Cyclic guanosine 5'-monophosphate binding to regulatory GAF domains of photoreceptor phosphodiesterase

Methods Mol Biol. 2005;307:141-54. doi: 10.1385/1-59259-839-0:141.

Abstract

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) photo receptor PDEs. Binding of cGMP to the rod PDE6 catalytic dimer (alphabeta) allosterically regulates the affinity of the inhibitory subunits of PDE6 (gamma) for the enzyme. Two nonidentical, high- affinity cGMP-binding sites exist on the nonactivated mammalian PDE6R holoenzyme (alphabetagammagamma). One of the sites does not readily exchange with free cGMP when the catalytic dimer is complexed with Pgamma. On dissociation of gamma 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / chemistry*
  • 3',5'-Cyclic-GMP Phosphodiesterases / metabolism
  • Animals
  • Catalytic Domain
  • Cyclic GMP / analysis*
  • Cyclic GMP / metabolism
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • Dimerization
  • Eye Proteins
  • Humans
  • Phosphoric Diester Hydrolases / chemistry*
  • Phosphoric Diester Hydrolases / metabolism
  • Retinal Cone Photoreceptor Cells / enzymology*
  • Retinal Cone Photoreceptor Cells / metabolism
  • Retinal Rod Photoreceptor Cells / enzymology*
  • Retinal Rod Photoreceptor Cells / metabolism

Substances

  • Eye Proteins
  • Phosphoric Diester Hydrolases
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Cyclic Nucleotide Phosphodiesterases, Type 6
  • PDE6A protein, human
  • PDE6B protein, human
  • Cyclic GMP