosberg A. D. Carazolol: a potent, selective beta 3-adrenoceptor agonist. Eur J Pharmacol. 1995 Nov 30;291(3):359–366. doi: 10.1016/0922-4106(95)90077-2. [DOI] [PubMed] [Google Scholar]
  31. Nakayama T. A., Khorana H. G. Mapping of the amino acids in membrane-embedded helices that interact with the retinal chromophore in bovine rhodopsin. J Biol Chem. 1991 Mar 5;266(7):4269–4275. [PubMed] [Google Scholar]
  32. Noda K., Saad Y., Graham R. M., Karnik S. S. The high affinity state of the beta 2-adrenergic receptor requires unique interaction between conserved and non-conserved extracellular loop cysteines. J Biol Chem. 1994 Mar 4;269(9):6743–6752. [PubMed] [Google Scholar]
  33. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  34. 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]
  35. Röper D., Jacoby E., Krüger P., Engels M., Grötzinger J., Wollmer A., Strassburger W. Modeling of G-protein coupled receptors with bacteriorhodopsin as a template. A novel approach based on interaction energy differences. J Recept Res. 1994 May;14(3-4):167–186. doi: 10.3109/10799899409066029. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Scheer A., Fanelli F., Costa T., De Benedetti P. G., Cotecchia S. Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO J. 1996 Jul 15;15(14):3566–3578. [PMC free article] [PubMed] [Google Scholar]
  39. Scheer A., Fanelli F., Costa T., De Benedetti P. G., Cotecchia S. The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):808–813. doi: 10.1073/pnas.94.3.808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schertler G. F., Villa C., Henderson R. Projection structure of rhodopsin. Nature. 1993 Apr 22;362(6422):770–772. doi: 10.1038/362770a0. [DOI] [PubMed] [Google Scholar]
  41. Schwartz T. W. Locating ligand-binding sites in 7TM receptors by protein engineering. Curr Opin Biotechnol. 1994 Aug;5(4):434–444. doi: 10.1016/0958-1669(94)90054-x. [DOI] [PubMed] [Google Scholar]
  42. Sheikh S. P., Zvyaga T. A., Lichtarge O., Sakmar T. P., Bourne H. R. Rhodopsin activation blocked by metal-ion-binding sites linking transmembrane helices C and F. Nature. 1996 Sep 26;383(6598):347–350. doi: 10.1038/383347a0. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Strader C. D., Gaffney T., Sugg E. E., Candelore M. R., Keys R., Patchett A. A., Dixon R. A. Allele-specific activation of genetically engineered receptors. J Biol Chem. 1991 Jan 5;266(1):5–8. [PubMed] [Google Scholar]
  45. Suryanarayana S., von Zastrow M., Kobilka B. K. Identification of intramolecular interactions in adrenergic receptors. J Biol Chem. 1992 Nov 5;267(31):21991–21994. [PubMed] [Google Scholar]
  46. Tota M. R., Candelore M. R., Dixon R. A., Strader C. D. Biophysical and genetic analysis of the ligand-binding site of the beta-adrenoceptor. Trends Pharmacol Sci. 1991 Jan;12(1):4–6. doi: 10.1016/0165-6147(91)90479-c. [DOI] [PubMed] [Google Scholar]
  47. Trumpp-Kallmeyer S., Hoflack J., Bruinvels A., Hibert M. Modeling of G-protein-coupled receptors: application to dopamine, adrenaline, serotonin, acetylcholine, and mammalian opsin receptors. J Med Chem. 1992 Sep 18;35(19):3448–3462. doi: 10.1021/jm00097a002. [DOI] [PubMed] [Google Scholar]
  48. Turcatti G., Nemeth K., Edgerton M. D., Meseth U., Talabot F., Peitsch M., Knowles J., Vogel H., Chollet A. Probing the structure and function of the tachykinin neurokinin-2 receptor through biosynthetic incorporation of fluorescent amino acids at specific sites. J Biol Chem. 1996 Aug 16;271(33):19991–19998. doi: 10.1074/jbc.271.33.19991. [DOI] [PubMed] [Google Scholar]
  49. Vauquelin G., Bottari S., Kanarek L., Strosberg A. D. Evidence for essential disulfide bonds in beta1-adrenergic receptors of turkey erythrocyte membranes. Inactivation by dithiothreitol. J Biol Chem. 1979 Jun 10;254(11):4462–4469. [PubMed] [Google Scholar]
  50. Wess J., Gdula D., Brann M. R. Site-directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding. EMBO J. 1991 Dec;10(12):3729–3734. doi: 10.1002/j.1460-2075.1991.tb04941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wieland K., Zuurmond H. M., Krasel C., Ijzerman A. P., Lohse M. J. Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9276–9281. doi: 10.1073/pnas.93.17.9276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zhang D., Weinstein H. Signal transduction by a 5-HT2 receptor: a mechanistic hypothesis from molecular dynamics simulations of the three-dimensional model of the receptor complexed to ligands. J Med Chem. 1993 Apr 2;36(7):934–938. doi: 10.1021/jm00059a021. [DOI] [PubMed] [Google Scholar]
  53. Zhou W., Flanagan C., Ballesteros J. A., Konvicka K., Davidson J. S., Weinstein H., Millar R. P., Sealfon S. C. A reciprocal mutation supports helix 2 and helix 7 proximity in the gonadotropin-releasing hormone receptor. Mol Pharmacol. 1994 Feb;45(2):165–170. [PubMed] [Google Scholar]

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