Structure of the Visual Signaling Complex between Transducin and Phosphodiesterase 6.

Heterotrimeric G proteins communicate signals from activated G protein-coupled receptors to downstream effector proteins. In the phototransduction pathway responsible for vertebrate vision, the G protein-effector complex is composed of the GTP-bound transducin α subunit (GαT·GTP) and the cyclic GMP (cGMP) phosphodiesterase 6 (PDE6), which stimulates cGMP hydrolysis, leading to hyperpolarization of the photoreceptor cell. Here we report a cryo-electron microscopy (cryoEM) structure of PDE6 complexed to GTP-bound GαT. The structure reveals two GαT·GTP subunits engaging the PDE6 hetero-tetramer at both the PDE6 catalytic core and the PDEγ subunits, driving extensive rearrangements to relieve all inhibitory constraints on enzyme catalysis. Analysis of the conformational ensemble in the cryoEM data highlights the dynamic nature of the contacts between the two GαT·GTP subunits and PDE6 that supports an alternating-site catalytic mechanism.

Synchronized Photoactivation of T4K Rhodopsin Causes a Chromophore-Dependent Retinal Degeneration That Is Moderated by Interaction with Phototransduction Cascade Components.

Multiple mutations in the Rhodopsin gene cause sector retinitis pigmentosa in humans and a corresponding light-exacerbated retinal degeneration (RD) in animal models. Previously we have shown that T4K rhodopsin requires photoactivation to exert its toxic effect. Here we further investigated the mechanisms involved in rod cell death caused by T4K rhodopsin in mixed male and female Xenopus laevis In this model, RD was prevented by rearing animals in constant darkness but surprisingly also in constant light. RD was maximized by light cycles containing at least 1 h of darkness and 20 min of light exposure, light intensities >750 lux, and by a sudden light onset. Under conditions of frequent light cycling, RD occurred rapidly and synchronously, with massive shedding of ROS fragments into the RPE initiated within hours and subsequent death and phagocytosis of rod cell bodies. RD was minimized by reduced light levels, pretreatment with constant light, and gradual light onset. RD was prevented by genetic ablation of the retinal isomerohydrolase RPE65 and exacerbated by ablation of phototransduction components GNAT1, SAG, and GRK1. Our results indicate that photoactivated T4K rhodopsin is toxic, that cell death requires synchronized photoactivation of T4K rhodopsin, and that toxicity is mitigated by interaction with other rod outer segment proteins regardless of whether they participate in activation or shutoff of phototransduction. In contrast, RD caused by P23H rhodopsin does not require photoactivation of the mutant protein, as it was exacerbated by RPE65 ablation, suggesting that these phenotypically similar disorders may require different treatment strategies.

Dual AAV-based PCDH15 gene therapy achieves sustained rescue of visual function in a mouse model of Usher syndrome 1F.

Mutations in the PCDH15 gene, encoding protocadherin-15, are among the leading causes of Usher syndrome type 1 (USH1F), and account for up to 12% USH1 cases worldwide. A founder truncating variant of PCDH15 has a ∼2% carrier frequency in Ashkenazi Jews accounting for nearly 60% of their USH1 cases. Although cochlear implants can restore hearing perception in USH1 patients, presently there are no effective treatments for the vision loss due to retinitis pigmentosa. We established a founder allele-specific Pcdh15 knockin mouse model as a platform to ascertain therapeutic strategies. Using a dual-vector approach to circumvent the size limitation of adeno-associated virus, we observed robust expression of exogenous PCDH15 in the retinae of Pcdh15KI mice, sustained recovery of electroretinogram amplitudes and key retinoid oxime, substantially improved light-dependent translocation of phototransduction proteins, and enhanced levels of retinal pigment epithelium-derived enzymes. Thus, our data raise hope and pave the way for future gene therapy trials in USH1F subjects.

Clinical and molecular analyses of isolated central congenital hypothyroidism based on a survey conducted in Japan.

Central congenital hypothyroidism (CH) can occur as an isolated deficiency or as part of combined pituitary hormone deficiency. Unlike primary CH, central CH cannot be detected by newborn screening (NBS) using dry filter paper blood TSH levels, and early diagnosis remains challenging. In this study, the clinical and genetic backgrounds of patients with isolated central CH were determined through a questionnaire-based survey among members of the Japanese Society for Pediatric Endocrinology. The known causes of isolated central CH were studied in 14 patients, including six with previously reported patient data. The results revealed IGSF1 and TBL1X pathogenic variants in nine and one patient, respectively. All six patients with low free thyroxine (FT4) levels detected in NBS carried IGSF1 pathogenic variants. Five patients with isolated central CH diagnosed after 3 months of age were variant-negative, except for one female patient with a heterozygous IGSF1 variant. Two of the four variant-negative patients and a variant-positive patient were diagnosed with pituitary hypoplasia. One and two patients with IGSF1 variant had obesity and intellectual disability, respectively. Left amblyopia was identified in the patient with a TBL1X variant. The study revalidated that IGSF1 variants comprise the most frequent pathogenic variant in patients with isolated central CH in Japan. The neonatal period is the optimal time for the diagnosis of central CH, particularly IGSF1 abnormalities, and the introduction of T4 screening should be considered in the future, taking cost-effectiveness into consideration.

Identification of potential biomarkers for papillary thyroid carcinoma by comprehensive bioinformatics analysis.

To perform bioinformatics analysis on the papillary thyroid carcinoma (PTC) gene chip dataset to explore new biological markers for PTC. The gene expression profiles of GSE3467 and GSE6004 chip data were collected by GEO2R, and the differentially expressed genes (DEGs) were selected for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Protein-protein interaction (PPI) relationship analysis was achieved using STRING, and the hub genes were obtained using the Cytoscape software. GEPIA was used to validate the expressions of the hub genes in the normal and tumor tissues and to conduct survival analyses. Pertinent genetic pathology results were fetched using the HPA database. Finally, the key genes were clinically verified by reverse transcription-polymerase chain reaction. 97 genes were jointly up-regulated and 107 genes were jointly down-regulated in GSE3467 and GSE6004. GO function enrichment analysis revealed that the DEGs were involved in the regulation of calcium ion transport into cytosol, integrin binding, and cell adhesion molecule binding. KEGG pathway enrichment analysis indicated that the DEGs were chiefly associated with thyroid cancer and non-small cell lung cancer. According to the PPI network, 30 key target genes were identified. Only the expressions of ANK2, TLE1, and TCF4 matched between the normal and tumor tissues, and were associated with disease prognosis. When compared with the normal thyroid tissues, the protein and mRNA expressions of ANK2, TLE1, and TCF4 were down-regulated in PTC. Significant differences exist in overall gene expression between the thyroid tissues of patients with PTC and those of healthy people. Furthermore, the differential genes ANK2, TLE1, and TCF4 are expected to be reliable molecular markers for the mechanism study and diagnosis of PTC.

Murburn model of vision: Precepts and proof of concept.

The classical paradigm of visual physiology comprises of the following features: (i) rod/cone cells located at the rear end of the retina serve as the primary transducers of incoming photo-information, (ii) cis-trans retinal (C20 H28 O) transformations on rhodopsin act as the transduction switch to generate a transmittable signal, (iii) signal amplification occurs via GDP-GTP exchange at transducin, and (iv) the amplified signal is relayed (as an action potential) as a flux-based ripple of Na-K ions along the axons of neurons. Fundamental physical principles, chemical kinetics, and awareness of architecture of eye/retina prompt a questioning of these classical assumptions. In lieu, based on experimental and in silico findings, a simple space-time resolved murburn model for the physiology of phototransduction in the retina is presented wherein molecular oxygen plays key roles. It is advocated that: (a) photo-induced oxygen to superoxide conversion serves as the key step in signal transduction in the visual cycle, (b) all photoactive cells of the retina serve as photoreceptors and rods/cones serve as the ultimate electron source in the retina (deriving oxygen and nutrients from retinal pigmented epithelium), (c) signal amplification is through superoxide mediated phosphorylation of GDP bound to inactive transducin, thereby activating a GDP-based cascade (a new mechanism for trimeric G-proteins), and (d) signal relay is primarily an electron movement along the neuron, from dendritic source to synaptic sink. In particular, we specify the roles for the various modules of transducin and GDP-based activation of phosphodiesterase-6 in the physiology of visual transduction.

Ubiquitin-specific protease 47 is associated with vascular calcification in chronic kidney disease by regulating osteogenic transdifferentiation of vascular smooth muscle cells.

Chronic kidney disease (CKD) has recently become a serious health and social concern. Vascular calcification, a common complication of CKD, is a risk factor that increases the incidence and mortality of cardiovascular events in patients with CKD. However, there are currently no effective therapeutic targets that can facilitate treatment with fewer side effects for vascular calcification in CKD. To identify potential therapeutic targets, we performed label-free quantification (LFQ) analyses of protein samples from rat aortic vascular smooth muscle cells (RASMCs) after high-phosphorus treatment by nano-UPLC-MS/MS. We determined that ubiquitin-specific protease 47 (USP47) may be associated with CKD vascular calcification by regulating the osteogenic transdifferentiation of the vascular smooth muscle cell (VSMC) phenotype, thus suggesting a novel and potentially effective therapeutic target for CKD vascular calcification. USP47 knockdown significantly reduced the expression of ß-transducin repeat-containing protein (BTRC), serine/threonine-protein kinase akt-1 (AKT1), Klotho, fibroblast growth factor (FGF23), and matrix Gla protein (MGP) in RASMCs after high-phosphorus treatment. Consistent with the results of protein-protein interaction (PPI) analyses, USP47 may be involved in regulating osteogenic transdifferentiation markers, such as runt-related transcription factor 2 (RUNX2), Klotho, FGF23, and MGP through the BTRC/AKT1 pathway upon CKD vascular calcification. These data indicate that USP47 may be associated with vascular calcification in CKD by regulating osteogenic differentiation of VSMCs. USP47 may regulate osteogenic transdifferentiation in VSMCs upon CKD vascular calcification through a process involving the BTRC/AKT1 pathway. This study identified a novel potential therapeutic target for the treatment of vascular calcification in CKD.

Immunohistochemical Characterization of the Development of Long Photoreceptor Cells in the Lamprey Retina.

The adult lamprey retina has two types of photoreceptor cells, short and long photoreceptor cells, which are equivalent to rods and cones of other vertebrates. In contrast, the retina of lamprey larvae only contains a single type of photoreceptor cell, which appears to correspond to the short photoreceptor cell. However, the developmental pattern of the long photoreceptor cell is unknown. Previously, we reported that antibodies against rhodopsin and iodopsin (the chicken red cone opsin) could discriminate between the outer segments of short and long photoreceptor cells, respectively, in the retina of adult Japanese river lamprey (Lethenteron camtschaticum). Here, we immunohistochemically investigate the appearance of long photoreceptor cells in the larval and adult retinas of the Far Eastern brook lamprey (Lethenteron reissneri), which is a close relative of the Japanese river lamprey, by using anti-iodopsin antibody. We found that iodopsin immunoreactivity was localized not only in the adult retina but also in the larval retina. Moreover, we examined the immunohistochemical localization of signal transduction molecules, such as transducin and arrestin, in the iodopsin-immunoreactive cells of the larval retina. The iodopsin-immunoreactive cells also contained both transducin and arrestin, suggesting that long photoreceptor cells are already functional in the larval stage before the acquisition of visual function. Our results suggest that the iodopsin-immunoreactive cells may be related to not only cone vision in the adult but also photoreception in the larval lamprey.

Retinal and extra-retinal photoreceptor responses and reproductive performance of Japanese quail (Coturnix coturnix japonica) following exposure to different photoperiodic regime.

Japanese quail is a truly photoperiodic avian species. In general long days are gonado-stimulatory and short days are gonado-inhibitory for this poultry bird. To investigate the correlation of retinal and extra-retinal photoreceptors with different photoperiodic conditions quail were divided into 2 groups and kept under long day (16L: 8D) and short day (8L: 16D) condition separately to develop photosensitivity and scotosensitivity respectively. Transfer of long day quail to intermediate day-length (13.5L: 10.5D) developed photorefractoriness (relative) and prolonged exposure to short photoperiodic conditions led the birds to develop scotorefractoriness. Increased expression of mRNA and immunosignaling of photoreceptors rhodopsin, transducin in eye and hypothalamus while decreased mRNA expression of melatonin receptors (Mel1b, Mel1c) were noted in the eyes of photosensitive (PS) and scotorefractory (SR) quail compared to photorefractory (PR) and scotosensitive (SS) birds respectively. Decreased expression of hypothalamic GnIH and melatonin receptors mRNA was observed in PS and SR birds compared to PR and SS birds respectively. Modulation of retinal and extra retinal photoreceptors leads to increased spermatogenesis as well as mRNA expression of steroidogenic genes and androgen receptor in the testis of sexually active PS and SR quail. These results led us to conclude that gonadal stimulation in PS as well as SR quail is outcome of activated retinal and extra retinal photoreceptors which lowered melatonin receptors and GnIH expression. Contrarily testicular inhibition in PR and SS is the outcome of decreased photoperception. It is suggested that decreased photoperception in SS quail increases after prolong exposure of the short day (in SR) leading to increased activity of HPG axis.

A 2-Benzylmalonate Derivative as STAT3 Inhibitor Suppresses Tumor Growth in Hepatocellular Carcinoma by Upregulating ß-TrCP E3 Ubiquitin Ligase.

The aberrant activation of a signal transducer and activator of transcription 3 (STAT3) restrains type I interferon (IFN) α/ß-induced antiviral responses and is associated with the development of cancer. Designing specific STAT3 inhibitors will thus provide new options for use as IFN therapy. Herein, we identified a novel small molecule, dimethyl 2-(4-(2-(methyl(phenyl(p-tolyl)methyl)amino)ethoxy)benzyl)malonate (CIB-6), which can inhibit the IFN-α-induced interferon stimulated response element (ISRE) luciferase reporter (IC50 value = 6.4 µM) and potentiate the antiproliferative effect of IFN-α in human hepatocellular carcinoma (HCC) cells. CIB-6 was found to bind to the STAT3 Src homology 2 (SH2) domain, thereby selectively inhibiting STAT3 phosphorylation without affecting Janus kinases and STAT1/2. CIB-6 also inhibited the migration and invasion of HCC cells by inhibiting the epithelial-mesenchymal transition (EMT) process. Mechanistically, CIB-6 reduced the expression of ß-catenin (an EMT key protein) via upregulating ß-transducin repeat-containing protein (ß-TrCP) and curbed nuclear factor kappa-B (NF-κB) activation through restricting the phosphorylation of the inhibitor of NF-κB (IκB) kinase (IKK) via STAT3 inhibition. Treatment with CIB-6 significantly retarded tumor growth in nude mice with SK-HEP-1 xenografts. In addition, clinical sample analysis revealed that lower ß-TrCP and higher ß-catenin expression could affect the median survival time of HCC patients. Our findings suggest that CIB-6 could be a new therapeutic strategy for HCC therapy through STAT3-mediated ß-TrCP/ß-catenin/NF-κB axis.

The molecular architecture of photoreceptor phosphodiesterase 6 (PDE6) with activated G protein elucidates the mechanism of visual excitation.

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαß) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gtα) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαß in its nonactivated state. Following reconstitution of PDE6 and activated Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

The N termini of the inhibitory γ-subunits of phosphodiesterase-6 (PDE6) from rod and cone photoreceptors differentially regulate transducin-mediated PDE6 activation.

Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαß) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.

Cryo-EM structure of the native rhodopsin dimer in nanodiscs.

Imaging of rod photoreceptor outer-segment disc membranes by atomic force microscopy and cryo-electron tomography has revealed that the visual pigment rhodopsin, a prototypical class A G protein-coupled receptor (GPCR), can organize as rows of dimers. GPCR dimerization and oligomerization offer possibilities for allosteric regulation of GPCR activity, but the detailed structures and mechanism remain elusive. In this investigation, we made use of the high rhodopsin density in the native disc membranes and of a bifunctional cross-linker that preserves the native rhodopsin arrangement by covalently tethering rhodopsins via Lys residue side chains. We purified cross-linked rhodopsin dimers and reconstituted them into nanodiscs for cryo-EM analysis. We present cryo-EM structures of the cross-linked rhodopsin dimer as well as a rhodopsin dimer reconstituted into nanodiscs from purified monomers. We demonstrate the presence of a preferential 2-fold symmetrical dimerization interface mediated by transmembrane helix 1 and the cytoplasmic helix 8 of rhodopsin. We confirmed this dimer interface by double electron-electron resonance measurements of spin-labeled rhodopsin. We propose that this interface and the arrangement of two protomers is a prerequisite for the formation of the observed rows of dimers. We anticipate that the approach outlined here could be extended to other GPCRs or membrane receptors to better understand specific receptor dimerization mechanisms.

Light deprivation reduces the severity of experimental diabetic retinopathy.

Illumination of the retina is a major determinant of energy expenditure by its neurons. However, it remains unclear whether light exposure significantly contributes to the pathophysiology of common retinal disease. Driven by the premise that light exposure reduces the metabolic demand of the retina, recent clinical trials failed to demonstrate a benefit for constant illumination in the treatment of diabetic retinopathy. Here, we instead ask whether light deprivation or blockade of visual transduction could modulate the severity of this common cause of blindness. We randomized adult mice with two different models of diabetic retinopathy to 1-3 months of complete dark housing. Unexpectedly, we find that diabetic mice exposed to short or prolonged light deprivation have reduced diabetes-induced retinal pathology, using measures of visual function, compared to control animals in standard lighting conditions. To corroborate these results, we performed assays of retinal vascular health in diabetic Gnat1-/- and Rpe65-/- mice, which lack phototransduction. Both mutants displayed less diabetes-associated retinal vascular disease compared to respective wild-type controls. Collectively, these results suggest that light-induced visual transduction promotes the development of diabetic retinopathy and implicate photoreceptors as an early source of visual pathology in diabetes.

TBL2 methylation is associated with hyper-low-density lipoprotein cholesterolemia: a case-control study.

BACKGROUND: HMGCR, SCAP, SREBF1, SREBF2 and TBL2 are well-known genes that are involved in the process of lipid metabolism. However, it is not known whether epigenetic changes of these genes are associated with lipid metabolism. In this study, the methylation levels of the HMGCR, SCAP, SREBF1, SREBF2 and TBL2 genes were analyzed between samples from a hyper-low-density lipoprotein cholesterolemia (hyper-LDL) group and a control group to examine the association between the methylation levels of these genes and the risk of hyper-LDL. METHODS: In this study, a case-control approach was used to explore the association between DNA methylation and hyper-LDL. The DNA methylation levels of HMGCR, SCAP, SREBF1, SREBF2 and TBL2 genes and 231 CpG sites in the promoter regions of these genes were measured in 98 hyper-LDL participants and 89 participants without hypo-LDL. RESULTS: Compared with participants without hyper-LDL, patients with hyper-LDL TBL2 gene had lower methylation levels (11.93 vs. 12.02, P = 0.004). The methylation haplotypes with significant abundance in the TBL2 gene are tcttttttttt (P = 0.034), ctttttttcct (P = 0.025), ctctttctttt (P = 0.040), ccttttttttt (P = 0.028), and tctttttttttttttt. CONCLUSION: The study demonstrates that participants with hyper-LDL have lower methylation of TBL2. The results suggest that DNA methylation of TBL2 can decrease the risk for hyper-LDL in humans.

Expanding the genetic and phenotypic spectrum of female infertility caused by TLE6 mutations.

PURPOSE: The present study was intended to identify genetic causes of infertile patients with recurrent failure of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) attempts. METHODS: Infertile patients with recurrent IVF/ICSI failure from Shanghai Ji Ai Genetics & IVF Institute and the Ninth Hospital affiliated with Shanghai Jiao Tong University were recruited. Genomic DNA samples were extracted from their peripheral blood. Whole-exome sequencing and Sanger validation were performed to identify candidate variants. RESULTS: We identified novel transducin-like enhancer of split 6 (TLE6) gene mutations in three patients with recurrent IVF/ICSI failure. One patient carried a homozygous missense mutation (c.1226G>A; p.Arg409Gln) with subsequent fertilization failure, while the other two patients carried either a homozygous missense mutation (c.1621G>A; p.Glu541Lys) or a compound heterozygous missense mutation (c.388G>A/c.1507G>A; p.Asp130Asn/p.Val503Ile) and had viable but low-quality embryos. CONCLUSIONS: Our study expands the mutational and phenotypic spectrum of TLE6 and suggests the important role of TLE6 during embryonic development. Our findings have implications for the genetic diagnosis of female infertility with recurrent IVF/ICSI failure.

Mutation spectrum and clinical investigation of achromatopsia patients with mutations in the GNAT2 gene.

Achromatopsia (ACHM) is a hereditary cone photoreceptor disorder characterized by the inability to discriminate colors, nystagmus, photophobia, and low-visual acuity. Six genes have been associated with this rare autosomal recessively inherited disease, including the GNAT2 gene encoding the catalytic α-subunit of the G-protein transducin which is expressed in the cone photoreceptor outer segment. Out of a cohort of 1,116 independent families diagnosed with a primary clinical diagnosis of ACHM, we identified 23 patients with ACHM from 19 independent families with likely causative mutations in GNAT2, representing 1.7% of our large ACHM cohort. In total 22 different potentially disease-causing variants, of which 12 are novel, were identified. The mutation spectrum also includes a novel copy number variation, a heterozygous duplication of exon 4, of which the breakpoint matches exactly that of the previously reported exon 4 deletion. Two patients carry just a single heterozygous variant. In addition to our previous study on GNAT2-ACHM, we also present detailed clinical data of these patients.

Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation.

Two regions on the α subunits of heterotrimeric GTP-binding proteins (G-proteins), the Switch II/α2 helix (which changes conformation upon GDP-GTP exchange) and the α3 helix, have been shown to contain the binding sites for their effector proteins. However, how the binding of Gα subunits to their effector proteins is translated into the stimulation of effector activity is still poorly understood. Here, we took advantage of a reconstituted rhodopsin-coupled phototransduction system to address this question and identified a distinct surface and an essential residue on the α subunit of the G-protein transducin (αT) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE). We started with a chimeric G-protein α subunit (αT*) comprising residues mainly from αT and a short stretch of residues from the Gi1 α subunit (αi1), which only weakly stimulates PDE activity. We then reinstated the αT residues by systematically replacing the corresponding αi1 residues within αT* with the aim of fully restoring PDE stimulatory activity. These experiments revealed that the αG/α4 loop and a phenylalanine residue at position 283 are essential for conferring the αT* subunit with full PDE stimulatory capability. We further demonstrated that this same region and amino acid within the α subunit of the Gs protein (αs) are necessary for full adenylyl cyclase activation. These findings highlight the importance of the αG/α4 loop and of an essential phenylalanine residue within this region on Gα subunits αT and αs as being pivotal for their selective and optimal stimulation of effector activity.

Evolution of the vertebrate phototransduction cascade activation steps.

We examine the molecular phylogeny of the proteins underlying the activation steps of vertebrate phototransduction, for both agnathan and jawed vertebrate taxa. We expand the number of taxa analysed and we update the alignment and tree building methodology from a previous analysis. For each of the four primary components (the G-protein transducin alpha subunit, GαT, the cyclic GMP phosphodiesterase, PDE6, and the alpha and beta subunits of the cGMP-gated ion channel, CNGC), the phylogenies appear consistent with expansion from an ancestral proto-vertebrate cascade during two rounds of whole-genome duplication followed by divergence of the agnathan and jawed vertebrate lineages. In each case, we consider possible scenarios for the underlying gene duplications and losses, and we apply relevant constraints to the tree construction. From tests of the topology of the resulting trees, we obtain a scenario for the expansion of each component during 2R that accurately fits the observations. Similar analysis of the visual opsins indicates that the only expansion to have occurred during 2R was the formation of Rh1 and Rh2. Finally, we propose a hypothetical scenario for the conversion of an ancestral chordate cascade into the proto-vertebrate phototransduction cascade, prior to whole-genome duplication. Together, our models provide a plausible account for the origin and expansion of the vertebrate phototransduction cascade.

Palmitoylation is a prerequisite for dimerization-dependent raftophilicity of rhodopsin.

The visual photopigment rhodopsin (Rh) is a prototypical G protein-coupled receptor (GPCR) responsible for initiation of the phototransduction cascade in rod photoreceptors. Similar to other GPCRs, Rh can form dimers or even higher oligomers and tends to have a supramolecular organization that is likely important in the dim light response. Rh also exhibits high affinity for lipid rafts (i.e. raftophilicity) upon light-dependent binding with the cognate G protein transducin (Gt), suggesting the presence of lipid raft-like domains in the retinal disk membrane and their importance in phototransduction. However, the relationship between Rh oligomerization and lipid rafts in the disk membrane remains to be explored. Given previous findings that Gt binds to dimeric Rh and that Rh is posttranslationally modified with two highly raftophilic palmitoyl moieties, we hypothesized that Rh becomes raftophilic upon dimerization. Here, using biochemical assays, we found that Rh*-Gt complexes in the detergent-resistant membrane are partially resistant to cholesterol depletion by methyl-ß-cyclodextrin and that the Rh-to-Gt stoichiometry in this methyl-ß-cyclodextrin-resistant complex is 2:1. Next, we found that IgG-mediated Rh-Rh cross-linking renders Rh highly raftophilic, supporting the premise that Rh becomes raftophilic upon dimerization. Rh depalmitoylation via reduction of thioester linkages blocked the translocation of IgG-cross-linked Rh to the detergent-resistant membrane, highlighting that the two palmitoyl moieties are important for the dimerization-dependent raftophilicity of Rh. These results indicate that palmitoylated GPCRs such as Rh can acquire raftophilicity upon G protein-stabilized dimerization and thereby organize receptor-cluster rafts by recruiting raftophilic lipids.