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Essential role for the second extracellular loop in C5a receptor activation

Nature Structural & Molecular Biology volume 12, pages 320–326 (2005)Cite this article

Abstract

More than 90% of G protein–coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, ∼80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor.

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Figure 1: Schematic diagram of the human C5aR.
Figure 2: Analysis of the second extracellular loop of the C5aR.
Figure 3: Cys188 is essential for ligand activation.
Figure 4: Activity of EC2-mutated C5aRs expressed in mammalian cells.
Figure 5: Identification of EC2 residues in the C5aR that confer constitutive activity.

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References

  1. Pierce, K.L., Premont, R.T. & Lefkowitz, R.J. Signalling: seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 3, 639–650 (2002).

    Article  CAS  Google Scholar 

  2. Gether, U. Uncovering molecular mechanisms involved in activation of G protein–coupled receptors. Endocr. Rev. 21, 90–113 (2000).

    Article  CAS  Google Scholar 

  3. Karnik, S.S., Gogonea, C., Patil, S., Saad, Y. & Takezako, T. Activation of G-protein-coupled receptors: a common molecular mechanism. Trends Endocrinol. Metab. 14, 431–437 (2003).

    Article  CAS  Google Scholar 

  4. Palczewski, K. et al. Crystal structure of rhodopsin: a G protein–coupled receptor. Science 289, 739–745 (2000).

    Article  CAS  Google Scholar 

  5. Okada, T. et al. Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Proc. Natl. Acad. Sci. USA 99, 5982–5987 (2002).

    Article  CAS  Google Scholar 

  6. Schertler, G.F. Structure of rhodopsin. Eye 12, 504–510 (1998).

    Article  Google Scholar 

  7. Baldwin, J.M., Schertler, G.F. & Unger, V.M. An α-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. J. Mol. Biol. 272, 144–164 (1997).

    Article  CAS  Google Scholar 

  8. Shi, L. & Javitch, J.A. The binding site of aminergic G protein–coupled receptors: the transmembrane segments and second extracellular loop. Annu. Rev. Pharmacol. Toxicol. 42, 437–467 (2002).

    Article  CAS  Google Scholar 

  9. Baranski, T.J. et al. C5a receptor activation. Genetic identification of critical residues in four transmembrane helices. J. Biol. Chem. 274, 15757–15765 (1999).

    Article  CAS  Google Scholar 

  10. Geva, A., Lassere, T.B., Lichtarge, O., Pollitt, S.K. & Baranski, T.J. Genetic mapping of the human C5a receptor. Identification of transmembrane amino acids critical for receptor function. J. Biol. Chem. 275, 35393–35401 (2000).

    Article  CAS  Google Scholar 

  11. Dayhoff, M.O., Schwartz, R.M. & Orcutt, B.C. In Atlas of Protein Sequence and Structure Vol. 5 (ed. Dayhoff, M.O.) 345–353 (National Biomedical Research Foundation, Silver Spring, Maryland, USA, 1978).

    Google Scholar 

  12. Fauchere, J.L., Charton, M., Kier, L.B., Verloop, A. & Pliska, V. Amino acid side chain parameters for correlation studies in biology and pharmacology. Int. J. Pept. Protein Res. 32, 269–278 (1988).

    Article  CAS  Google Scholar 

  13. Gerard, C. & Gerard, N.P. C5A anaphylatoxin and its seven transmembrane-segment receptor. Annu. Rev. Immunol. 12, 775–808 (1994).

    Article  CAS  Google Scholar 

  14. Cain, S.A., Higginbottom, A. & Monk, P.N. Characterisation of C5a receptor agonists from phage display libraries. Biochem. Pharmacol. 66, 1833–1840 (2003).

    Article  CAS  Google Scholar 

  15. Kolakowski, L.F., Jr., Lu, B., Gerard, C. & Gerard, N.P. Probing the “message:address” sites for chemoattractant binding to the C5a receptor. Mutagenesis of hydrophilic and proline residues within the transmembrane segments. J. Biol. Chem. 270, 18077–18082 (1995).

    Article  CAS  Google Scholar 

  16. Karnik, S.S., Sakmar, T.P., Chen, H.B. & Khorana, H.G. Cysteine residues 110 and 187 are essential for the formation of correct structure in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 85, 8459–8463 (1988).

    Article  CAS  Google Scholar 

  17. Davidson, F.F., Loewen, P.C. & Khorana, H.G. Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. Proc. Natl. Acad. Sci. USA 91, 4029–4033 (1994).

    Article  CAS  Google Scholar 

  18. Zeng, F.Y., Soldner, A., Schoneberg, T. & Wess, J. Conserved extracellular cysteine pair in the M3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but not for G protein coupling. J. Neurochem. 72, 2404–2414 (1999).

    Article  CAS  Google Scholar 

  19. Reddy, P.S. & Corley, R.B. Assembly, sorting, and exit of oligomeric proteins from the endoplasmic reticulum. Bioessays 20, 546–554 (1998).

    Article  CAS  Google Scholar 

  20. Floyd, D.H. et al. C5a receptor oligomerization II: fluorescence resonance energy transfer studies of a human G protein–coupled receptor expressed in yeast. J. Biol. Chem. 278, 35354–35361 (2003).

    Article  CAS  Google Scholar 

  21. Whistler, J.L. et al. Constitutive activation and endocytosis of the complement factor 5a receptor: evidence for multiple activated conformations of a G protein–coupled receptor. Traffic 3, 866–877 (2002).

    Article  CAS  Google Scholar 

  22. Cook, J.V. & Eidne, K.A. An intramolecular disulfide bond between conserved extracellular cysteines in the gonadotropin-releasing hormone receptor is essential for binding and activation. Endocrinology 138, 2800–2806 (1997).

    Article  CAS  Google Scholar 

  23. Shi, L. & Javitch, J.A. The second extracellular loop of the dopamine D2 receptor lines the binding-site crevice. Proc. Natl. Acad. Sci. USA 101, 440–445 (2004).

    Article  CAS  Google Scholar 

  24. Parnot, C., Miserey-Lenkei, S., Bardin, S., Corvol, P. & Clauser, E. Lessons from constitutively active mutants of G protein–coupled receptors. Trends Endocrinol. Metab. 13, 336–343 (2002).

    Article  CAS  Google Scholar 

  25. Milligan, G. Constitutive activity and inverse agonists of G protein–coupled receptors: a current perspective. Mol. Pharmacol. 64, 1271–1276 (2003).

    Article  CAS  Google Scholar 

  26. abu Alla, S. et al. Extracellular domains of the bradykinin B2 receptor involved in ligand binding and agonist sensing defined by anti-peptide antibodies. J. Biol. Chem. 271, 1748–1755 (1996).

    Article  CAS  Google Scholar 

  27. Ott, T.R. et al. Two mutations in extracellular loop 2 of the human GnRH receptor convert an antagonist to an agonist. Mol. Endocrinol. 16, 1079–1088 (2002).

    Article  CAS  Google Scholar 

  28. Mobini, R. et al. Probing the immunological properties of the extracellular domains of the human β(1)-adrenoceptor. J. Autoimmun. 13, 179–186 (1999).

    Article  CAS  Google Scholar 

  29. Lebesgue, D. et al. An agonist-like monoclonal antibody against the human β2- adrenoceptor. Eur. J. Pharmacol. 348, 123–133 (1998).

    Article  CAS  Google Scholar 

  30. Wang, W. et al. Stimulatory activity of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptors. Chin. Med. J. (Engl.) 113, 867–871 (2000).

    CAS  Google Scholar 

  31. Filipek, S. et al. A concept for G protein activation by G protein–coupled receptor dimers: the transducin/rhodopsin interface. Photochem. Photobiol. Sci. 3, 628–638 (2004).

    Article  CAS  Google Scholar 

  32. Pease, J.E., Burton, D.R. & Barker, M.D. Generation of chimeric C5a/formyl peptide receptors: towards the identification of the human C5a receptor binding site. Eur. J. Immunol. 24, 211–215 (1994).

    Article  CAS  Google Scholar 

  33. Crass, T. et al. Chimeric receptors of the human C3a receptor and C5a receptor (CD88). J. Biol. Chem. 274, 8367–8370 (1999).

    Article  CAS  Google Scholar 

  34. DeMartino, J.A. et al. The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs. J. Biol. Chem. 269, 14446–14450 (1994).

    CAS  PubMed  Google Scholar 

  35. Siciliano, S.J. et al. Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein–coupled receptors. Proc. Natl. Acad. Sci. USA 91, 1214–1218 (1994).

    Article  CAS  Google Scholar 

  36. Gerber, B.O., Meng, E.C., Dotsch, V., Baranski, T.J. & Bourne, H.R. An activation switch in the ligand binding pocket of the C5a receptor. J. Biol. Chem. 276, 3394–3400 (2001).

    Article  CAS  Google Scholar 

  37. Polak, M. Hyperfunctioning thyroid adenoma and activating mutations in the TSH receptor gene. Arch. Med. Res. 30, 510–513 (1999).

    Article  CAS  Google Scholar 

  38. Shenker, A. Activating mutations of the lutropin choriogonadotropin receptor in precocious puberty. Receptors Channels 8, 3–18 (2002).

    Article  CAS  Google Scholar 

  39. Parma, J. et al. Somatic mutations causing constitutive activity of the thyrotropin receptor are the major cause of hyperfunctioning thyroid adenomas: identification of additional mutations activating both the cyclic adenosine 3′,5′-monophosphate and inositol phosphate-Ca2+ cascades. Mol. Endocrinol. 9, 725–733 (1995).

    CAS  PubMed  Google Scholar 

  40. Li, S., Liu, X., Min, L. & Ascoli, M. Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization. J. Biol. Chem. 276, 7968–7973 (2001).

    Article  CAS  Google Scholar 

  41. Ryu, K. et al. Modulation of high affinity hormone binding. Human choriogonadotropin binding to the exodomain of the receptor is influenced by exoloop 2 of the receptor. J. Biol. Chem. 273, 6285–6291 (1998).

    Article  CAS  Google Scholar 

  42. Decaillot, F.M. et al. Opioid receptor random mutagenesis reveals a mechanism for G protein–coupled receptor activation. Nat. Struct. Biol. 10, 629–636 (2003).

    Article  CAS  Google Scholar 

  43. Parnot, C. et al. Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay. Proc. Natl. Acad. Sci. USA 97, 7615–7620 (2000).

    Article  CAS  Google Scholar 

  44. Holst, B. & Schwartz, T.W. Molecular mechanism of agonism and inverse agonism in the melanocortin receptors: Zn(2+) as a structural and functional probe. Ann. NY Acad. Sci. 994, 1–11 (2003).

    Article  CAS  Google Scholar 

  45. Nanevicz, T., Wang, L., Chen, M., Ishii, M. & Coughlin, S.R. Thrombin receptor activating mutations. Alteration of an extracellular agonist recognition domain causes constitutive signaling. J. Biol. Chem. 271, 702–706 (1996).

    Article  CAS  Google Scholar 

  46. Altenbach, C., Klein-Seetharaman, J., Cai, K., Khorana, H.G. & Hubbell, W.L. Structure and function in rhodopsin: mapping light-dependent changes in distance between residue 316 in helix 8 and residues in the sequence 60–75, covering the cytoplasmic end of helices TM1 and TM2 and their connection loop CL1. Biochemistry 40, 15493–15500 (2001).

    Article  CAS  Google Scholar 

  47. Brown, A.J. et al. Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein α-subunit chimeras. Yeast 16, 11–22 (2000).

    Article  CAS  Google Scholar 

  48. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position–specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank G. Nikiforovich, H. Bourne, K. Blumer, E. Meng and members of the Baranski lab for helpful discussions and review of the manuscript. This work was supported by an award from the American Heart Association (J.M.K.) and by grants from the American Cancer Society IRG-58-010-43 (T.J.B.), the Culpeper Award, Rockefeller Brothers Fund (T.J.B.), and the US National Institutes of Health, GM63720-01 (T.J.B.).

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  1. Departments of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, Campus Box 8127, 660 S. Euclid Avenue, St. Louis, 63110, Missouri, USA

    Jeffery M Klco, Christina B Wiegand, Kirk Narzinski & Thomas J Baranski

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Correspondence to Thomas J Baranski.

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Supplementary information

Supplementary Fig. 1

Localization of C5aR cysteine mutants in yeast. (PDF 851 kb)

Supplementary Fig. 2

Expression of NQ receptors in yeast. (PDF 367 kb)

Supplementary Fig. 3

Alignment of EC2 residues in C5aR and rhodopsin. (PDF 42 kb)

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Klco, J., Wiegand, C., Narzinski, K. et al. Essential role for the second extracellular loop in C5a receptor activation. Nat Struct Mol Biol 12, 320–326 (2005). https://doi.org/10.1038/nsmb913

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