Phosphodiesterase 6 (PDE6) is highly concentrated in the retina. It is most abundant in the internal membranes of retinal photoreceptors, where it reduces cytoplasmic levels of cyclic guanosine monophosphate (cGMP) in rod and cone outer segments in response to light. The rod PDE6 holoenzyme comprises a and b catalytic subunits and two identical inhibitory c subunits. Each catalytic subunit contains three distinct globular domains corresponding to the catalytic domain and two GAF domains (responsible for allosteric cGMP binding). The PDE6 catalytic subunits resemble PDE5 in amino-acid sequence as well as in three-dimensional structure of the catalytic dimer; preference for cGMP over cyclic adenosine monophosphate (cAMP) as a substrate; and the ability to bind cGMP at the regulatory GAF domains. Most PDE5 inhibitors inhibit PDE6 with similar potency, and electroretinogram studies show modest effects of PDE5 inhibitors on visual function-an observation potentially important in designing PDE5-specific therapeutic agents.

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (␣␤) to which low molecular weight inhibitory ␥ subunits bind to form the nonactivated PDE holoenzyme (␣␤␥ 2 ). Although it is known that ␥ binds tightly to ␣␤, the binding affinity for each ␥ subunit to ␣␤, the domains on ␥ that interact with ␣␤, and the allosteric interactions between ␥ and the regulatory and catalytic regions on ␣␤ are not well understood. We show here that the ␥ subunit binds to two distinct sites on the catalytic ␣␤ dimer (K D1 < 1 pM, K D2 ‫؍‬ 3 pM) when the regulatory GAF domains of bovine rod PDE6 are occupied by cGMP. Binding heterogeneity of ␥ to ␣␤ is absent when cAMP occupies the noncatalytic sites. Two major domains on ␥ can interact independently with ␣␤ with the N-terminal half of ␥ binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of ␥ is responsible for the positive cooperativity between ␥ and cGMP binding sites on ␣␤ but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of ␥ that bind to ␣␤, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of ␥ interaction with ␣␤, and positive cooperativity of ␥ with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors.The extent and lifetime of activation of the photoreceptor cGMP PDE 1 (PDE6; EC 3.1.4.35) must be precisely regulated in rod and cone cells to control the exquisite sensitivity, speed, and adaptational properties of the visual transduction pathway in vertebrate photoreceptors. The membrane-associated rod photoreceptor PDE6 consists of a dimer of two homologous catalytic subunits (P␣␤) to which two low molecular weight inhibitory subunits (P␥) bind (holoenzyme stoichiometry, ␣␤␥ 2 ). The catalytic subunits contain GAF domains that are responsible for high affinity, noncatalytic binding of two cGMP molecules/holoenzyme. It is well established that relief of the inhibitory constraint on PDE6 arises from the binding of activated heterotrimeric G protein (transducin) to P␥ following photoactivation of the visual pigment, rhodopsin (reviewed in Refs. 1-4). However, the strength of the interaction between P␥ and P␣␤ has been difficult to quantitate, and K D values vary widely (from picomolar (5-7) up to nanomolar values (8, 9)). In addition, it has not been conclusively demonstrated whether both P␥ molecules bind with equal affinity to P␣␤ to form the nonactivated holoenzyme (although two different binding sites on P␣␤ have been inferred using mutant P␥ (10)). Finally, recent evidence suggests that binding of activated transducin to PDE6 relieves inhibition at only one of the two active sites, further supporting the idea of catalytic subunit heterogeneity with respect to ...

A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transducin lowers the cGMP binding afnit for noncatalytic sites on PDE and accelerates the disation of cGMP from these sites. These sites can exist in three states: gh affinity (Kd =60 nM) for the nonactivated PDE, intermediate affinity (Kd 180 nM) when the enzyme is activated in a complex with trasducin, and low aMnity (Kd > 1 FM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by htrnducin causes a 10-fold increase in the rate of cGMP discation from one of the two noncatalyc sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP disiation rate from both sites >50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of liht-adapted photoreceptors.The chain ofevents responsible for visual excitation in retinal rod photoreceptors is well documented (reviewed in refs. 1-4). Photoexcitation of rhodopsin results in activation of transducin, the heterotrimeric guanine nucleotide-binding protein (G protein) of photoreceptor cells, which then binds and activates its effector enzyme, cGMP phosphodiesterase (PDE). The acceleration of PDE activity results in a drop in the cytoplasmic concentration of cGMP, which causes the closure of cGMP-gated plasma membrane cationic channels and cell hyperpolarization. The visual excitation cascade is inactivated by several processes that serve to restore the dark-adapted condition. These events include phosphorylation of rhodopsin by rhodopsin kinase and binding of arrestin to phosphorylated rhodopsin, hydrolysis of GTP on transducin by its intrinsic GTPase activity, restoration of PDE inhibition by its y subunits, and resynthesis of cGMP by guanylate cyclase.Nonactivated rod PDE is a heterotetramer consisting of two similar a and 13 catalytic subunits and two identical fy subunits which serve as a protein inhibitor of the enzyme (reviewed in refs. 2, 5, and 6). Each of the a and 1 subunits contains one catalytic site for cGMP hydrolysis as well as one or, probably, two noncatalytic cGMP binding sites ( Fig. 1A; refs. 7-10). The inhibitory constraint of the PDE y subunits is released when activated transducin a subunit (a-GTP) binds to the PDE heterotetramer and either displaces PDE Y subunits or causes their physical removal from the PDE catalytic subunits (11)(12)(13)(14)(15)(16)...

Several classes of PDE inhibitors inhibit PDE6 equally as well as the PDE family to which they are targeted. In intact ROS, high PDE6 concentrations, binding of the gamma-subunit to the active site, and calcium feedback mechanisms attenuate the effectiveness of PDE inhibitors to inhibit PDE6 and disrupt the cGMP signaling pathway during visual transduction.

The visual transduction pathway in vertebrate photoreceptors transforms a light stimulus entering the photoreceptor outer segments into an electrical response at the synapses of rod and cone photoreceptor cells. This process is mediated by complex biochemical pathways that precisely regulate cGMP levels, thereby controlling the extent, duration, and adaptation of the photoreceptor to the light stimulus. This review first summarizes the major mechanisms of regulating cytoplasmic cGMP levels (synthesis, degradation, buffering, and efflux) as well as the primary targets of action of cGMP (cyclic nucleotide-gated ion channels, cGMP-dependent protein kinase, and cGMPregulated phosphodiesterases). This information is applied to our current understanding of how these processes operate in the signal-transducing outer segment of rod and cone photoreceptors to carry out visual excitation, recovery, and adaptation in response to light stimulation.