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.

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.

Unique interface and dynamics of the complex of HSP90 with a specialized cochaperone AIPL1.

Photoreceptor phosphodiesterase PDE6 is central for visual signal transduction. Maturation of PDE6 depends on a specialized chaperone complex of HSP90 with aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1). Disruption of PDE6 maturation underlies a severe form of retina degeneration. Here, we report a 3.9 Å cryoelectron microscopy (cryo-EM) structure of the complex of HSP90 with AIPL1. This structure reveals a unique interaction of the FK506-binding protein (FKBP)-like domain of AIPL1 with HSP90 at its dimer interface. Unusually, the N terminus AIPL1 inserts into the HSP90 lumen in a manner that was observed previously for HSP90 clients. Deletion of the 7 N-terminal residues of AIPL1 decreased its ability to cochaperone PDE6. Multi-body refinement of the cryo-EM data indicated large swing-like movements of AIPL1-FKBP. Modeling the complex of HSP90 with AIPL1 using crosslinking constraints indicated proximity of the mobile tetratricopeptide repeat (TPR) domain with the C-terminal domain of HSP90. Our study establishes a framework for future structural studies of PDE6 maturation.

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.

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.

Transducin Partners Outside the Phototransduction Pathway.

Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin α (Gαt 1) and ßγ (Gß1γ1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows Gαt1 and/or Gß1γ1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed Gαt1 and Gß1γ1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of Gα subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with Gα subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of Gαt1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of Gαt1.

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.

A nonhuman primate model of inherited retinal disease.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

False-Positive Monospot in a Returning Traveler with Dengue Fever.

The heterophile antibody (Monospot), initial test of choice for Epstein-Barr virus (EBV)-associated infectious mononucleosis, is both sensitive (70-92%) and specific (96-100%). False positives have been demonstrated in cases of viral hepatitis, human immunodeficiency virus, leukemia, lymphoma, pancreatic cancer, systemic lupus erythematosus, and rubella. We present a case of a 46-yr-old male who developed fever, chills, headaches, myalgia, fatigue, and photophobia 1 d after returning from the Philippines. He demonstrated a mild transaminitis and significant thrombocytopenia (12,000 cells/µL). His initial evaluation revealed a positive heterophile antibody test. Without a classic EBV presentation, a fever in returning traveler evaluation was instituted resulting in a positive dengue test by direct fluorescence IgM (8.82 IU) and IgG (7.13 IU), respectively. Both his EBV DNA polymerase chain reaction and IgM by viral capsid antigen were negative. Dengue, an RNA flavivirus, and the dengue antibody have demonstrated cross-reactivity with other flaviviruses including Japanese encephalitis virus, yellow fever virus, West Nile virus, and St. Louis encephalitis. However, EBV is a double-helix DNA herpesvirus and structurally very different. To our knowledge, this is the first reported case of cross-reactivity between dengue and EBV that describes a potential false positive for the heterophile antibody test.

Mechanisms of mutant PDE6 proteins underlying retinal diseases.

Mutations in PDE6 genes encoding the effector enzymes in rods and cones underlie severe retinal diseases including retinitis pigmentosa (RP), autosomal dominant congenital stationary night blindness (adCSNB), and achromatopsia (ACHM). Here we examined a spectrum of pathogenic missense mutations in PDE6 using the system based on co-expression of cone PDE6C with its specialized chaperone AIPL1 and the regulatory Pγ subunit as a potent co-chaperone. We uncovered two mechanisms of PDE6C mutations underlying ACHM: (a) folding defects leading to expression of catalytically inactive proteins and (b) markedly diminished ability of Pγ to co-chaperone mutant PDE6C proteins thereby dramatically reducing the levels of functional enzyme. The mechanism of the Rambusch adCSNB associated with the H258N substitution in PDE6B was probed through the analysis of the model mutant PDE6C-H262N. We identified two interrelated deficits of PDE6C-H262N: disruption of the inhibitory interaction of Pγ with mutant PDE6C that markedly reduced the ability of Pγ to augment the enzyme folding. Thus, we conclude that the Rambusch adCSNB is triggered by low levels of the constitutively active PDE6. Finally, we examined PDE6C-L858V, which models PDE6B-L854V, an RP-linked mutation that alters the protein isoprenyl modification. This analysis suggests that the type of prenyl modifications does not impact the folding of PDE6, but it modulates the enzyme affinity for its trafficking partner PDE6D. Hence, the pathogenicity of PDE6B-L854V likely arises from its trafficking deficiency. Taken together, our results demonstrate the effectiveness of the PDE6C expression system to evaluate pathogenicity and elucidate the mechanisms of PDE6 mutations in retinal diseases.

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.

Fasting Serum Glucose and Cholesterol as Predictors of Cardiovascular Reactivity to Acute Stress in a Sample of African American College Students.

OBJECTIVE: African Americans are at a greater risk of cardiovascular hyperactivity to stress than Caucasians; however the risk factors for this activity are not clearly delineated for African Americans. The purpose of this study was to determine the ability of fasting serum cholesterol concentration and fasting serum glucose (FSG) to predict cardiovascular reactivity to stress in African Americans. DESIGN, SETTING, PARTICIPANTS: Serum cholesterol concentration and FSG levels were measured in 48 (40 women, 8 men) African American college students aged 18-30 years. Heart rate, cardiac output, stroke volume, mean arterial pressure and systolic and diastolic blood pressure were measured as the participants viewed a racially noxious scene on a digital video disc. Measurements were taken prior to the scene (pre stressor period), during the scene (stressor period), and while the participant recovered from the scene (recovery period). RESULTS: A multiple regression analysis revealed that total serum cholesterol and LDL significantly predicted diastolic blood pressure during the pre-stressor period. FSG significantly predicted mean arterial pressure during the recovery period, and predicted stroke volume during the pre-stressor period, stressor period, and the recovery period. CONCLUSIONS: FSG was a better predictor of cardiovascular reactivity to stress than serum cholesterol concentration, predicting mean arterial pressure and stroke volume. This finding may be due to the association of glucose with diabetes, which is more prevalent in African Americans.

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.

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.

Comparative analysis of cone and rod transducins using chimeric Gα subunits.

The molecular nature of transducin-α subunits (Gα(t)) may contribute to the distinct physiology of cone and rod photoreceptors. Biochemical properties of mammalian cone Gα(t2) subunits and their differences with rod Gα(t1) are largely unknown. Here, we examined properties of chimeric Gα(t2) in comparison with its rod counterpart. The key biochemical difference between the rod- and cone-like Gα(t) was ~10-fold higher intrinsic nucleotide exchange on the chimeric Gα(t2). Presented mutational analysis suggests that weaker interdomain interactions between the GTPase (Ras-like) domain and the helical domain in Gα(t2) are in part responsible for its increased spontaneous nucleotide exchange. However, the rates of R*-dependent nucleotide exchange of chimeric Gα(t2) and Gα(t1) were equivalent. Furthermore, chimeric Gα(t2) and Gα(t1) exhibited similar rates of intrinsic GTPase activity as well as similar acceleration of GTP hydrolysis by the RGS domain of RGS9. Our results suggest that the activation and inactivation properties of cone and rod Gα(t) subunits in an in vitro reconstituted system are comparable.

Atypical retinal degeneration 3 in mice is caused by defective PDE6B pre-mRNA splicing.

Mutations in the key rod phototransduction enzyme phosphodiesterase 6 (PDE6) are known to cause recessive retinitis pigmentosa in humans. Mouse models of mutant PDE6 represent a common approach to understanding the mechanisms of visual disorders related to PDE6 defects. Mutation N605S in the PDE6B subunit is linked to atypical retinal degeneration 3 (atrd3) in mice. We examined PDE6 in atrd3 mice and an atrd3 mutant counterpart of human cone PDE6C expressed in rods of transgenic Xenopus laevis. These animal models revealed remarkably different phenotypes. In contrast to dramatic downregulation of the mutant rod PDE6 protein and activity levels in mice, expression and localization of the cone PDE6C in X. laevis were essentially unaffected by this mutation. Examination of the PDE6B mRNA in atrd3 retina showed that the mutation-carrying exon 14 was spliced-out in the majority of the transcript. Thus, retinal degeneration in atrd3 mice is caused by low levels of PDE6 protein due to defective processing of PDE6B pre-mRNA rather than by deleterious effects of the N605S mutation on PDE6 folding, stability or function.

Classroom-based science research at the introductory level: changes in career choices and attitude.

Our study, focused on classroom-based research at the introductory level and using the Phage Genomics course as the model, shows evidence that first-year students doing research learn the process of science as well as how scientists practice science. A preliminary but notable outcome of our work, which is based on a small sample, is the change in student interest in considering different career choices such as graduate education and science in general. This is particularly notable, as previous research has described research internships as clarifying or confirming rather than changing undergraduates' decisions to pursue graduate education. We hypothesize that our results differ from previous studies of the impact of engaging in research because the students in our study are still in the early stages of their undergraduate careers. Our work builds upon the classroom-based research movement and should be viewed as encouraging to the Vision and Change in Undergraduate Biology Education movement advocated by the American Association for the Advancement of Science, the National Science Foundation, and other undergraduate education stakeholders.

Interaction of transducin with uncoordinated 119 protein (UNC119): implications for the model of transducin trafficking in rod photoreceptors.

The key visual G protein, transducin undergoes bi-directional translocations between the outer segment (OS) and inner compartments of rod photoreceptors in a light-dependent manner thereby contributing to adaptation and neuroprotection of rods. A mammalian uncoordinated 119 protein (UNC119), also known as Retina Gene 4 protein (RG4), has been recently implicated in transducin transport to the OS in the dark through its interaction with the N-acylated GTP-bound transducin-α subunit (Gα(t1)). Here, we demonstrate that the interaction of human UNC119 (HRG4) with transducin is dependent on the N-acylation, but does not require the GTP-bound form of Gα(t1). The lipid specificity of UNC119 is unique: UNC119 bound the myristoylated N terminus of Gα(t1) with much higher affinity than a prenylated substrate, whereas the homologous prenyl-binding protein PrBP/δ did not interact with the myristoylated peptide. UNC119 was capable of interacting with Gα(t1)GDP as well as with heterotrimeric transducin (G(t)). This interaction of UNC119 with G(t) led to displacement of Gß(1)γ(1) from the heterotrimer. Furthermore, UNC119 facilitated solubilization of G(t) from dark-adapted rod OS membranes. Consistent with these observations, UNC119 inhibited rhodopsin-dependent activation of G(t), but had no effect on the GTP-hydrolysis by Gα(t1). A model for the role of UNC119 in the IS→OS translocation of G(t) is proposed based on the UNC119 ability to dissociate G(t) subunits from each other and the membrane. We also found that UNC119 inhibited activation of G(o) by D2 dopamine receptor in cultured cells. Thus, UNC119 may play conserved inhibitory role in regulation of GPCR-G protein signaling in non-visual tissues.

Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent.

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K(m) (20-23 µM) and k(cat) (4200-5100 s(-1)) values. Likewise, the K(i) values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific PDE6 γ-subunits (Pγ) were comparable. Recombinant cone transducin-α (Gα(t2)) and native rod Gα(t1) fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of Gα(t2) or Gα(t1). Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with Pγ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.

Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the gamma-subunit.

The inhibitory interaction of phosphodiesterase-6 (PDE6) with its gamma-subunit (Pgamma) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pgamma-inhibitory peptide Pgamma(70-87) have been determined at 2.9 and 3.0 A, respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pgamma and suggest the conformational change of Pgamma on transducin activation. Two variable H- and M-loops of PDE5/6cd form a distinct interface that contributes to the Pgamma-binding site. This allows the Pgamma C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pgamma-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pgamma(70-87)-binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.