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Gael Manes - One of the best experts on this subject based on the ideXlab platform.
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a truncated form of rod photoreceptor pde6 β subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the γ subunit
PLOS ONE, 2014Co-Authors: Pallavi Cheguru, Audrey Senechal, Beatrice Bocquet, Gael Manes, Anurima Majumder, Christian P. HamelAbstract:Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod Phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the Phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the Phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the Phototransduction thus leading to the absence of rod adaptation.
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a truncated form of rod photoreceptor pde6 β subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the γ subunit
PLOS ONE, 2014Co-Authors: Pallavi Cheguru, Audrey Senechal, Beatrice Bocquet, Gael Manes, Anurima Majumder, Christian P. HamelAbstract:Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod Phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the Phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the Phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the Phototransduction thus leading to the absence of rod adaptation.
Wolfgang Baehr - One of the best experts on this subject based on the ideXlab platform.
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membrane protein transport in photoreceptors the function of pdeδ
Investigative Ophthalmology & Visual Science, 2014Co-Authors: Wolfgang BaehrAbstract:Photoreceptors are polarized neurons, with specific subcellular compartmentalization and unique requirements for protein synthesis and trafficking. Each photoreceptor contains an outer segment (OS) housing the Phototransduction machinery, an inner segment (IS) where proteins are biosynthesized, and a synaptic terminal for signal transmission. Outer and inner segment are joined by a connecting cilium (CC), corresponding to the transition zone of primary cilia. Proteins that participate in Phototransduction (rhodopsin, transducin, cGMP phosphodiesterase 6 [PDE6], cyclic nucleotide-gated [CNG] channel subunits) and accessory proteins (rhodopsin kinase or GRK1, arrestins, guanylate cyclase [GC], GC-activating proteins or GCAPs, and the GTPase-activating protein [GAP] complex) are synthesized in the IS and must be transported through the CC to the OS. These proteins are either transmembrane (TM) proteins or peripherally associated membrane proteins that are attached to the membrane surface. How TM proteins (e.g., rhodopsin) and peripherally associated proteins (e.g., transducin and PDE6) traffic through the IS to incorporate eventually in the nascent disc membrane is unknown.
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a functional kinase homology domain is essential for the activity of photoreceptor guanylate cyclase 1
Journal of Biological Chemistry, 2010Co-Authors: Grzegorz Bereta, Wolfgang Baehr, Benlian Wang, Philip D Kiser, Geeng Fu Jang, Krzysztof PalczewskiAbstract:Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of Phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of Phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg2+ binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.
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Trafficking of Membrane Proteins to Cone But Not Rod Outer Segments Is Dependent on Heterotrimeric Kinesin-II
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2009Co-Authors: Prachee Avasthi, Chingkang Chen, Carl B Watt, Jeanne M Frederick, David S. Williams, Robert E. Marc, Wolfgang BaehrAbstract:Heterotrimeric kinesin-II is a molecular motor localized to the inner segment, connecting cilium and axoneme of mammalian photoreceptors. Our purpose was to identify the role of kinesin-II in anterograde intraflagellar transport by photoreceptor-specific deletions of kinesin family member 3A (KIF3A), its obligatory motor subunit. In cones lacking KIF3A, membrane proteins involved in Phototransduction did not traffic to the outer segments resulting in complete absence of a photopic electroretinogram and progressive cone degeneration. Rod photoreceptors lacking KIF3A degenerated rapidly between 2 and 4 weeks postnatally, but the Phototransduction components including rhodopsin trafficked to the outer segments during the course of degeneration. Furthermore, KIF3A deletion did not affect synaptic anterograde trafficking. The results indicate that trafficking of membrane proteins to the outer segment is dependent on kinesin-II in cone, but not rod photoreceptors, even though rods and cones share similar structures, and closely related Phototransduction polypeptides.
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the function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors
Journal of Biological Chemistry, 2007Co-Authors: Wolfgang Baehr, Sukanya Karan, Tadao Maeda, Dong Gen Luo, Darin J Bronson, Carl B Watt, Kingwai Yau, Jeanne M Frederick, Krzysztof PalczewskiAbstract:Abstract Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to Phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1–/– and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated Phototransduction proteins.
Christian P. Hamel - One of the best experts on this subject based on the ideXlab platform.
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a truncated form of rod photoreceptor pde6 β subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the γ subunit
PLOS ONE, 2014Co-Authors: Pallavi Cheguru, Audrey Senechal, Beatrice Bocquet, Gael Manes, Anurima Majumder, Christian P. HamelAbstract:Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod Phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the Phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the Phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the Phototransduction thus leading to the absence of rod adaptation.
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a truncated form of rod photoreceptor pde6 β subunit causes autosomal dominant congenital stationary night blindness by interfering with the inhibitory activity of the γ subunit
PLOS ONE, 2014Co-Authors: Pallavi Cheguru, Audrey Senechal, Beatrice Bocquet, Gael Manes, Anurima Majumder, Christian P. HamelAbstract:Autosomal dominant congenital stationary night blindness (adCSNB) is caused by mutations in three genes of the rod Phototransduction cascade, rhodopsin (RHO), transducin α-subunit (GNAT1), and cGMP phosphodiesterase type 6 β-subunit (PDE6B). In most cases, the constitutive activation of the Phototransduction cascade is a prerequisite to cause adCSNB. The unique adCSNB-associated PDE6B mutation found in the Rambusch pedigree, the substitution p.His258Asn, leads to rod photoreceptors desensitization. Here, we report a three-generation French family with adCSNB harboring a novel PDE6B mutation, the duplication, c.928-9_940dup resulting in a tyrosine to cysteine substitution at codon 314, a frameshift, and a premature termination (p.Tyr314Cysfs*50). To understand the mechanism of the PDE6β1-314fs*50 mutant, we examined the properties of its PDE6-specific portion, PDE6β1-313. We found that PDE6β1-313 maintains the ability to bind noncatalytic cGMP and the inhibitory γ-subunit (Pγ), and interferes with the inhibition of normal PDE6αβ catalytic subunits by Pγ. Moreover, both truncated forms of the PDE6β protein, PDE6β1-313 and PDE6β1-314fs*50 expressed in rods of transgenic X. laevis are targeted to the Phototransduction compartment. We hypothesize that in affected family members the p.Tyr314Cysfs*50 change results in the production of the truncated protein, which binds Pγ and causes constitutive activation of the Phototransduction thus leading to the absence of rod adaptation.
Rameshwar K Sharma - One of the best experts on this subject based on the ideXlab platform.
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differential ca 2 sensor guanylate cyclase activating protein modes of photoreceptor rod outer segment membrane guanylate cyclase signaling
Biochemistry, 2012Co-Authors: Teresa Duda, Alexandre Pertzev, Rameshwar K SharmaAbstract:Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of Phototransduction. It is a bimodal Ca2+ signal transduction switch, operating between 20 and ∼1000 nM. Modulated by Ca2+ sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca2+]i from 200 to 20 nM progressively turns it “on”, as does the modulation by the Ca2+ sensor S100B, increasing [Ca2+]i from 100 to 1000 nM. The GCAP mode plays a vital role in Phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca2+ signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 ups...
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rod outer segment membrane guanylate cyclase type 1 ros gc1 gene structure organization and regulation by phorbol ester a protein kinase c activator
Molecular and Cellular Biochemistry, 1998Co-Authors: Teresa Duda, Venkateswar Venkataraman, Anuradha Krishnan, Rameshwar K SharmaAbstract:At present there are two recognized members of the ROS-GC subfamily of membrane guanylate cyclases. They are ROS-GC1 and ROS-GC2. A distinctive feature of this family is that its members are not switched on by the extracellular peptide hormones; instead, they are modulated by intracellular Ca2+ signals, consistent to their linkage with Phototransduction. An intriguing feature of ROS-GC1, which distinguishes it from ROS-GC2, is that it has two Ca2+ switches. One switch inhibits the enzyme at micromolar concentrations of Ca2+, as in Phototransduction; the other, stimulates. The stimulatory switch, most likely, is linked to retinal synaptic activity. Thus, ROS-GC1 is linked to both Phototransduction and the synaptic activity. The present study describes (1) the almost complete structural identity of 18.5 kb ROS-GC1 gene; (2) its structural organization: the gene is composed of 20 exons and 19 introns with classical GT/AG boundaries; (3) the activity of the ROS-GC1 promoter assayed through luciferase reporter in COS cells; and (4) induction of the gene by phorbol ester, a protein kinase C (PKC) activator. The co-presence of PKC and ROS-GC1 in photoreceptors suggests that regulation of the ROS-GC1 gene by PKC might be a physiologically relevant phenomenon.
Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.
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a functional kinase homology domain is essential for the activity of photoreceptor guanylate cyclase 1
Journal of Biological Chemistry, 2010Co-Authors: Grzegorz Bereta, Wolfgang Baehr, Benlian Wang, Philip D Kiser, Geeng Fu Jang, Krzysztof PalczewskiAbstract:Phototransduction is carried out by a signaling pathway that links photoactivation of visual pigments in retinal photoreceptor cells to a change in their membrane potential. Upon photoactivation, the second messenger of Phototransduction, cyclic GMP, is rapidly degraded and must be replenished during the recovery phase of Phototransduction by photoreceptor guanylate cyclases (GCs) GC1 (or GC-E) and GC2 (or GC-F) to maintain vision. Here, we present data that address the role of the GC kinase homology (KH) domain in cyclic GMP production by GC1, the major cyclase in photoreceptors. First, experiments were done to test which GC1 residues undergo phosphorylation and whether such phosphorylation affects cyclase activity. Using mass spectrometry, we showed that GC1 residues Ser-530, Ser-532, Ser-533, and Ser-538, located within the KH domain, undergo light- and signal transduction-independent phosphorylation in vivo. Mutations in the putative Mg2+ binding site of the KH domain abolished phosphorylation, indicating that GC1 undergoes autophosphorylation. The dramatically reduced GC activity of these mutants suggests that a functional KH domain is essential for cyclic GMP production. However, evidence is presented that autophosphorylation does not regulate GC1 activity, in contrast to phosphorylation of other members of this cyclase family.
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the function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors
Journal of Biological Chemistry, 2007Co-Authors: Wolfgang Baehr, Sukanya Karan, Tadao Maeda, Dong Gen Luo, Darin J Bronson, Carl B Watt, Kingwai Yau, Jeanne M Frederick, Krzysztof PalczewskiAbstract:Abstract Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to Phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin α-subunit were mostly unaffected. Outer segment membranes of GC1–/– and GC double knock-out cones were destabilized and devoid of cone transducin (α- and γ-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the down-regulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated Phototransduction proteins.
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rhodopsin phosphorylation and dephosphorylation in vivo
Journal of Biological Chemistry, 1995Co-Authors: Hiroshi Ohguro, Preston J Van Hooser, Ann H Milam, Krzysztof PalczewskiAbstract:Abstract Rhodopsin is an important member of the superfamily of G protein-coupled receptors. In vitro studies have suggested that multiphosphorylation of rhodopsin is a pivotal step in Phototransduction. Because the in vitro biochemical experiments were conducted under non-physiological conditions, we investigated the phosphorylation of mouse rhodopsin in vivo and determined the sites of phosphorylation and the time course of dephosphorylation. We found that a single phosphate group is incorporated into the rhodopsin molecule in a light-dependent manner, primarily at Ser after flashes and at Ser after continuous illumination. Dephosphorylation of these sites had different kinetics and spatial distribution in rod outer segments. Dephosphorylation of Ser was complete within 30 min, while Ser was dephosphorylated much slower (requiring up to 60 min), correlating with the regeneration of rhodopsin. These results suggest that phosphorylation of Ser and Ser plays different roles in Phototransduction.