PDE6 REGULATION

The extent of PDE6 activation and the lifetime of activated PDE6 following photic stimulation are the rate-limiting steps in the excitation and recovery phases of the phototransduction pathway. While binding of activated transducin (specifically the α-subunit with GTP bound; α*-GTP) to relieve the inhibitory constraint of the γ-subunit is central to the PDE6 activation/inactivation mechanism, other factors (such as allosteric regulation by the GAF domains and binding of other PDE6 regulatory proteins) are likely to modulate the light sensitivity, extent of amplification, and duration of the activated state of PDE6.

Transducin Activation of Rod PDE6 During Visual Excitation

It is generally agreed that transducin activation of PDE6 results from the binding of the activated transducin α-subunit (α*-GTP) to the nonactivated PDE6 holoenzyme (αβγ2) and the displacement of the γ-subunit from its binding site at the entrance to the PDE6 catalytic site (Fig. 4). However, the detailed mechanism of this process remains surprisingly unclear.

The prevailing model of visual excitation (Fig. 4A) asserts that one molecule of α*- GTP binds to each PDE6 catalytic subunit, displacing both γ-subunits and enhancing cGMP hydrolysis at each catalytic site [45, 158]. However, evidence for the stoichiometry of αt-GTP binding to PDE6 and the extent to which PDE6 can be activated is not consistent with this model. For example, in those instances when physical removal of the γ-subunit from the PDE6 holoenzyme was directly compared to the maximal extent of transducin activation of PDE6, the hydrolytic activity was up to twofold higher for the PDE6 αβ catalytic dimer (devoid of γ-subunit) compared to the α*-GTP–PDE6 activated complex [73, 159–161]. Furthermore, correlations of α*-GTP binding to PDE6 with activation of cGMP hydrolysis demonstrate that a single α*-GTP was able to maximally activate the PDE6 αβ catalytic dimer [161–163]. These observations are consistent with the idea that α*-GTP relieves inhibition at only one of the two active sites on the PDE6 catalytic dimer (Fig. 4B). Although evidence for a second α*-GTP binding to PDE6 (Fig. 4C) has been reported [164], its affinity is likely much weaker and nonproductive under physiological conditions.

A description of the mechanism of PDE6 reinhibition following transducin deactivation was presented in this chapter in the sections on deactivation of transducin and PDE6.

Functions of the Regulatory cGMP-Binding GAF Domains of PDE6

Whereas allosteric regulation of catalysis has been demonstrated for the PDE2 and PDE5 cGMP-binding GAF domains [165–169], no evidence for intramolecular allosteric communication between the GAF and catalytic domains has been reported for PDE6 [142, 154, 170]. There are, however, inherent experimental difficulties in quantifying cGMP binding to the GAF domains when cGMP is itself a substrate for catalysis at the active site.

Nonetheless, cGMP binding to the PDE6 GAF domains must induce a conformational change in the catalytic subunits since the affinity with which the γ-subunit binds to the catalytic dimer is markedly enhanced when cGMP is bound (Fig. 5). This can be seen as a decrease in the basal activity of PDE6 holoenzyme when cGMP occupies

Fig. 5. Positive cooperativity between cyclic guanosine monophosphate (cGMP) and γ- subunit binding to the rod phosphodiesterase 6 (PDE6) catalytic dimer. The enzyme active sites of PDE6 are denoted by notches, while the cGMP-binding sites are represented by circular pockets. A When γ-subunit (shown with its catalytic-interacting and GAF-interacting subdomains) is mixed with PDE6 catalytic dimer lacking bound cGMP at the GAF domains, the binding affinity of both γ-subunits is equal (dissociation constant, KD = 3 pM). On occupancy of the GAF domains by cGMP (black circles), one γ-subunit-binding site now binds to the catalytic subunit with more than tenfold higher affinity, while the other is unchanged. B Addition of cGMP to the catalytic dimer results in high-affinity (KD = 60 nM) binding to one GAF domain. Only at high cGMP concentrations will the second site become occupied (KD > 1 µM). On addition of stoichiometric amounts of γ-subunit, the cGMP-binding affinity is greatly increased at the low-affinity site and modestly increased at the high-affinity site. (KD values are for bovine rod PDE6 [96, 142].)

the GAF domain [154]. Furthermore, when cGMP is bound to the PDE6 catalytic dimer, the intrinsic γ-subunit binding affinity is enhanced for one, but not both, of its binding sites on the catalytic dimer; the second γ-subunit binding site retains the same affinity for PDE6 regardless of the state of occupancy of the GAF domains by cGMP [142]. When transducin activates the PDE6 holoenzyme, the γ-subunit remains associated with the PDE6 catalytic dimer when cGMP is present but is released in a complex with transducin α-subunit when the GAF domains are unoccupied [170, 171].

This allosteric change in the GAFa domain is reciprocal in that addition of γ-subunit to PDE6 catalytic dimers greatly enhances the binding affinity of cGMP for PDE6 [95, 143]. The two cGMP-binding sites have intrinsically different binding affinities: One GAF domain binds cGMP with high affinity, while the other GAF domain is a low-affinity site in the absence of γ-subunit (Fig. 5B). The cGMP-binding affinity at both sites is increased more than 100-fold when γ-subunit recombines with the catalytic dimer [73, 96, 142].

The reciprocal positive cooperativity between cGMP and γ-subunit binding to PDE6 catalytic dimer is also relevant to transducin-activated PDE6. Not only does displacement of the γ-subunit by α*-GTP relieve inhibition at the active site, cGMP-binding

affinity to one of the GAF domains is lowered about tenfold. Once cGMP has dissociated from the catalytic subunit, the γ-subunit affinity is concomitantly reduced, causing its dissociation from PDE6, presumably in a complex of with α*-GTP. The second GAF domain retains high affinity for cGMP, and the second γ-subunit remains associated with the PDE6 catalytic dimer [73, 95].

Taken together, the interplay between the γ-subunit and the GAF domains suggests a unique physiological role for the PDE6 GAF domains. In dark-adapted photoreceptors, cytoplasmic free cGMP levels are several micromolar, and the cGMP-binding GAF domains would be occupied with cGMP while two γ-subunits block catalysis at the active sites. On light activation of PDE6, displacement of one γ-subunit by transducin will relieve inhibition at the active site and lower cGMP affinity for one binding site. For transient light activation, cGMP dissociation from the GAFa domains is unlikely because recovery of cGMP levels is fast, thereby promoting tight reassociation of the γ-subunit and the return of PDE6 holoenzyme to its dark-adapted state. For prolonged illumination (i.e., during light adaptation) during which cGMP levels remain low, cGMP dissociation from the GAFa domain might occur, lowering the γ-subunit affinity for its PDE6 catalytic subunit and permitting the γ-subunit (complexed with transducin α-subunit and RGS9) to serve as a GTPase accelerating factor [64]. This would increase the rate of transducin inactivation and help restore PDE6 to its nonactivated state. In this way, the GAFa domains of PDE6 might be sensors of cytoplasmic cGMP and respond to sustained decreases in cGMP levels with a negative-feedback mechanism to help restore the ability to detect light stimuli. An alternative hypothesis that the GAF domains buffer cellular cGMP and release it during photoresponse recovery [67, 97] has not been supported by the kinetics of cGMP binding and dissociation with the GAFa domains [68, 73].

Potential PDE6 Regulatory Binding Proteins

Two photoreceptor proteins, a glutamic acid-rich protein 2 (GARP2) and a 17-kDa prenyl-binding protein (PrBP/δ; originally referred to as the PDE “δ-subunit”) have been shown to bind to PDE6 [172, 173], but their roles in regulating PDE6 activity or its subcellular localization are currently unknown.

Glutamic Acid-Rich Protein 2

GARP2 is a truncated, alternative splice product of the β-subunit of the rod cGMPgated ion channel (CNGB1). GARP2 has a unique eight amino acid C-terminus (compared to the CNGB1 sequence), a high content of proline and glutamate residues, and a natively unfolded structure [173–176]. GARP2 is specifically expressed in rods but not cones [173] and is concentrated at the rims of the outer segment disk membranes [173, 175]. The GARP2 content in rod outer segments is roughly stoichiometric with the PDE6 content [176, 177], making it an attractive candidate as a PDE6 regulatory protein.

The few studies of the ability of GARP2 to regulate PDE6 differ in their conclusions. One study reported that addition of recombinant GARP2 was able to deactivate transducin-activated PDE6, but had no effect on nonactivated PDE6 holoenzyme or the catalytic dimer [173]. In contrast, purified, native GARP2 failed to deactivate trans- ducin-activated PDE6 [177], and it is now believed that this inhibitory action of GARP2 on activated PDE6 can be attributed to the fusion tag present on the recombinant