Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

Abstract

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.

Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

“…However, the chimeric PDE5/6 catalytic domain contains most of the PDE6 residues that interact with the C-terminal domain of PDE␥. In addition, PDE␥ 63-87 inhibits PDE5/6 as potently as full-length PDE␥ with a K i of 1.7 M, comparable with that for PDE6 from bovine retina (18,31). Thus the PDE5/6 construct recapitulates the contacts between PDE6 and the PDE␥ C-terminal domain and offers a model for the native PDE␥/PDE6 interaction.…”

“…These conformations were calculated from a large set of long-range structural constraints generated by introducing a paramagnetic spin-label, 3-maleimido-PROXYL (mPROXYL), into each of 10 single cysteine-containing PDE␥ variants. We also used NMR spectroscopy to investigate the interaction between the PDE␥ C-terminal fragment (PDE␥ ) and the PDE5/6 chimeric catalytic domain, as a mimic of the PDE6 catalytic core (18). Our results demonstrate that the full-length PDE␥ contains transiently populated structural elements that provide insight into the regulatory mechanism of PDE␥ in the signaling cascade of phototransduction.…”

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

“…PDE Activity Assay and Data Analysis-PDE activity was measured using 5 M [ 3 H]cGMP and 1 pM PDE6 or 200 M [ 3 H]cGMP and 20 pM PDE6 as described (15,25). To determine K m values for cGMP, PDE activity was measured using 10 -1000 M cGMP, and the data were fit to the equation, Y ϭ V max ⅐X/(K m ϩ X).…”

“…The problem of PDE6 expression was partially addressed through generation and characterization of chimeras between PDE6 and PDE5 (cGMP-binding, cGMP-specific PDE) (12)(13)(14)(15). The two PDE enzymes share similar domain organization, a relatively high homology of the catalytic domains, specificity for cGMP relative to cAMP, and sensitivity to common catalytic site inhibitors (1,3).…”

“…The two PDE enzymes share similar domain organization, a relatively high homology of the catalytic domains, specificity for cGMP relative to cAMP, and sensitivity to common catalytic site inhibitors (1,3). The chimeric PDE5/PDE6 approach facilitated delineation of the P␥-binding sites of PDE6, but it has severe limitations in identification of the unique catalytic determinants of the visual effector (15). The reason for the inability of PDE6 to fold correctly in various cell types, besides photoreceptor cells, is unknown.…”