Cyclic Guanosine 5′-Monophosphate Binding to Regulatory GAF Domains of Photoreceptor Phosphodiesterase

  • Rick H. Cote
Part of the Methods In Molecular Biology™ book series (MIMB, volume 307)


Of the 11 families of mammalian cyclic nucleotide phosphodiesterases (PDEs), 5 contain regulatory domains capable of binding cyclic guanosine 5′-monophosphate (cGMP). The best understood of the GAF-containing PDEs is the family of rod (PDE6R) and cone (PDE6C) photoreceptor PDEs. Binding of cGMP to the rod PDE6 catalytic dimer (αβ) allosterically regulates the affinity of the inhibitory subunits of PDE6 (γ) for the enzyme. Two nonidentical, highaffinity cGMP-binding sites exist on the nonactivated mammalian PDE6R holoenzyme (αβγγ). One of the sites does not readily exchange with free cGMP when the catalytic dimer is complexed with Pγ. On dissociation of γ from the catalytic dimer, one of the two cGMP-binding sites undergoes a transition from high to low affinity. This chapter describes techniques to quantify cGMP binding to PDE6 in order to study the regulatory significance of the GAF domains. For high-affinity cGMP binding sites on PDE6, membrane filtration is the method of choice because of its speed, simplicity, and sensitivity. However, lower-affinity cGMP-binding sites require a method that does not perturb the equilibrium between bound and free ligand. The use of ammonium sulfate solutions during filtration extends to lower-binding affinities the useful range of membrane filtration. However, a centrifugal separation technique that minimizes perturbation of the cGMP-binding equilibrium is also presented for measuring lower-affinity cGMP-binding sites. These methods are applicable to understanding the regulatory mechanisms regulating other GAF-containing PDEs as well.

Key Words

Photoreceptor phosphodiesterase cyclic guanosine 5′-monophosphate GAF domain transducin membrane filtration ligand binding 



I am grateful to the past and present members of my laboratory for developing the procedures and conducting the experiments reported in this chapter. This work was supported by the National Institutes of Health (National Eye Institute, EY-05798). This is Scientific Contribution #2191 from the New Hampshire Agricultural Experiment Station.


  1. 1.
    Taylor, W. R. and Baylor, D. A. (1995) Conductance and kinetics of single cGMP-activated channels in salamander rod outer segments. J. Physiol. (Lond.) 483, 567–582.Google Scholar
  2. 2.
    Artemyev, N. O., Arshavsky, V. Y., and Cote, R. H. (1998) Photoreceptor phosphodiesterase: interaction of inhibitory γ subunit and cyclic GMP with specific binding sites on catalytic subunits. Methods 14, 93–104.PubMedCrossRefGoogle Scholar
  3. 3.
    Yamazaki, A., Sen, I., Bitensky, M. W., Casnellie, J. E., and Greengard, P. (1980) Cyclic GMP-specific, high affinity, noncatalytic binding sites on light-activated phosphodiesterase. J. Biol. Chem. 255, 11,619–11,624.PubMedGoogle Scholar
  4. 4.
    Cote, R. H. and Brunnock, M. A. (1993) Intracellular cGMP concentration in rod photoreceptors is regulated by binding to high and moderate affinity cGMP binding sites. J. Biol. Chem. 268, 17,190–17,198.PubMedGoogle Scholar
  5. 5.
    Cote, R. H., Bownds, M. D., and Arshavsky, V. Y. (1994) cGMP binding sites on photoreceptor phosphodiesterase: role in feedback regulation of visual transduction. Proc. Natl. Acad. Sci. USA 91, 4845–4849.PubMedCrossRefGoogle Scholar
  6. 6.
    Mou, H., Grazio, H. J., Cook, T. A., Beavo, J. A., and Cote, R. H. (1999) cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its γ and δ subunits. J. Biol. Chem. 274, 18,813–18,820.PubMedCrossRefGoogle Scholar
  7. 7.
    Kincaid, R. L. and Manganiello, V. C. (1988) Assay of cyclic nucleotide phosphodiesterase using radiolabeled and fluorescent substrates. Methods Enzymol. 159, 457–470.PubMedCrossRefGoogle Scholar
  8. 8.
    Norton, A. W., D’Amours, M. R., Grazio, H. J., Hebert, T. L., and Cote, R. H. (2000) Mechanism of transducin activation of frog rod photoreceptor phosphodiesterase: allosteric interactions between the inhibitory γ subunit and the noncatalytic cGMP binding sites. J. Biol. Chem. 275, 38,611–38,619.PubMedCrossRefGoogle Scholar
  9. 9.
    Bennett, J. P. Jr. and Yamamura, H. I. (1985) Neurotransmitter, hormone, or drug receptor binding methods, in Neurotransmitter Receptor Binding, 2nd ed. (Yamamura, H. I., ed.), Raven, New York, pp. 61–89.Google Scholar
  10. 10.
    Doskeland, S. O., Ueland, P. M., and Haga, H. J. (1977) Factors affecting the binding of [3H] adenosine 3′:5′-cyclic monophosphate to protein kinase from bovine adrenal cortex. Biochem. J. 161, 653–665.PubMedGoogle Scholar
  11. 11.
    Corbin, J. D. and Doskeland, S. O. (1983) Studies of two different intrachain cGMP-binding sites of cGMP-dependent protein kinase. J. Biol. Chem. 258, 11,391–11,397.PubMedGoogle Scholar
  12. 12.
    Doskeland, S. O. and Ogreid, D. (1988) Ammonium sulfate precipitation assay for the study of cyclic nucleotide binding to proteins. Methods Enzymol. 159, 147–151.PubMedCrossRefGoogle Scholar
  13. 13.
    Miot, F., Van Haastert, P. J. M., and Erneux, C. (1985) Specificity of cGMP binding to a purified cGMP stimulated phosphodiesterase from bovine adrenal tissue. Eur. J. Biochem. 149, 59–65.PubMedCrossRefGoogle Scholar
  14. 14.
    Coquil, J. F., Franks, D. J., Wells, J. N., Dupuis, M., and Hamet, P. (1980) Characteristics of a new binding protein distinct from the kinase for guanosine 3′-5′-monophosphate in rat platelets. Biochim. Biophys. Acta 631, 148–165.PubMedGoogle Scholar
  15. 15.
    Hulme, E. C. (1990) Receptor binding studies, a brief outline, in Receptor-Effector Coupling, A Practical Approach (Hulme, E. C., ed.), Oxford University Press, UK, pp. 203–215.Google Scholar
  16. 16.
    Forget, R. S., Martin, J. E., and Cote, R. H. (1993) A centrifugal separation procedure detects moderate affinity cGMP binding sites in membrane-associated proteins and permeabilized cells. Anal. Biochem. 215, 159–161.PubMedCrossRefGoogle Scholar
  17. 17.
    Bownds, D., Gordon-Walker, A., Gaide Huguenin, A. C., and Robinson, W. (1971) Characterization and analysis of frog photoreceptor membranes. J. Gen. Physiol. 58, 225–237.PubMedCrossRefGoogle Scholar
  18. 18.
    Leatherbarrow, R. J. (1990) Using linear and non-linear regression to fit biochemical data. Trends Biochem. Sci. 15, 455–458.PubMedCrossRefGoogle Scholar
  19. 19.
    McPherson, G. A. (1985) Analysis of radioligand binding experiments on a microcomputer system. J. Pharmacol. Meth. 14, 213–228.CrossRefGoogle Scholar
  20. 20.
    Munson, P. J. (1983) LIGAND: a computerized analysis of ligand binding data. Methods Enzymol. 92, 543–576.PubMedCrossRefGoogle Scholar
  21. 21.
    Munson, P. J. and Rodbard, D. (1980) LIGAND: a versatile computerized approach for the characterization of ligand binding systems. Anal. Biochem. 107, 220–239.PubMedCrossRefGoogle Scholar
  22. 22.
    Gillespie, P. G. and Beavo, J. A. (1989) cGMP is tightly bound to bovine retinal rod phosphodiesterase. Proc. Natl. Acad. Sci. USA 86, 4311–4315.PubMedCrossRefGoogle Scholar
  23. 23.
    Mou, H. and Cote, R. H. (2001) The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory γ subunit. J. Biol. Chem. 276, 27,527–27,534.PubMedCrossRefGoogle Scholar
  24. 24.
    Gillespie, P. G. and Beavo, J. A. (1988) Characterization of a bovine cone photoreceptor phosphodiesterase purified by cyclic GMP-Sepharose chromatography. J. Biol. Chem. 263, 8133–8141.PubMedGoogle Scholar
  25. 25.
    Cote, R. H., Daly, A. E., Valeriani, B. A., and Vardi, N. (2002) Regulation of cone photoreceptor phosphodiesterase (PDE6C) by its inhibitory γ′ subunit and by cGMP binding. Invest. Ophthalmol. Vis. Sci. 43, ARVO E-Abstract 1960.Google Scholar
  26. 26.
    D’Amours, M. R., Granovsky, A. E., Artemyev, N. O., and Cote, R. H. (1999) The potency and mechanism of action of E4021, a PDE5-selective inhibitor, on the photoreceptor phosphodiesterase depends on its state of activation. Mol. Pharmacol. 55, 508–514.Google Scholar
  27. 27.
    Hebert, M. C., Schwede, F., Jastorff, B., and Cote, R. H. (1998). Structural features of the noncatalytic cGMP binding sites of frog photoreceptor phosphodiesterase using cGMP analogs. J. Biol. Chem. 273, 5557–5565.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Rick H. Cote
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of New HampshireDurham

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