Reconstitution of the phosphodiesterase 6 maturation process important for photoreceptor cell function.

The sixth family phosphodiesterases (PDE6) are principal effector enzymes of the phototransduction cascade in rods and cones. Maturation of nascent PDE6 protein into a functional enzyme relies on a coordinated action of ubiquitous chaperone HSP90, its specialized cochaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and the regulatory Pγ-subunit of PDE6. Deficits in PDE6 maturation and function underlie severe visual disorders and blindness. Here, to elucidate the roles of HSP90, AIPL1, and Pγ in the maturation process, we developed the heterologous expression system of human cone PDE6C in insect cells allowing characterization of the purified enzyme. We demonstrate that in the absence of Pγ, HSP90, and AIPL1 convert the inactive and aggregating PDE6C species into dimeric PDE6C that is predominantly misassembled. Nonetheless, a small fraction of PDE6C is properly assembled and fully functional. From the analysis of mutant mice that lack both rod Pγ and PDE6C, we conclude that, in contrast to the cone enzyme, no maturation of rod PDE6AB occurs in the absence of Pγ. Co-expression of PDE6C with AIPL1 and Pγ in insect cells leads to a fully mature enzyme that is equivalent to retinal PDE6. Lastly, using immature PDE6C and purified chaperone components, we reconstituted the process of the client maturation in vitro. Based on this analysis we propose a scheme for the PDE6 maturation process.

Reproducibility of the Rod Photoreceptor Response Depends Critically on the Concentration of the Phosphodiesterase Effector Enzyme.

The high sensitivity of night vision requires that rod photoreceptors reliably and reproducibly signal the absorption of single photons, a process that depends on tight regulation of intracellular cGMP concentration through the phototransduction cascade. Here in the mouse (Mus musculus), we studied a single-site D167A mutation of the gene for the α subunit of rod photoreceptor phosphodiesterase (PDEA), made with the aim of removing a noncatalytic binding site for cGMP. This mutation unexpectedly eliminated nearly all PDEA expression and reduced expression of the ß subunit (PDEB) to ∼5%-10% of WT. The remaining PDE had nearly normal specific activity; degeneration was slow, with 50%-60% of rods remaining after 6 months. Responses were larger and more sensitive than normal but slower in rise and decay, probably from slower dark turnover of cGMP. Remarkably, responses became much less reproducible than WT, with response variance increasing for amplitude by over 10-fold, and for latency and time-to-peak by >100-fold. We hypothesize that the increase in variance is the result of greater variability in the dark-resting concentration of cGMP, produced by spatial and temporal nonuniformity in spontaneous PDE activity. This variability decreased as stimuli were made brighter, presumably because of greater spatial uniformity of phototransduction and the approach to saturation. We conclude that the constancy of the rod response depends critically on PDE expression to maintain adequate spontaneous PDE activity, so that the concentration of second messenger is relatively uniform throughout the outer segment.SIGNIFICANCE STATEMENT Rod photoreceptors in the vertebrate retina reliably signal the absorption of single photons of light by generating responses that are remarkably reproducible in amplitude and waveform. We show that this reproducibility depends critically on the concentration of the effector enzyme phosphodiesterase (PDE), which metabolizes the second messenger cGMP and generates rod light responses. In rods with the D167A mutation of the α subunit of PDE, only 5%-10% of PDE is expressed. Single-photon responses then become much more variable than in WT rods. We think this variability is caused by spatial and temporal inhomogeneity in the concentration of cGMP in darkness, so that photons absorbed in different parts of the cell produce responses of greatly varying amplitude and waveform.

Molecular insights into the maturation of phosphodiesterase 6 by the specialized chaperone complex of HSP90 with AIPL1.

Phosphodiesterase 6 (PDE6) is a key effector enzyme in vertebrate phototransduction, and its maturation and function are known to critically depend on a specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Defects in PDE6 and AIPL1 underlie several severe retinal diseases, including retinitis pigmentosa and Leber congenital amaurosis. Here, we characterize the complex of AIPL1 with HSP90 and demonstrate its essential role in promoting the functional conformation of nascent PDE6. Our analysis suggests that AIPL1 preferentially binds to HSP90 in the closed state with a stoichiometry of 1:2, with the tetratricopeptide repeat domain and the tetratricopeptide repeat helix 7 extension of AIPL1 being the main contributors to the AIPL1/HSP90 interface. We demonstrate that mutations of these determinants markedly diminished both the affinity of AIPL1 for HSP90 and the ability of AIPL1 to cochaperone the maturation of PDE6 in a heterologous expression system. In addition, the FK506-binding protein (FKBP) domain of AIPL1 encloses a unique prenyl-binding site that anchors AIPL1 to posttranslational lipid modifications of PDE6. A mouse model with rod PDE6 lacking farnesylation of its PDE6A subunit revealed normal expression, trafficking, and signaling of the enzyme. Furthermore, AIPL1 was unexpectedly capable of inducing the maturation of unprenylated cone PDE6C, whereas mutant AIPL1 deficient in prenyl binding competently cochaperoned prenylated PDE6C. Thus, we conclude neither sequestration of the prenyl modifications is required for PDE6 maturation to proceed, nor is the FKBP-lipid interaction involved in the conformational switch of the enzyme into the functional state.

A Novel Role for UNC119 as an Enhancer of Synaptic Transmission.

Mammalian UNC119 is a ciliary trafficking chaperone highly expressed in the inner segment of retinal photoreceptors. Previous research has shown that UNC119 can bind to transducin, the synaptic ribbon protein RIBEYE, and the calcium-binding protein CaBP4, suggesting that UNC119 may have a role in synaptic transmission. We made patch-clamp recordings from retinal slices in mice with the UNC119 gene deleted and showed that removal of even one gene of UNC119 has no effect on the rod outer segment photocurrent, but acted on bipolar cells much like background light: it depolarized membrane potential, decreased sensitivity, accelerated response decay, and decreased the Hill coefficient of the response-intensity relationship. Similar effects were seen on rod bipolar-cell current and voltage responses, and after exposure to bright light to translocate transducin into the rod inner segment. These findings indicate that UNC119 deletion reduces the steady-state glutamate release rate at rod synapses, though no change in the voltage dependence of the synaptic Ca current was detected. We conclude that UNC119, either by itself or together with transducin, can facilitate the release of glutamate at rod synapses, probably by some interaction with RIBEYE or other synaptic proteins rather than by binding to CaBP4 or calcium channels.

Ric-8A, a GEF, and a Chaperone for G Protein α-Subunits: Evidence for the Two-Faced Interface.

Resistance to inhibitors of cholinesterase 8A (Ric-8A) is a prominent non-receptor GEF and a chaperone of G protein α-subunits (Gα). Recent studies shed light on the structure of Ric-8A, providing insights into the mechanisms underlying its interaction with Gα. Ric-8A is composed of a core armadillo-like domain and a flexible C-terminal tail. Interaction of a conserved concave surface of its core domain with the Gα C-terminus appears to mediate formation of the initial Ric-8A/GαGDP intermediate, followed by the formation of a stable nucleotide-free complex. The latter event involves a large-scale dislocation of the Gα α5-helix that produces an extensive primary interface and disrupts the nucleotide-binding site of Gα. The distal portion of the C-terminal tail of Ric-8A forms a smaller secondary interface, which ostensibly binds the switch II region of Gα, facilitating binding of GTP. The two-site Gα interface of Ric-8A is distinct from that of GPCRs, and might have evolved to support the chaperone function of Ric-8A.

Large-scale conformational rearrangement of the α5-helix of Gα subunits in complex with the guanine nucleotide exchange factor Ric8A.

Resistance to inhibitors of cholinesterase 8A (Ric8A) protein is an important G protein-coupled receptor (GPCR)-independent regulator of G protein α-subunits (Gα), acting as a guanine nucleotide exchange factor (GEF) and a chaperone. Insights into the complex between Ric8A and Gα hold the key to understanding the mechanisms underlying noncanonical activation of G-protein signaling as well as the folding of nascent Gα proteins. Here, we examined the structure of the complex of Ric8A with minimized Gαi (miniGαi) in solution by small-angle X-ray scattering (SAXS) and exploited the scattering profile in modeling of the Ric8A/miniGαi complex by steered molecular dynamics (SMD) simulations. A small set of models of the complex featured minimal clash scores, excellent agreement with the experimental SAXS data, and a large-scale rearrangement of the signal-transducing α5-helix of Gα away from its ß-sheet core. The resulting interface involved the Gα α5-helix bound to the concave surface of Ric8A and the Gα ß-sheet that wraps around the C-terminal part of the Ric8A armadillo domain, leading to a severe disruption of the GDP-binding site. Further modeling of the flexible C-terminal tail of Ric8A indicated that it interacts with the effector surface of Gα. This smaller interface may enable the Ric8A-bound Gα to interact with GTP. The two-interface interaction with Gα described here distinguishes Ric8A from GPCRs and non-GPCR regulators of G-protein signaling.

Interaction of the tetratricopeptide repeat domain of aryl hydrocarbon receptor-interacting protein-like 1 with the regulatory Pγ subunit of phosphodiesterase 6.

Phosphodiesterase-6 (PDE6) is key to both phototransduction and health of rods and cones. Proper folding of PDE6 relies on the chaperone activity of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), and mutations in both PDE6 and AIPL1 can cause a severe form of blindness. Although AIPL1 and PDE6 are known to interact via the FK506-binding protein domain of AIPL1, the contribution of the tetratricopeptide repeat (TPR) domain of AIPL1 to its chaperone function is poorly understood. Here, we demonstrate that AIPL1-TPR interacts specifically with the regulatory Pγ subunit of PDE6. Use of NMR chemical shift perturbation (CSP) mapping technique revealed the interface between the C-terminal portion of Pγ and AIPL1-TPR. Our solution of the crystal structure of the AIPL1-TPR domain provided additional information, which together with the CSP data enabled us to generate a model of this interface. Biochemical analysis of chimeric AIPL1-AIP proteins supported this model and also revealed a correlation between the affinity of AIPL1-TPR for Pγ and the ability of Pγ to potentiate the chaperone activity of AIPL1. Based on these results, we present a model of the larger AIPL1-PDE6 complex. This supports the importance of simultaneous interactions of AIPL1-FK506-binding protein with the prenyl moieties of PDE6 and AIPL1-TPR with the Pγ subunit during the folding and/or assembly of PDE6. This study sheds new light on the versatility of TPR domains in protein folding by describing a novel TPR-protein binding partner, Pγ, and revealing that this subunit imparts AIPL1 selectivity for its client.

Unique structural features of the AIPL1-FKBP domain that support prenyl lipid binding and underlie protein malfunction in blindness.

FKBP-domain proteins (FKBPs) are pivotal modulators of cellular signaling, protein folding, and gene transcription. Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a distinctive member of the FKBP superfamily in terms of its biochemical properties, and it plays an important biological role as a chaperone of phosphodiesterase 6 (PDE6), an effector enzyme of the visual transduction cascade. Malfunction of mutant AIPL1 proteins triggers a severe form of Leber congenital amaurosis and leads to blindness. The mechanism underlying the chaperone activity of AIPL1 is largely unknown, but involves the binding of isoprenyl groups on PDE6 to the FKBP domain of AIPL1. We solved the crystal structures of the AIPL1-FKBP domain and its pathogenic mutant V71F, both in the apo form and in complex with isoprenyl moieties. These structures reveal a module for lipid binding that is unparalleled within the FKBP superfamily. The prenyl binding is enabled by a unique "loop-out" conformation of the ß4-α1 loop and a conformational "flip-out" switch of the key W72 residue. A second major conformation of apo AIPL1-FKBP was identified by NMR studies. This conformation, wherein W72 flips into the ligand-binding pocket and renders the protein incapable of prenyl binding, is supported by molecular dynamics simulations and appears to underlie the pathogenicity of the V71F mutant. Our findings offer critical insights into the mechanisms that underlie AIPL1 function in health and disease, and highlight the structural and functional diversity of the FKBPs.

α2δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses.

α2δ-4 is an auxiliary subunit of voltage-gated Cav1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the CACNA2D4 gene encoding α2δ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of α2δ-4, we used genome editing to generate an α2δ-4 knock-out (α2δ-4 KO) mouse. In male and female α2δ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in α2δ-4 KO retina. In reconstructions of α2δ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in α2δ-4 KO mice correlates with a progressive loss of Cav1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in α2δ-4 KO mice and better under photopic than scotopic conditions. We conclude that α2δ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with CACNA2D4 mutations.SIGNIFICANCE STATEMENT In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for α2δ-4, a subunit of voltage-gated Ca2+ channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca2+ channels are progressively lost and that rod and cone synapses are disrupted in mice that lack α2δ-4. Our results suggest that alterations in presynaptic Ca2+ signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the CACNA2D4 gene encoding α2δ-4.

The PDE6 mutation in the rd10 retinal degeneration mouse model causes protein mislocalization and instability and promotes cell death through increased ion influx.

The retinal degeneration model rd10 contains a missense mutation of the catalytic PDE6 ß subunit, which hydrolyzes cGMP in response to light. This model produces cell death more slowly than others caused by PDE6 loss of function, making it of particular interest for studying potential therapeutics. We used morphology, biochemistry, and single-cell physiology to examine the mechanism of rd10 degeneration. Our results show that the mutation produces no alteration of Pde6b RNA but does dramatically decrease maximal and basal PDE6 activity, apparently caused by a decrease in protein stability and transport. The enzymatic properties of the remaining mutant PDE6 appear to be nearly normal. We demonstrate that an increase in free cGMP, which would result from decreased PDE6 activity and serve to increase opening of the cGMP-gated channels and calcium influx, is an underlying cause of cell death: degeneration of rd10/Cngb1-/- double mutants is slower than the parent rd10 line. Paradoxically, degeneration in rd10/Cngb1-/- is also slower than in Cngb1-/- This rescue is correlated with a lowering of cGMP content in Cngb1-/- retinas and suggests that it may be caused by mislocalization of active PDE6. Single-cell recordings from rd10 rods show that the rates of rise and decay of the response are significantly slower; simulations indicate that these changes are primarily the result of the decrease in PDE6 concentration and rod collecting area. Together, these results provide insights into the complex mechanisms that underlie rd10-mediated retinal degeneration and a cautionary note for analysis of therapeutic interventions.

Aryl Hydrocarbon Receptor-interacting Protein-like 1 Is an Obligate Chaperone of Phosphodiesterase 6 and Is Assisted by the γ-Subunit of Its Client.

Phosphodiesterase 6 (PDE6) is the effector enzyme in the phototransduction cascade and is critical for the health of both rod and cone photoreceptors. Its dysfunction, caused by mutations in either the enzyme itself or AIPL1 (aryl hydrocarbon receptor-interacting protein-like 1), leads to retinal diseases culminating in blindness. Progress in research on PDE6 and AIPL1 has been severely hampered by failure to express functional PDE6 in a heterologous expression system. Here, we demonstrated that AIPL1 is an obligate chaperone of PDE6 and that it enables low yield functional folding of cone PDE6C in cultured cells. We further show that the AIPL1-mediated production of folded PDE6C is markedly elevated in the presence of the inhibitory Pγ-subunit of PDE6. As illustrated in this study, a simple and sensitive system in which AIPL1 and Pγ are co-expressed with PDE6 represents an effective tool for probing structure-function relationships of AIPL1 and reliably establishing the pathogenicity of its variants.

Exchange of Cone for Rod Phosphodiesterase 6 Catalytic Subunits in Rod Photoreceptors Mimics in Part Features of Light Adaptation.

Despite the expression of homologous phototransduction components, the molecular basis for differences in light-evoked responses between rod and cone photoreceptors remains unclear. We examined the role of cGMP phosphodiesterase (PDE6) in this difference by expressing cone PDE6 (PDE6C) in rd1/rd1 rods lacking rod PDE6 (PDE6AB) using transgenic mice. The expression of PDE6C rescues retinal degeneration observed in rd1/rd1 rods. Double-transgenic rods (PDE6C++) were compared with rd1/+ rods based on similar PDE6 expression. PDE6C increased the basal PDE activity and speeded the rate-limiting step for phototransduction deactivation, causing rod photoresponses to appear light adapted, with reduced dark current and sensitivity and faster response kinetics. When PDE6C++ and rd1/+ rods were exposed to similar background light, rd1/+ rods displayed greater desensitization. These results indicate an increased spontaneous activity and faster deactivation of PDE6C compared with PDE6AB in darkness, but that background light increases steady PDE6C activity to a lesser extent. In addition to accelerating the recovery of the photoresponse, faster PDE6C deactivation may blunt the rise in background-induced steady PDE6C activity. Therefore, higher basal PDE6C activity and faster deactivation together partially account for faster and less sensitive cone photoresponses in darkness, whereas a reduced rise of steady PDE6C activity in background light may allow cones to avoid saturation. SIGNIFICANCE STATEMENT: Cones are the primary photoreceptors responsible for most of our visual experience. Cone light responses are less sensitive and display speeded responses compared with rods. Despite the fact that rods and cones use a G-protein signaling cascade with similar organization, the mechanistic basis for these differences remains unclear. Here, we examined the role of distinct isoforms of PDE6, the effector enzyme in phototransduction, in these differences. We developed a transgenic mouse model that expresses cone PDE6 in rods and show that the cone PDE6 isoform is partially responsible for the difference in sensitivity and response kinetics between rods and cones.

Luteinizing Hormone Causes Phosphorylation and Activation of the cGMP Phosphodiesterase PDE5 in Rat Ovarian Follicles, Contributing, Together with PDE1 Activity, to the Resumption of Meiosis.

The meiotic cell cycle of mammalian oocytes in preovulatory follicles is held in prophase arrest by diffusion of cGMP from the surrounding granulosa cells into the oocyte. Luteinizing hormone (LH) then releases meiotic arrest by lowering cGMP in the granulosa cells. The LH-induced reduction of cGMP is caused in part by a decrease in guanylyl cyclase activity, but the observation that the cGMP phosphodiesterase PDE5 is phosphorylated during LH signaling suggests that an increase in PDE5 activity could also contribute. To investigate this idea, we measured cGMP-hydrolytic activity in rat ovarian follicles. Basal activity was due primarily to PDE1A and PDE5, and LH increased PDE5 activity. The increase in PDE5 activity was accompanied by phosphorylation of PDE5 at serine 92, a protein kinase A/G consensus site. Both the phosphorylation and the increase in activity were promoted by elevating cAMP and opposed by inhibiting protein kinase A, supporting the hypothesis that LH activates PDE5 by stimulating its phosphorylation by protein kinase A. Inhibition of PDE5 activity partially suppressed LH-induced meiotic resumption as indicated by nuclear envelope breakdown, but inhibition of both PDE5 and PDE1 activities was needed to completely inhibit this response. These results show that activities of both PDE5 and PDE1 contribute to the LH-induced resumption of meiosis in rat oocytes, and that phosphorylation and activation of PDE5 is a regulatory mechanism.

Distinct patterns of compartmentalization and proteolytic stability of PDE6C mutants linked to achromatopsia.

Phosphodiesterase-6 (PDE6) is an essential effector enzyme in vertebrate photoreceptor cells. Mutations in rod and cone PDE6 cause recessive retinitis pigmentosa and achromatopsia, respectively. The mechanisms of missense PDE6 mutations underlying severe visual disorders are poorly understood. To probe these mechanisms, we expressed seven known missense mutants of cone PDE6C in rods of transgenic Xenopus laevis and examined their stability and compartmentalization. PDE6C proteins with mutations in the catalytic domain, H602L and E790K, displayed modestly reduced proteolytic stability, but they were properly targeted to the outer segment of photoreceptor cells. Mutations in the regulatory GAF domains, R104W, Y323N, and P391L led to a proteolytic degradation of the proteins involving a cleavage in the GAFb domain. Lastly, the R29W and M455V mutations residing outside the conserved PDE6 domains produced a pattern of subcellular compartmentalization different from that of PDE6C. Thus, our results suggest a spectrum of mechanisms of missense PDE6C mutations in achromatopsia including catalytic defects, protein mislocalization, or a specific sequence of proteolytic degradation.

Transducin translocation contributes to rod survival and enhances synaptic transmission from rods to rod bipolar cells.

In rod photoreceptors, several phototransduction components display light-dependent translocation between cellular compartments. Notably, the G protein transducin translocates from rod outer segments to inner segments/spherules in bright light, but the functional consequences of translocation remain unclear. We generated transgenic mice where light-induced transducin translocation is impaired. These mice exhibited slow photoreceptor degeneration, which was prevented if they were dark-reared. Physiological recordings showed that control and transgenic rods and rod bipolar cells displayed similar sensitivity in darkness. After bright light exposure, control rods were more strongly desensitized than transgenic rods. However, in rod bipolar cells, this effect was reversed; transgenic rod bipolar cells were more strongly desensitized than control. This sensitivity reversal indicates that transducin translocation in rods enhances signaling to rod bipolar cells. The enhancement could not be explained by modulation of inner segment conductances or the voltage sensitivity of the synaptic Ca(2+) current, suggesting interactions of transducin with the synaptic machinery.

Extended conformation of the proline-rich domain of human aryl hydrocarbon receptor-interacting protein-like 1: implications for retina disease.

Mutations in the primate-specific proline-rich domain (PRD) of aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are thought to cause Leber congenital amaurosis or dominant cone-rod dystrophy. The role of PRD and the mechanisms of PRD mutations are poorly understood. Here, we have examined properties of hAIPL1 and effects of the PRD mutations on protein structure and function. Solution structures of hAIPL1, hAIPL11-316 with PRD truncation, and the P351Δ12 and P376S mutants were examined by small angle X-ray scattering. Our analysis suggests that PRD assumes an extended conformation and does not interact with the FK506-binding and tetratricopeptide domains. The PRD truncation, but not PRD mutations, reduced the molecule's radius of gyration and maximum dimension. We demonstrate that hAIPL1 is a monomeric protein, and its secondary structure and stability are not affected by the PRD mutations. PRD itself is an extended monomeric random coil. The PRD mutations caused little or no changes in hAIPL1 binding to known partners, phosphodiesterase-6A and HSP90. We also identified the γ-subunit of phosphodiesterase-6 as a novel partner of hAIPL1 and hypothesize that this interaction is altered by P351Δ12. Our results highlight the complexity of mechanisms of PRD mutations in disease and the possibility that certain mutations are benign variants. Mutations in the proline-rich domain (PRD) of human AIPL1 cause severe retinal diseases, yet the role of PRD and the mechanisms of PRD mutations are unknown. Here, we describe a SAXS-derived solution structure of AIPL1 and functional properties of disease-linked AIPL1-PRD mutants. This structure and functional analyses provide a framework for understanding the mechanisms of PRD in disease.

Interaction of aryl hydrocarbon receptor-interacting protein-like 1 with the farnesyl moiety.

Aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) is a photoreceptor specific chaperone of the visual effector enzyme phosphodiesterase-6 (PDE6). AIPL1 has been shown to bind the farnesylated PDE6A subunit. Mutations in AIPL1 are thought to destabilize PDE6 and thereby cause Leber congenital amaurosis type 4 (LCA4), a severe form of childhood blindness. Here, we examined the solution structure of AIPL1 by small angle x-ray scattering. A structural model of AIPL1 with the best fit to the scattering data features two independent FK506-binding protein (FKBP)-like and tetratricopeptide repeat domains. Guided by the model, we tested the hypothesis that AIPL1 directly binds the farnesyl moiety. Our studies revealed high affinity binding of the farnesylated-Cys probe to the FKBP-like domain of AIPL1, thus uncovering a novel function of this domain. Mutational analysis of the potential farnesyl-binding sites on AIPL1 identified two critical residues, Cys-89 and Leu-147, located in close proximity in the structure model. The L147A mutation and the LCA-linked C89R mutation prevented the binding of the farnesyl-Cys probe to AIPL1. Furthermore, Cys-89 and Leu-147 flank the unique insert region of AIPL1, deletion of which also abolished the farnesyl interaction. Our results suggest that the binding of PDE6A farnesyl is essential to normal function of AIPL1 and its disruption is one of the mechanisms underlying LCA.

The GAFa domain of phosphodiesterase-6 contains a rod outer segment localization signal.

Photoreceptor phosphodiesterase-6 (PDE6) is a peripheral membrane protein synthesized in the inner segment of photoreceptor cells. Newly synthesized PDE6 is transported to the outer segment (OS) where it serves as a key effector enzyme in the phototransduction cascade. Proper localization of PDE6 in photoreceptors is critically important to the function and survival of photoreceptor cells. The mechanism of PDE6 transport to the OS remains largely unknown. In this study, we investigated potential OS targeting signals of PDE6 by constructing cGMP-binding, cGMP-specific phosphodiesterase-5/PDE6 chimeric proteins and analyzing their localization in rods of transgenic Xenopus laevis. We found that efficient OS localization of chimeric isoprenylated PDE enzymes required the presence of a targeting motif within the PDE6 GAFa domain. Furthermore, the GAFa-dependent localization signal was sufficient to target GAFa fusion protein to the OS. Our results support the idea that effective trafficking of the peripheral membrane proteins to the OS of photoreceptor cells requires a sorting/targeting motif in addition to a membrane-binding signal.

Molecular basis of CRX/DNA recognition and stoichiometry at the Ret4 response element.

Two retinal transcription factors, cone-rod homeobox (CRX) and neural retina leucine zipper (NRL), cooperate functionally and physically to control photoreceptor development and homeostasis. Mutations in CRX and NRL cause severe retinal diseases. Despite the roles of NRL and CRX, insight into their functions at the molecular level is lacking. Here, we have solved the crystal structure of the CRX homeodomain in complex with its cognate response element (Ret4) from the rhodopsin proximal promoter region. The structure reveals an unexpected 2:1 stoichiometry of CRX/Ret4 and unique orientation of CRX molecules on DNA, and it explains the mechanisms of pathogenic mutations in CRX. Mutations R41Q and E42K disrupt the CRX protein-protein contacts based on the structure and reduce the CRX/Ret4 binding stoichiometry, suggesting a novel disease mechanism. Furthermore, we show that NRL alters the stoichiometry and increases affinity of CRX binding at the rhodopsin promoter, which may enhance transcription of rod-specific genes and suppress transcription of cone-specific genes.

Decreased catalytic activity and altered activation properties of PDE6C mutants associated with autosomal recessive achromatopsia.

Mutations in the gene encoding the catalytic subunit of the cone photoreceptor phosphodiesterase (PDE6C) have been recently reported in patients with autosomal recessive inherited achromatopsia (ACHM) and early-onset cone photoreceptor dysfunction. Here we present the results of a comprehensive study on PDE6C mutations including the mutation spectrum, its prevalence in a large cohort of ACHM/cone dysfunction patients, the clinical phenotype and the functional characterization of mutant PDE6C proteins. Twelve affected patients from seven independent families segregating PDE6C mutations were identified in our total patient cohort of 492 independent families. Eleven different PDE6C mutations were found including two nonsense mutations, three mutations affecting transcript splicing as shown by minigene assays, one 1 bp-insertion and five missense mutations. We also performed a detailed functional characterization of six missense mutations applying the baculovirus system to express recombinant mutant and wildtype chimeric PDE6C/PDE5 proteins in Sf9 insect cells. Purified proteins were analyzed using Western blotting, phosphodiesterase (PDE) activity measurements as well as inhibition assays by zaprinast and Pγ. Four of the six PDE6C missense mutations led to baseline PDE activities and most likely represent functional null alleles. For two mutations, p.E790K and p.Y323N, we observed reduction in PDE activity of approximately 60% and 80%, respectively. We also observed differences for Pγ inhibition. The p.E790K mutant, with an IC50 value of 2.7 nm is 20.7-fold more sensitive for Pγ inhibition, whereas the p.Y323N mutant with an IC50 of 158 nm is 3-fold less sensitive when compared with the wildtype control.