Other mechanisms for inactivating PDE6 independent of the hydrolysis of GTP by transducin α-subunit have been proposed [69–71]. For example, free γ-subunit (i.e., not associated with transducin or PDE6) has been shown to reinhibit transducin-activated PDE6 in vitro even though the transducin α-subunit remains persistently activated [72, 73]. Using a transgenic animal overexpressing the γ-subunit further supports the idea that the free γ-subunit is able to bind to and inhibit transducin-activated PDE6 [71], but the physiological significance of this is unclear since the concentration of γ-subunit in rod outer segments is equal to the concentration of PDE6 catalytic subunits, and all γ-subunit is found membrane associated [73]. Other potential regulatory mechanisms for PDE6 involving novel binding proteins are considered in the section Potential PDE6 Regulatory Binding Proteins.

Activation of GC

Rapid restoration of cGMP levels following PDE6 deactivation requires calciumdependent activation of photoreceptor GCs by their GCAPs. When the cytoplasmic concentration drops below about 500 nM, calcium dissociates from the GCAPs, relieving inhibition of GC and accelerating cGMP synthesis [4, 6, 7]. The powerful calcium feedback mechanism involving GCAP-GC regulation of cGMP synthesis helps determine the amplitude and temporal characteristics of the photoresponse and regulates the cGMP metabolic flux that is modulated during light adaptation [54].

Regulation of the CNG Ion Channel

In rod photoreceptors, calcium/calmodulin binding to the β-subunit of the CNG channel serves to decrease the cGMP sensitivity of the channel [74]. In cone photoreceptors, modulation of cGMP sensitivity by calcium is greater than in rods, but the identity of calcium regulatory protein is not certain [75, 76].

PHOTORECEPTOR PDE (PDE6) STRUCTURE AND FUNCTION

The Cyclic Nucleotide Phosphodiesterase Superfamily

The photoreceptor PDE6 enzyme found in retinal rods and cones is a member of the class I PDE superfamily, of which 11 distinct gene families exist in vertebrates (Fig. 2). Class I PDEs all contain the characteristic PDEase I catalytic domain. The 11 PDE families are readily distinguished by comparison of their primary amino acid sequences as well as by their regulatory mechanisms, substrate preference, pharmacological inhibitor specificity, and expression patterns [8]. Three PDE families (PDE5, PDE6, and PDE9) strongly prefer cGMP as the substrate, three are cAMP specific (PDE4, PDE7, PDE8), and the rest do not discriminate between the two substrates. Five PDE families (PDE2, PDE5, PDE6, PDE10, PDE11) contain two tandem GAF domains in their N-terminal regulatory domain, which, in the case of PDE6, bind cGMP with high affinity [77]. Other PDE families are subject to regulation by calcium/calmodulin, phosphorylation, and extrinsic regulatory proteins [8]. The photoreceptor PDE6 is most closely related to PDE5 in its amino acid sequence, substrate preference, and pharmacological profile but differs in its mechanism of regulation [9].

Fig. 2. The superfamily of cyclic nucleotide phosphodiesterases (PDEs). In humans, there are 21 PDE genes that are grouped into 11 different families, PDE1 though PDE11. Many of the PDE families exist as multiple splice variants (not shown), although this is not the case for PDE6. All class I PDEs share a highly homologous catalytic domain (PDEase I) in the C-terminal region of the linear sequence. The N-terminal half contains regulatory elements and targeting elements (defined in the box). The PDE6 family is unique in having a C-terminal membrane-targeting domain and in being directly regulated by an inhibitory γ-subunit (not shown). Abbreviations: PAS, Per-Arnt-Sim; GAF, see text; Cam, Calmodulin

Subunit Composition of Rod and Cone PDE6 Holoenzyme

Although all class I PDEs are believed to exist as a dimer of two catalytic subunits, rod PDE6 is the only one in which the catalytic dimer consists of two nonidentical α (PDE6A) and β (PDE6B) subunits [78–81]. In contrast, cone PDE6 is a homodimeric enzyme composed of α′ (PDE6C) subunits [82, 83]. A second distinctive trait of PDE6 (this one shared by rod and cone isoforms) is the association of high-affinity inhibitory rod or cone γ-subunits to the corresponding catalytic dimer [84, 85]. The rod PDE6 holoenzyme (Fig. 3A) has been conclusively shown to be composed of two γ-subunits bound to the rod αβ-dimer [80, 86], and the cone enzyme is also assumed to be a heterotetramer.

Catalytic Subunit

The primary sequence of the α, β, and α′ catalytic subunits of PDE6 [87–89] consist of an N-terminal region of unknown function, two regulatory GAF domains (GAFa and GAFb) arranged in tandem, the catalytic domain, and a C-terminal motif that is subject to isoprenylation (Fig. 3B).

Regulatory GAF Domain

The tandem GAF domains (named for their occurrence in cGMP binding PDEs, certain adenylate cyclases, and the Escherichia coli FhlA protein [77, 90]) serve several

Fig. 3. Domain organization of the catalytic and inhibitory subunits of phosphodiesterase 6 (PDE6). A The rod PDE6 catalytic dimer (left) consists of nonidentical α- and β-subunits that are farnesylated (α-subunit) or geranylgeranylated (β-subunit). The enzyme-active site is denoted by the notch, while the cGMP-binding site is indicated by a circular pocket. Right: The γ-subunit binds to both the active site (to inhibit catalysis) and to the GAF domains (to stabilize cyclic guanosine monophosphate [cGMP] binding). B The ~100-kDa catalytic subunit of PDE6 consists of two tandem GAF domains, a catalytic domain, and a prenylated C-terminus. C The 10-kDa γ-subunit has several functional domains: a proline-rich (Pro-rich) region and a polycationic region (PC region) that both interact with the cGMP-binding site on PDE6, an α-helical region, and the C-terminal residues (CT), which bind to the active site of PDE6. Activated transducin α-subunit also interacts with the γ-subunit at both its PC region and the α-helical region.

important functions. First, the GAF domains of PDE6 contain nucleotide-binding pockets (distinct from the enzyme’s active sites) that bind cGMP with high affinity [91]. The PDE6 GAF domains also serve a function unique to the PDE6 family, namely, to bind the inhibitory γ-subunit in a cGMP-dependent manner. Finally, the GAF domains of PDE6 are responsible for catalytic subunit dimerization [92, 93].

Although the rod PDE6 holoenzyme (αβγ2) has two GAF domains per catalytic subunit, rod PDE6 binds only two cGMP molecules with high affinity [94–96]. A second class of low-affinity cGMP-binding sites found in rod outer segments [97] might also represent cGMP binding to the remaining PDE6 GAF domains of the catalytic dimer, but this has not been experimentally verified to date. The high-affinity cGMP-binding sites on PDE6 catalytic subunits discriminate cGMP over cAMP by about 106-fold [98, 99]. The cGMP-binding site in PDE6 has been localized to the GAFa domain in rod and cone

isoforms, and several amino acid residues important for high-affinity interactions with cGMP have been defined [99, 100].

The GAFa domain is also a major binding site for the inhibitory γ-subunit of PDE6, as judged by cross-linking and site-directed mutagenesis work [100–102]. Differences in the binding of the γ-subunit to the α- and β-subunit reveal potential structural differences between α-GAFa and β-GAFa that may account for heterogeneity in cGMP or γ-subunit affinity for the holoenzyme [102]. The proximity of γ-subunit interacting sites with the cGMP-binding pocket in GAFa provides structural support for the positive cooperativity between cGMP and γ-subunit binding (discussed in section “Functions of the Regulatory cGMP-binding GAF domains of PDE6”).

The GAFa domains of rod and cone PDE6 catalytic subunits serve as the primary dimerization domain between two individual subunits. This is evident in the structural model of the PDE6 holoenzyme [103]. The N-terminal region of the GAFa was shown by a mutagenesis study to be essential for dimerization of rod PDE6 [92]; these studies also support the view that rod PDE6 exists primarily or exclusively as αβ heterodimers, consistent with earlier biochemical evidence [81].

Catalytic Domain

The PDE6 catalytic domain contains the same invariant catalytic site residues that typify all class I PDEs, referred to as PDEase_I in the Conserved Domain Database (CDD) at the National Center for Biotechnology Information (NCBI). Multiple-sequence alignment of the catalytic domains of three human rod and cone PDE6 genes and the PDE5 gene reveals 84% amino acid identity between the PDE6A and PDE6B rod subunit isoforms, about 75% amino acid identity between rod and cone PDE6C subunits, and 43% identity between PDE5 and PDE6. Because of the difficulty in expressing functional PDE6 catalytic subunits in heterologous systems [104–108], site-directed mutagenesis of PDE6 has not been feasible. Instead, progress in understanding the structure and function of the PDE6 catalytic domain has relied on constructing chimeric proteins containing both PDE5 and PDE6 sequences in conjunction with structural homology modeling with known catalytic domain crystal structures [105]. This approach has identified some of the important residues responsible for the very high turnover number (kcat) of PDE6 compared to PDE5 [108, 109]. The histidine residues responsible for binding divalent cations in the active site of PDE5 [110, 111] are present in PDE6 and bind zinc with high affinity and magnesium with lower affinity [112]. These divalent cations are not only critical for the catalytic mechanism but also confer structural stability to the enzyme.

As mentioned, the nonactivated PDE6 enzyme is inhibited by direct binding of its inhibitory γ-subunit. The γ-subunit exerts its inhibitory action by directly binding to the catalytic pocket, thereby blocking access of cGMP to its binding site [113, 114]. PDE5/6 chimeras have also proved useful in identifying specific γ-interacting amino acid residues (PDE6 α′: M758, Q752, F777, and F781) within the M loop of the catalytic domain structure [108, 109, 115].

C-Terminal Prenylation

Although several PDE families contain signaling motifs that target the enzyme to the membrane, PDE6 is the only vertebrate PDE family that is either farnesylated (PDE6A) or geranylgeranylated (PDE6B and, probably, PDE6C) at a C-terminal CAAX (C = cysteine,

A = aliphatic, X = any amino acid) motif [116, 117]. The prenylated, carboxymethylated C-termini are responsible for anchoring PDE6 to the outer segment disk membrane [118], thereby facilitating two-dimensional collisions with transducin during visual excitation. PDE6 is membrane bound, except when the 17-kDa PrBP/δ protein (originally termed PDEδ) is present to bind to the prenyl groups and solubilize PDE6 from the membrane (see section “Potential PDE6 regulatory binding proteins” for discussion). Disk membrane association of PDE6 can be disrupted in vitro by altering the ionic, nucleotide, divalent cation, or illumination conditions, which is useful for purification of the enzyme [119].

Inhibitory γ-Subunit

The γ-subunit of rod and cone PDE6 [120, 121] serves a remarkable number of functions considering its small size (~10 kDa). Most likely, the γ-subunit exists in solution as an unfolded or intrinsically disordered protein due to its minimal secondary structure [122, 123]. This may account for the ability of this small protein to span the distance from the GAFa domain to the active site on the catalytic domain of the catalytic dimer [102]. Important regions of the γ-subunit include the following:

1.A proline-rich region (amino acids [a.a.] 22–28 of the rod sequence) is a potential site of interaction with proteins containing SH3 (src homology3) domains [124] as well as a site for protein phosphorylation by proline-directed kinases [125–127].

2.The polycationic region (a.a. 29–45) is a major site of interaction with the transducin α-subunit during light activation of PDE6 [128–133], as well as a substrate for phosphorylation by serine/threonine kinases [127, 134, 135] and adenosine diphosphate (ADP) ribosylation at two arginine residues [136, 137].

3.The combined proline-rich and polycationic regions of the γ-subunit is the strongest site of interaction with the PDE6 catalytic dimer [102, 128, 138–142] and enhances the binding of cGMP to the regulatory GAF domains [142, 143].

4.The region between the polycationic and α-helical segments of the γ-subunit contains a site of interaction (a.a. 66) with RGS9-1 [66], which serves to accelerate the GTPase activity of activated transducin [61, 64, 66].

5.An α-helical region of the γ-subunit (a.a. 62–83) is a major site of interaction with activated transducin α-subunit and is primarily responsible for the GTPase accelerating activity of the γ-subunit [66, 144–150]. In addition, amino acids F73, N74, H75, and L78 of the γ-subunit interact with the PDE6 catalytic subunits [102, 150], even though the α-helical region itself does not directly block the active site. It is thus likely that transducin binds to one face of the γ-subunit α-helical region and disrupts contacts between the opposite face of the α-helical domain and the catalytic domain of PDE6.

6.The last five C-terminal residues (a.a. 83–87) directly bind to the active site to cover the catalytic pocket and block cyclic nucleotide entry [113–115, 128, 131, 146].

It must also be emphasized that the γ-subunit interacts differently with the α- and β-subunits of rod PDE6, as judged by both biochemical and structural evidence [73, 96, 102, 142, 151]. Because the GAFa domains of the α- and β-subunits of rod PDE6 are more dissimilar than the GAFb or catalytic domains, it is possible that differences in γ-subunit binding affinity or functional properties may result from differences in interactions of γ with the GAFa domains.