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Retinoids pp 85-94 | Cite as

In Vitro Assays of Rod and Cone Opsin Activity: Retinoid Analogs as Agonists and Inverse Agonists

  • Masahiro Kono
  • Rosalie K. Crouch
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 652)

Abstract

Upon absorption of a photon, the bound 11-cis-retinoid isomerizes to the all-trans form resulting in a protein conformational change that enables it to activate its G protein, transducin, to begin the visual signal transduction cascade. The native ligand, 11-cis-retinal, acts as an inverse agonist to both the apoproteins of rod and cone visual pigments (opsins); all-trans-retinal is an agonist. Truncated analogs of retinal have been used to characterize structure–function relationships with rod opsins, but little has been done with cone opsins. Activation of transducin by an opsin is one method to characterize the conformational state of the opsin. This chapter describes an in vitro transducin activation assay that can be used with cone opsins to determine the degree to which different ligands can act as an agonist or an inverse agonist to gain insight into the ligand-binding pocket of cone opsins and differences between the different classes of opsins. The understanding of the effects of ligands on cone opsin activity can potentially be applied to future therapeutic agents targeting opsins.

Key words

Retinal analog cone opsin G protein-coupled receptor transducin cone pigment rhodopsin 

References

  1. 1.
    Lou, J., Tan, Q., Karnaukhova, E., Berova, N., Nakanishi, K., Crouch, R.K. (2000) Synthetic retinals: Convenient probes of rhodopsin and visual transduction process. Methods Enzymol. 315, 219–237.PubMedCrossRefGoogle Scholar
  2. 2.
    Cohen, G.B., Yang, T., Robinson, P.R., Oprian, D.D. (1993) Constitutive activation of opsin: Influence of charge at position 134 and size at position 296. Biochemistry 32, 6111–6115.PubMedCrossRefGoogle Scholar
  3. 3.
    Surya, A., Foster, K.W., Knox, B.E. (1995) Transducin activation by the bovine opsin apoprotein. J. Biol. Chem. 270, 5024–5031.PubMedCrossRefGoogle Scholar
  4. 4.
    Isayama, T., Chen, Y., Kono, M., DeGrip, W.J., Ma, J.-X., Crouch, R.K., Makino, C.L. (2006) Differences in the pharmacological activation of visual opsins. Vis. Neurosci. 23, 899–908.PubMedCrossRefGoogle Scholar
  5. 5.
    Kono, M. (2006) Constitutive activity of a UV cone opsin. FEBS Lett. 580, 229–232.PubMedCrossRefGoogle Scholar
  6. 6.
    Melia, T.J., Jr., Cowan, C.W., Angleson, J.K., Wensel, T.G. (1997) A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin. Biophys. J. 73, 3182–3191.PubMedCrossRefGoogle Scholar
  7. 7.
    Bartl, F.J., Fritze, O., Ritter, E., Herrmann, R., Kuksa, V., Palczewski, K., Hofmann, K.P., Ernst, O.P. (2005) Partial agonism in a G protein-coupled receptor. Role of the retinal ring structure in rhodopsin activation. J. Biol. Chem. 280, 34259–34267.PubMedCrossRefGoogle Scholar
  8. 8.
    Han, M., Groesbeek, M., Sakmar, T.P., Smith, S.O. (1997) The C9 methyl group of retinal interacts with glycine-121 in rhodopsin. Proc. Natl. Acad. Sci. USA 94, 13442–13447.PubMedCrossRefGoogle Scholar
  9. 9.
    Buczylko, J., Saari, J.C., Crouch, R.K., Palczewski, K. (1996) Mechanisms of opsin activation. J. Biol. Chem. 271, 20621–20630.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhukovsky, E.A., Robinson, P.R., Oprian, D.D. (1991) Transducin activation by rhodopsin without a covalent bond to the 11-cis-retinal chromophore. Science 251, 558–560.PubMedCrossRefGoogle Scholar
  11. 11.
    Ramon, E., Mao, X., Ridge, K.D. (2009) Studies on the stability of the human cone visual pigments. Photochem. Photobiol. 85, 509–516.PubMedCrossRefGoogle Scholar
  12. 12.
    Crescitelli, F. (1984) The gecko visual pigment: The dark exchange of chromophore. Vision Res. 24, 1551–1553.PubMedCrossRefGoogle Scholar
  13. 13.
    Kefalov, V.J., Estevez, M.E., Kono, M., Goletz, P.W., Crouch, R.K., Cornwall, M.C., Yau, K.-W. (2005) Breaking the covalent bond – a pigment property that contributes to desensitization in cones. Neuron 46, 879–890.PubMedCrossRefGoogle Scholar
  14. 14.
    Matsumoto, H., Tokunaga, F., Yoshizawa, T. (1975) Accessibility of the iodopsin chromophore. Biochim. Biochem. Acta 404, 300–308.CrossRefGoogle Scholar
  15. 15.
    Ala-Laurila, P., Cornwall, M.C., Crouch, R.K., Kono, M. (2009) The action of 11-cis-retinol on cone opsins and intact cone photoreceptors. J. Biol. Chem. 284, 16492–16500.PubMedCrossRefGoogle Scholar
  16. 16.
    Das, J., Crouch, R.K., Ma, J.-X., Oprian, D.D., Kono, M. (2004) Role of the 9-methyl group of retinal in cone visual pigments. Biochemistry 43, 5532–5538.PubMedCrossRefGoogle Scholar
  17. 17.
    Jones, G.J., Crouch, R.K., Wiggert, B., Cornwall, M.C., Chader, G.J. (1989) Retinoid requirements for recovery of sensitivity after visual-pigment bleaching in isolated photoreceptors. Proc. Natl. Acad. Sci. USA 86, 9606–9610.PubMedCrossRefGoogle Scholar
  18. 18.
    Kono, M., Goletz, P.W., Crouch, R.K. (2008) 11-cis and all-trans retinols can activate rod opsin: Rational design of the visual cycle. Biochemistry 47, 7567–7571.PubMedCrossRefGoogle Scholar
  19. 19.
    Oprian, D.D. (1993) Expression of opsin genes in COS cells. Methods Neurosci. 15, 301–306.Google Scholar
  20. 20.
    Kono, M., Crouch, R.K., Oprian, D.D. (2005) A dark and constitutively active mutant of the tiger salamander UV pigment. Biochemistry 44, 799–804.PubMedCrossRefGoogle Scholar
  21. 21.
    Robinson, P.R. (2000) Assays for the detection of constitutively active opsins. Methods Enzymol. 315, 207–218.PubMedCrossRefGoogle Scholar
  22. 22.
    Baehr, W., Morita, E.A., Swanson, R.J., Applebury, M.L. (1982) Characterization of bovine rod outer segment G-protein. J. Biol. Chem. 257, 6452–6460.PubMedGoogle Scholar
  23. 23.
    Wessling-Resnick, M., Johnson, G.L. (1987) Allosteric behavior in transducin activation mediated by rhodopsin. J. Biol. Chem. 262, 3697–3705.PubMedGoogle Scholar
  24. 24.
    Yu, H., Kono, M., McKee, T.D., Oprian, D.D. (1995) A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins. Biochemistry 34, 14963–14969.PubMedCrossRefGoogle Scholar
  25. 25.
    Nathans, J. (1990) Determinants of visual pigment absorbance: Identification of the retinylidene Schiff’s base counterion in bovine rhodopsin. Biochemistry 29, 9746–9752.PubMedCrossRefGoogle Scholar
  26. 26.
    Tsutsui, K., Imai, H., Shichida, Y. (2007) Photoisomerization efficiency in UV-absorbing visual pigments: Protein-directed isomerization of an unprotonated retinal Schiff base. Biochemistry 46, 6437–6445.PubMedCrossRefGoogle Scholar
  27. 27.
    Vissers, P.M.A.M., Bovee-Geurts, P.H.M., Portier, M.D., Klaassen, C.H.W., DeGrip, W.J. (1998) Large-scale production and purification of the human green cone pigment: Characterization of late photo-intermediates. Biochem. J. 330, 1201–1208.PubMedGoogle Scholar
  28. 28.
    Rim, J., Oprian, D.D. (1995) Constitutive activation of opsin: Interaction of mutants with rhodopsin kinase and arrestin. Biochemistry 34, 11938–11945.PubMedCrossRefGoogle Scholar
  29. 29.
    Han, M., Groesbeek, M., Smith, S.O., Sakmar, T.P. (1998) Role of the C9 methyl group in rhodopsin activation: Characterization of mutant opsins with the artificial chromophore 11-cis-9-demethylretinal. Biochemistry 37, 538–545.PubMedCrossRefGoogle Scholar
  30. 30.
    Okano, T., Fukada, Y., Artamonov, I.D., Yoshizawa, T. (1989) Purification of cone visual pigments from chicken retina. Biochemistry 28, 8848–8856.PubMedCrossRefGoogle Scholar
  31. 31.
    Liang, J., Govindjee, R., Ebrey, T.G. (1993) Metarhodopsin intermediates of the gecko cone pigment P521. Biochemistry 32, 14187–14193.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Masahiro Kono
    • 1
  • Rosalie K. Crouch
    • 1
  1. 1.Department of OphthalmologyMedical University of South CarolinaCharlestonUSA

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