Rick H. Cote

Evaluation of the 17-kDa Prenyl-binding Protein as a Regulatory Protein for Phototransduction in Retinal Photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEδ) tightly bound. This protein, here termed PrBP/δ, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/δ in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/δ in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/δ was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/δ. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/δ can solubilize PDE6 and prevent its activation by transducin. PrBP/δ also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/δ content of purified ROS reveals insufficient amounts of PrBP/δ (<0.1 PrBP/δ per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/δ in frog and bovine retina shows greatest PrBP/δ immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/δ labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/δ as a PDE6 subunit and implicate PrBP/δ in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.

cGMP Binding to Noncatalytic Sites on Mammalian Rod Photoreceptor Phosphodiesterase Is Regulated by Binding of Its γ and δ Subunits

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, αβγ2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, αβγ2δ) can release and bind cGMP at both noncatalytic sites; the δ subunit is likely responsible for this difference in cGMP exchange rates. Removal of the δ and/or γ subunits yields a catalytic αβ dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (K D >1 μm); cGMP binding affinity at the second site (K D ∼60 nm) is reduced 3–4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of γ subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the γ subunit with the αβ dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.

The glutamic acid-rich protein-2 (GARP2) is a high affinity rod photoreceptor phosphodiesterase (PDE6)-binding protein that modulates its catalytic properties.

The glutamic acid-rich protein-2 (GARP2) is a splice variant of the β-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor “dark noise” and allowing these sensory cells to operate at the single photon detection limit.

Structural features of the noncatalytic cGMP binding sites of frog photoreceptor phosphodiesterase using cGMP analogs.

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE. None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2′-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2′ regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in theanti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.

Rod and cone opsin families differ in spectral tuning domains but not signal transducing domains as judged by saturated evolutionary trace analysis

The visual receptor of rods and cones is a covalent complex of the apoprotein, opsin, and the light-sensitive chromophore, 11-cis-retinal. This pigment must fulfill many functions including photoactivation, spectral tuning, signal transmission, inactivation, and chromophore regeneration. Rod and cone photoreceptors employ distinct families of opsins. Although it is well known that these opsin families provide unique ranges in spectral sensitivity, it is unclear whether the families have additional functional differences. In this study, we use evolutionary trace (ET) analysis of 188 vertebrate opsin sequences to identify functionally important sites in each opsin family. We demonstrate the following results. (1) The available vertebrate opsin sequences produce a definitive description of all five vertebrate opsin families. This is the first demonstration of sequence saturation prior to ET analysis, which we term saturated ET (SET). (2) The cone opsin classes have class-specific sites compared to the rod opsin class. These sites reside in the transmembrane region and tune the spectral sensitivity of each opsin class to its characteristic wavelength range. (3) The cytoplasmic loops, primarily responsible for signal transmission and inactivation, are essentially invariant in rod versus cone opsins. This indicates that the electrophysiological differences between rod and cone photoreceptors cannot be ascribed to differences in the protein interaction regions of the opsins. SET shows that chromophore binding and regeneration are the only aspects of opsin structure likely to have functionally significant differences between rods and cones, whereas excitatory and adaptational properties of the opsin families appear to be functionally invariant.

Regulation of Photoreceptor Phosphodiesterase (PDE6) by Phosphorylation of Its Inhibitory γ Subunit Re-evaluated

Phosphorylation of the inhibitory γ subunit (Pγ) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pγ phosphorylation for PDE6 regulation by preparing Pγ stoichiometrically phosphorylated at Thr22 or at Thr35. Phosphorylation of Pγ at either residue caused a minor decrease—not the previously reported increase—in the ability of Pγ to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pγ phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pγ subunits. The strength of Pγ interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pγ phosphorylation at Thr22 (but not Thr35), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527–27534). In contrast, the effects of Pγ phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pγ at either Thr22 or Thr35 greatly diminished its ability to bind activated transducin, consistent with earlier work. In situphosphorylation of Pγ by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only ∼10% of the endogenous Pγ was phosphorylated. This is attributed to Pγ being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pγ phosphorylation at either Thr22 or Thr35modestly weakens its direct interactions with PDE6. However, Pγ phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pγ available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802–37808) in photoreceptor cells.

Molecular architecture of photoreceptor phosphodiesterase elucidated by chemical cross-linking and integrative modeling

Photoreceptor phosphodiesterase (PDE6) is the central effector enzyme in visual excitation pathway in rod and cone photoreceptors. Its tight regulation is essential for the speed, sensitivity, recovery and adaptation of visual detection. Although major steps in the PDE6 activation/deactivation pathway have been identified, mechanistic understanding of PDE6 regulation is limited by the lack of knowledge about the molecular organization of the PDE6 holoenzyme (αβγγ). Here, we characterize the PDE6 holoenzyme by integrative structural determination of the PDE6 catalytic dimer (αβ), based primarily on chemical cross-linking and mass spectrometric analysis. Our models built from high-density cross-linking data elucidate a parallel organization of the two catalytic subunits, with juxtaposed α-helical segments within the tandem regulatory GAF domains to provide multiple sites for dimerization. The two catalytic domains exist in an open configuration when compared to the structure of PDE2 in the apo state. Detailed structural elements for differential binding of the γ-subunit to the GAFa domains of the α- and β-subunits are revealed, providing insight into the regulation of the PDE6 activation/deactivation cycle.

Identification of Amino Acid Residues Responsible for the Selectivity of Tadalafil Binding to Two Closely Related Phosphodiesterases, PDE5 and PDE6

Background: Most PDE5-selective inhibitors also potently inhibit photoreceptor PDE6. Results: Evolutionary trace analysis predicted amino acids responsible for the selectivity of tadalafil binding to the PDE6 catalytic site without altering vardenafil binding. Conclusion: A limited number of amino acid residues account for drug selectivity of PDE inhibitors. Significance: This work will help identify more selective PDE5 inhibitors lacking adverse side effects on vision. The 11 families of the Class I cyclic nucleotide phosphodiesterases (PDEs) are critical for regulation of cyclic nucleotide signaling. PDE5 (important in regulating vascular smooth muscle contraction) and PDE6 (responsible for regulating visual transduction in vertebrate photoreceptors) are structurally similar but have several functional differences whose structural basis is poorly understood. Using evolutionary trace analysis and structural homology modeling in conjunction with site-directed mutagenesis, we have tested the hypothesis that class-specific differences between PDE5 and PDE6 account for the biochemical and pharmacological differences in the two enzyme families. Replacing human PDE5 residues in the M-loop region of the binding site for the PDE5-selective inhibitor tadalafil (Cialis®) with the corresponding class-specific cone PDE6 residues (P773E, I778V, E780L, F787W, E796V, D803P, L804M, N806D, I813L, S815K) reduces tadalafil binding affinity to levels characteristic of PDE6. These mutations fail to alter vardenafil (Levitra®) affinity for the active site. Class-specific differences in PDE5 versus cone PDE6 that contribute to the accelerated catalytic efficiency of PDE6 were identified but required heterologous expression of full-length PDE5 constructs. Introduction of PDE6 residues into the background of the PDE5 protein sequence often led to loss of catalytic activity and reduced protein solubility, supporting the idea that multiple structural elements of PDE6 are highly susceptible to misfolding during heterologous expression. This work validates the use of PDE5 as a template to identify functional differences between PDE5 and PDE6 that will accelerate efforts to develop the next generation of PDE5-selective inhibitors with fewer adverse side effects resulting from PDE6 inhibition.

Purification of PDE6 Isozymes From Mammalian Retina

The photoreceptor phosphodiesterase (PDE6) is the central effector of visual transduction in vertebrate retinal photoreceptors. Distinct isozymes of PDE6 exist in rods and cones. Mammalian retina serves as an abundant source of tissue for PDE6 purification. Methods are described for the isolation and purification of membrane-associated PDE6 from rod outer segment membranes. Purification of cone PDE6 from the soluble fraction of retinal extracts is also described. Several procedures that can purify the rod and cone isozymes to homogeneity, including anion exchange, hydrophobic interaction, gel filtration, hydroxyapatite, and immunoaffinity chromatography, are presented. A method to activate PDE6 by limited proteolysis of its inhibitory gamma-subunit is also provided.

Assay and Functional Properties of PrBP(PDEδ), a Prenyl‐Binding Protein Interacting with Multiple Partners

A 17-kDa prenyl-binding protein, PrBP(PDEdelta), is highly conserved among various species from human to Caenorhabditis elegans. First identified as a putative regulatory delta subunit of the cyclic nucleotide phosphodiesterase (PDE6) purified from mammalian photoreceptor cells, PrBP(PDEdelta) has been hypothesized to reduce activation of PDE6 by the heterotrimeric G-protein, transducin, thereby desensitizing the photoresponse. However, recent work shows that PrBP(PDEdelta) interacts with numerous prenylated proteins at their farnesylated or geranylgeranylated C-termini, as well as with non-prenylated proteins. These polypeptides include small GTPases such as Rab13, Ras, Rap, and Rho6, as well as components involved in phototransduction (e.g., rod and cone PDE6, rod and cone opsin kinases). Expression of PrBP(PDEdelta) in tissues and organisms not expressing PDE6, the demonstration of multiple interacting partners with PrBP(PDEdelta), and its low abundance in rod outer segments all argue against it being a regulatory PDE6 subunit. This raises intriguing questions as to its physiological functions. In this chapter, we review the current status of PrBP(PDEdelta) and describe some of the assays used to determine these interactions in detail. In mammalian photoreceptors, the results are consistent with a role of PrBP(PDEdelta) in the transport of prenylated proteins from their site of synthesis in the inner segment to the outer segment where phototransduction occurs.