A. Structure, function and allosteric regulation of rod PDE6

A collaboration of our lab with Dr. Patrick Schultz (CNRS, IGBMC, Illkirch) and with Dr. Claire Lugnier (CNRS, Pharmacy, Illkirch) provided the first 3-dimensional model for the rod PDE6 holoenzyme (Kameni Tcheudji et al., 2001). While these images placed the catalytic and GAF domains on a structural model, we now seek to understand how the inhibitory γ subunit other PDE6-binding proteins, or transducin activation lead to conformational changes in the quaternary structure of the enzyme.

Previous work from our lab has contributed to understanding the role of the GAF domains in PDE6 regulation. It is well established that cGMP binds to two high-affinity sites on amphibian (Cote et al., 1994) and mammalian (Gillespie and Beavo, 1989; Mou et al., 1999) rod PDE. There is also evidence for another, lower affinity class of cGMP binding sites on PDE6 (Cote and Brunnock, 1993). cGMP binding enhances the affinity of the inhibitory γ subunit in a reciprocal manner (Yamazaki et al., 1982; Arshavsky et al., 1992; Cote et al., 1994). This positive cooperativity between γ and cGM binding to PDE6 is also observed in PDE6 upon transducin activation (Norton et al., 2000) and upon PDE6 phosphorylation (unpublished results). Our lab is continuing to investigate the structural basis and functional significance of rod PDE6 having non-identical cGMP binding and γ binding sites on the ro catalytic αβ heterodime

References

Arshavsky,V.Y., Dumke,C.L., and Bownds,M.D. (1992). Noncatalytic cGMP binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory γ-subunits. A putative regulatory mechanism of the rod photoresponse. J. Biol. Chem. 267, 24501-24507.

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. U. S. A. 91, 4845-4849.

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, 17190-17198.

Gillespie,P.G. and Beavo,J.A. (1989). cGMP is tightly bound to bovine retinal rod phosphodiesterase. Proc. Natl. Acad. Sci. U. S. A. 86, 4311-4315.

Kameni Tcheudji,J.F., Lebeau,L., Virmaux,N., Maftei,C.G., Cote,R.H., Lugnier,C., and Schultz,P. (2001). Molecular organization of bovine rod cGMP-phosphodiesterase 6. J. Mol. Biol. 310, 781-791.

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, 18813-18820.

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, 27527-27534.

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, 38611-38619.

Yamazaki,A., Bartucci,F., Ting,A., and Bitensky,M.W. (1982). Reciprocal effects of an inhibitory factor on catalytic activity and noncatalytic cGMP binding sites of rod phosphodiesterase. Proc. Natl. Acad. Sci. U. S. A. 79, 3702-3706.