Binding of cGMP to GAF Domains in Amphibian Rod Photoreceptor cGMP Phosphodiesterase (PDE)

• Published in: The Journal of biological chemistry Vol. 277; no. 43; pp. 40675 - 40686
• Main Authors: Yamazaki, Matsuyo, Li, Ning, Bondarenko, Vladimir A., Yamazaki, Russell K., Baehr, Wolfgang, Yamazaki, Akio
• Format: Journal Article
• Language: English
• Published: American Society for Biochemistry and Molecular Biology 25.10.2002
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M203469200

Retinal cGMP phosphodiesterase (PDE6) is a key enzyme in vertebrate phototransduction. Rod PDE contains two homologous catalytic subunits (Pαβ) and two identical regulatory subunits (Pγ). Biochemical studies have shown that amphibian Pαβ has high affinity, cGMP-specific, non-catalytic binding sites and that Pγ stimulates cGMP binding to these sites. Here we show by molecular cloning that each catalytic subunit in amphibian PDE, as in its mammalian counterpart, contains two homologous tandem GAF domains in its N-terminal region. In Pγ-depleted membrane-bound PDE (20–40% Pγ still present), a single type of cGMP-binding site with a relatively low affinity ( K d ∼ 100 n m ) was observed, and addition of Pγ increased both the affinity for cGMP and the level of cGMP binding. We also show that mutations of amino acid residues in four different sites in Pγ reduced its ability to stimulate cGMP binding. Among these, the site involved in Pγ phosphorylation by Cdk5 (positions 20–23) had the largest effect on cGMP binding. However, except for the C terminus, these sites were not involved in Pγ inhibition of the cGMP hydrolytic activity of Pαβ. In addition, the Pγ concentration required for 50% stimulation of cGMP binding was much greater than that required for 50% inhibition of cGMP hydrolysis. These results suggest that the Pαβ heterodimer contains two spatially and functionally distinct types of Pγ-binding sites: one for inhibition of cGMP hydrolytic activity and the second for activation of cGMP binding to GAF domains. We propose a model for the Pαβ-Pγ interaction in which Pγ, by binding to one of the two sites in Pαβ, may preferentially act either as an inhibitor of catalytic activity or as an activator of cGMP binding to GAF domains in frog PDE.