B. Novel PDE6-binding proteins involved in light adaptation

In the rod outer segment, the highly organized stacks of disk membranes serve to localize the major components of visual excitation: rhodopsin, transducin, and PDE6. The co-localization of PDE6 with rhodopsin and transducin on the surface of the disk membrane has obvious implications for the speed and effectiveness of the activation pathway once rhodopsin is bleached (Lamb, 1996). In addition to the central players in the activation process, many other proteins co-localize to the disk membrane, either as peripheral or integral membrane proteins, or undergo light-induced changes in membrane attachment. Some of these proteins may interact with rod PDE6 to modulate the extent of its activation or the lifetime of its activated state.

ROS contain a set of glutamic acid-rich proteins (GARPs) that exist in three forms: as a domain of the β subunit of the cGMP-gated ion channel, and a two proteins (GARP1 and GARP2) which associate with the rims of the disk membranes (Molday and Molday, 1998). The predominant GARP2 form (Sugimoto et al., 1991) has been shown to bind to the rim protein, peripherin, the cGMP-gated ion channel, and PDE6 (Körschen et al., 1999; Poetsch et al. 2001). GARP2 binding at its proline-rich repeats (R1-R4) to PDE6 was able to reverse activation of transducin-activated PDE6, but not trypsin-activated P αβ catalytic dimers, suggesting a specific interaction of GARP2 with th PDE-transducin complex (Körschen et al., 1999). We have demonstrate light-induced changes in binding of GARP2 to PDE6, and hypothesize that GARP2 may represent a transducin-independent mechanism to inactivate PDE6. In collaboration with Dr. Benjamin Kaupp (Inst. Biol. Information Processing, Julich), we are characterizing the interaction of GARP2 with PDE6, and the ability of this protein to alter the catalytic and allosteric properties of PDE6 during visual transduction.

A 17 kDa prenyl binding protein (PrBP) was originally identified as a fourth (δ) subunit of PDE6, co-purifying with a soluble form of bovine PDE (Gillespie et al., 1989). Outside the retina, PrBP is likely involved in vesicular transport (particularly, but not exclusively, monomeric G-proteins) (Marzesco et al., 1998), and its function in retinal photoreceptors remains unclear. We have shown that PrBP/δ has no direct effect on rod PDE catalysis, but does enhance cGMP exchange at the GAF domains upon binding to the PDE6 holoenzyme (Mou et al., 1999). The hypothesis that PrBP/δ uncouples transducin activation of PDE6 during light adaptation by releasing PDE6 from the disk membrane has experimental support in vitro (Cook et al., 2001). We are investigating the abundance, subcellular localization [in collaboration with Dr. Wolfgang Baehr (Moran Eye Inst., Univ. Utah), and Dr. Noga Vardi, (Neuroscience, Univ. Penn.], and regulatory significance for phototransduction of PrBP/δ.

References

Cook,T.A., Ghomashchi,F., Gelb,M.H., Florio,S.K., and Beavo,J.A. (2001). The δ subunit of type 6 phosphodiesterase reduces light-induced cGMP hydrolysis in rod outer segments. J. Biol. Chem. 276, 5248-5255.

Gillespie,P.G., Prusti,R.K., Apel,E.D., and Beavo,J.A. (1989). A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15 kDa subunit. J. Biol. Chem. 264, 12187-12193.

Körschen,H.G., Beyermann,M., Müller,F., Heck,M., Vantler,M., Koch,K.W. , Kellner,R., Wolfrum,U., Bode,C., Hofmann,K.P., and Kaupp,U.B. (1999). Interaction of glutamic-acid-rich proteins with the cGMP signalling pathway in rod photoreceptors. Nature 400, 761-766.

Lamb,T.D. (1996). Gain and kinetics of activation in the G-protein cascade of phototransduction. Proc. Natl. Acad. Sci. U. S. A. 93, 566-570.

Marzesco,A.M., Galli,T., Louvard,D., and Zahraoui,A. (1998). The rod cGMP phosphodiesterase delta subunit dissociates the small GTPase Rab13 from membranes. J. Biol. Chem. 273, 22340-22345.

Molday,R.S. and Molday,L.L. (1998). Molecular properties of the cGMP-gated channel of rod photoreceptors. Vision Res. 38, 1315-1323.

Poetsch,A., Molday,L.L., and Molday,R.S. (2001). The cGMP-gated channel and related glutamic acid rich proteins interact with peripherin-2 at the rim region of rod photoreceptor disc membranes. J. Biol. Chem. 276, 48009-48016.

Sugimoto,Y., Yatsunami,K., Tsujimoto,M., Khorana,H.G., and Ichikawa,A. (1991). The amino acid sequence of a glutamic acid-rich protein from bovine retina as deduced from the cDNA sequence. Proc. Natl. Acad. Sci. U. S. A. 88, 3116-3119.