Interactions Between Catalytic and Inhibitory Subunits of PDE6

  • Nikolai O. Artemyev
Part of the Methods In Molecular Biology™ book series (MIMB, volume 307)

Abstract

Rod and cone photoreceptor guanosine 3′5′-cyclic-monophosphate phosphodiesterases (PDEs) are classified into the PDE6 family of cyclic nucleotide PDEs. A unique feature of the PDE6 enzymes is the presence of inhibitory γ-subunits (Pγ). The inhibitory interaction between Pγ and the rod PDE6αβ catalytic subunits is critically important for understanding the mechanism of phototransduction. Recent insights into the molecular interface between Pγ and PDE6αβ have been achieved using mutagenesis of Pγ, fluorescence labeling, and crosslinking approaches.

Key Words

PDE6 guanosine 3′5′-cyclic-monophosphate phototransduction mutagenesis polymerase chain reaction fluorescence labeling crosslinking 

Notes

Acknowledgment

This work was supported by National Institutes of Health grant EY-10843.

References

  1. 1.
    Chabre, M. and Deterre, P. (1989) Molecular mechanism of visual transduction. Eur. J. Biochem. 179, 255–266.PubMedCrossRefGoogle Scholar
  2. 2.
    Yarfitz, S. and Hurley, J. B. (1994) Transduction mechanisms of vertebrate and invertebrate photoreceptors. J. Biol. Chem. 269, 14,329–14,332.PubMedGoogle Scholar
  3. 3.
    Ovchinnikov, Y. A., Lipkin, V. M., Kumarev, V. P., Gubanov, V. V., Khramtsov, N. V., Akhmedov, N. B., Zagranichny, V. E., and Muradov, K. G. (1986) Cyclic GMP phosphodiesterase from cattle retina: amino acid sequence of the γ-subunit and nucleotide sequence of the corresponding cDNA. FEBS Lett. 204, 288–292.PubMedCrossRefGoogle Scholar
  4. 4.
    Granovsky, A. E., Natochin, M., and Artemyev, N. O. (1997) The γ-subunit of rod cGMP-phosphodiesterase blocks the enzyme catalytic site. J. Biol. Chem. 272, 11,686–11,689.PubMedCrossRefGoogle Scholar
  5. 5.
    Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Mechanism of photoreceptor PDE inhibition by its γ-subunits. Proc. Natl. Acad. Sci. USA 93, 5407–5412.PubMedCrossRefGoogle Scholar
  6. 6.
    Thompson, W. J. and Appleman, M. M. (1971) Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10, 311–316.PubMedCrossRefGoogle Scholar
  7. 7.
    Liebman, P. A. and Evanczuk, A. T. (1982) Real time assay of rod disk membrane cGMP phosphodiesterase and its controller enzymes. Methods Enzymol. 81, 532–542.PubMedCrossRefGoogle Scholar
  8. 8.
    Gillespie, P. G. and Beavo, J. A. (1989) Inhibition and stimulation of photoreceptor phosphodiesterases by dipyridamole and M&B 22,948. Mol. Pharmacol. 36, 773–781.PubMedGoogle Scholar
  9. 9.
    Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.PubMedCrossRefGoogle Scholar
  10. 10.
    Brown, R. L. and Stryer, L. (1989) Expression in bacteria of functional inhibitory subunit of retinal rod cGMP phosphodiesterase. Proc. Natl. Acad. Sci. USA 86, 4922–4926.PubMedCrossRefGoogle Scholar
  11. 11.
    Slepak, V. Z., Artemyev, N. O., Yun, Z. Dumke, C. L., Sabacan, L., Sondek, J., Hamm, H. E., Bownds, M. D., and Arshavsky, V. Y. (1995) An effector’s site that stimulates G-protein GTPase in photoreceptors. J. Biol. Chem. 270, 14,319–14,324.PubMedCrossRefGoogle Scholar
  12. 12.
    Granovsky, A. E., McEntaffer, R., and Artemyev, N. O. (1998) Probing functional interfaces of rod PDE γ-subunit using scanning fluorescent labeling. Cell Biochem. Biophys. 28, 115–133.PubMedCrossRefGoogle Scholar
  13. 13.
    Granovsky, A. E. and Artemyev, N. O. (2001) A conformational switch in the inhibitory G-subunit of PDE6 upon the enzyme activation by transducin. Biochemistry 40, 13,209–13,215.PubMedCrossRefGoogle Scholar
  14. 14.
    Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1993) Escherichia Coli, plasmids and bacteriophages, in, Current Protocols in Molecular Biology (Jansen, K., ed.), John Wiley & Sons, New York, pp. 1.8.1.–1.8.8.Google Scholar
  15. 15.
    Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  16. 16.
    Hurley, J. B. and Stryer, L. (1982) Purification and characterization of the γ regulatory subunit of the cyclic GMP phosphodiesterase from retinal rod outer segments. J. Biol. Chem. 257, 11,094–11,099.PubMedGoogle Scholar
  17. 17.
    Artemyev, N. O. and Hamm, H. E. (1992) Two-site high-affinity interaction between inhibitory and catalytic subunuts of rod cyclic GMP phosphodiesterase. Biochem. J. 283, 273–279.PubMedGoogle Scholar
  18. 18.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  19. 19.
    Cheng, Y.-C. and Prusoff, W. H. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108.PubMedCrossRefGoogle Scholar
  20. 20.
    Linden, J. (1982) Calculating the dissociation constant of an unlabeled compound from the concentration required to displace radiolabel binding by 50%. J. Cyclic Nuc. Res. 8, 163–172.Google Scholar
  21. 21.
    Muradov, K. G., Granovsky, A. E., and Artemyev, N. O. (2002) Direct interaction of the inhibitory γ-subunit of rod cGMP phosphodiesterase (PDE6) with the PDE6 GAF domains. Biochemistry 41, 3884–3890.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Nikolai O. Artemyev
    • 1
  1. 1.Department of Physiology and BiophysicsUniversity of Iowa College of MedicineIowa City

Personalised recommendations