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Shahrokh C. Khani - One of the best experts on this subject based on the ideXlab platform.

  • Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase
    The Journal of biological chemistry, 2015
    Co-Authors: Keisuke Sakurai, Shahrokh C. Khani, Jeannie Chen, Vladimir J Kefalov
    Abstract:

    Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of Rhodopsin Kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light.

  • variation in Rhodopsin Kinase expression alters the dim flash response shut off and the light adaptation in rod photoreceptors
    Investigative Ophthalmology & Visual Science, 2011
    Co-Authors: Keisuke Sakurai, Shahrokh C. Khani, Joyce E Young, Vladimir J Kefalov
    Abstract:

    Rod photoreceptors are exquisitely sensitive light detectors that are perfectly suited for function in dim light.1 The detection of light and the conversion of its energy into an electric signal take place at the membrane discs in the outer segments of rod photoreceptors. Phototransduction is initiated by the absorption of a photon by a molecule of visual pigment, Rhodopsin.2 In its active state, Rhodopsin (R*) binds to a G protein, transducin, triggering the exchange of guanosine-5′-triphosphate (GTP) for guanosine-5′-diphosphate (GDP) on its α-subunit (Tα). In turn, the activated Tα-GTP binds to an effector enzyme, cGMP phosphodiesterate (PDE) which eventually results in closure of cGMP-gated channels in the outer segment. The amplification of the rod signal is produced by two phototransduction components: a single R* activating multiple Tα subunits, and a single Tα/PDE complex hydrolyzing multiple cGMP molecules. Response termination requires the timely inactivation of both R* and Tα/PDE. First, R* is partially inactivated on phosphorylation by Rhodopsin Kinase (GRK1),3,4 a reaction inhibited in darkness by recoverin.5 Phosphorylated Rhodopsin is then completely inactivated on binding to arrestin.6 Transducin is inactivated in a GTP hydrolysis reaction catalyzed by regulator of G protein signalling 9 (RGS9)7 which returns transducin into its inactive GDP-bound state. The molecular mechanisms that rate-limit the inactivation of the transduction cascade and dominate the light response shut off have been an active area of research but are still subjects of debate. While early studies indicated that shut off of the transduction cascade is controlled by the inactivation of R*8, a recent study demonstrated that the inactivation of the Tα/PDE complex is the rate-limiting step in the shut off of the light response in mouse rods.9 The same study stated that overexpression of Rhodopsin Kinase does not affect the termination of the light response and concluded that the inactivation of R* is very rapid (≤80 ms) and substantially faster than that of Tα/PDE (see also Ref. 10). However, whether the inactivation of R* by Rhodopsin Kinase is slow enough to modulate the overall response kinetics in rods remains controversial.11 More importantly, it is not known whether Rhodopsin phosphorylation affects the function of rods during light adaptation. We recently generated transgenic mice with rods and cones overexpressing GRK1 driven by the full length Rhodopsin Kinase promoter12 in preparation for studying how GRK1 expression modulates cone function. We performed initial recordings from the rods of these mice to confirm that, as previously suggested, overexpression of GRK1 in mouse rods does not affect the kinetics of their responses.9 Surprisingly, we observed a notable acceleration of rod response shut off in rods overexpressing GRK1. We proceeded to characterize in detail the effect of GRK1 expression level on the function of mouse rods in darkness and during background adaptation. Our results demonstrate that R* inactivation by Rhodopsin Kinase affects the kinetics of the single-photon response and plays a role in the background adaptation of mammalian rods.

  • conserved interactions of a compact highly active enhancer promoter upstream of the Rhodopsin Kinase grk1 gene
    Genomics, 2007
    Co-Authors: Joyce E Young, Eileen M. Kasperek, Agnieszka Lis, Todd Vogt, Shahrokh C. Khani
    Abstract:

    Rhodopsin Kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  • Conserved interactions of a compact highly active enhancer/promoter upstream of the Rhodopsin Kinase (GRK1) gene.
    Genomics, 2007
    Co-Authors: Joyce E Young, Todd M. Vogt, Eileen M. Kasperek, Agnieszka Lis, Shahrokh C. Khani
    Abstract:

    Rhodopsin Kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  • Conserved structure and spatiotemporal function of the compact Rhodopsin Kinase (GRK1) enhancer/promoter.
    Molecular vision, 2005
    Co-Authors: Joyce E Young, Kenneth W. Gross, Shahrokh C. Khani
    Abstract:

    Purpose: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of Rhodopsin Kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites. Methods: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development. Results: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening. Conclusions: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.

Krzysztof Palczewski - One of the best experts on this subject based on the ideXlab platform.

  • Structures of Rhodopsin Kinase in different ligand states reveal key elements involved in G protein-coupled receptor Kinase activation.
    The Journal of biological chemistry, 2008
    Co-Authors: Puja Singh, Krzysztof Palczewski, Benlian Wang, Tadao Maeda, John J.g. Tesmer
    Abstract:

    G protein-coupled receptor (GPCR) Kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins. Here we report six crystal structures of Rhodopsin Kinase (GRK1), revealing not only three distinct nucleotide-binding states of a GRK but also two key structural elements believed to be involved in the recognition of activated GPCRs. The first is the C-terminal extension of the Kinase domain, which was observed in all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N terminus, observed in one of the GRK1·(Mg2+)2·ATP structures. The N terminus was also clearly phosphorylated, leading to the identification of two novel phosphorylation sites by mass spectral analysis. Co-localization of the N terminus and the C-terminal extension near the hinge of the Kinase domain suggests that activated GPCRs stimulate Kinase activity by binding to this region to facilitate full closure of the Kinase domain.

  • null mutation in the Rhodopsin Kinase gene slows recovery kinetics of rod and cone phototransduction in man
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Artur V Cideciyan, Shahrokh C. Khani, Xinyu Zhao, Lori Nielsen, Samuel G Jacobson, Krzysztof Palczewski
    Abstract:

    Rhodopsin Kinase (RK), a specialized G-protein-coupled receptor Kinase expressed in retina, is involved in quenching of light-induced signal transduction in photoreceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recovery phase abnormalities of rod-isolated photoresponses by electroretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated Rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably constitute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated Rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.

  • Null mutation in the Rhodopsin Kinase gene slows recovery kinetics of rod and cone phototransduction in man (dark adaptationynightblindnessyOguchi diseaseyphotoreceptoryretina)
    1998
    Co-Authors: Artur V Cideciyan, Lori Nielsen, Samuel G Jacobson, C. Khani, Krzysztof Palczewski
    Abstract:

    Rhodopsin Kinase (RK), a specialized G- protein-coupled receptor Kinase expressed in retina, is involved in quenching of light-induced signal transduction in photore- ceptors. The role of RK in recovery after photoactivation has been explored in vitro and in vivo experimentally but has not been specifically defined in humans. We investigated the effects on human vision of a mutation in the RK gene causing Oguchi disease, a recessively inherited retinopathy. In vitro experiments demonstrated that the mutation, a deletion of exon 5, abolishes the enzymatic activity of RK and is likely a null. Both a homozygote and heterozygote with this RK mutation had recov- ery phase abnormalities of rod-isolated photoresponses by elec- troretinography (ERG); photoactivation was normal. Kinetics of rod bleaching adaptation by psychophysics were dramatically slowed in the homozygote but normal final thresholds were attained. Light adaptation was normal at low backgrounds but became abnormal at higher backgrounds. A slight slowing of cone deactivation kinetics in the homozygote was detected by ERG. Cone bleaching adaptation and background adaptation were normal. In this human in vivo condition without a functional RK and probable lack of phosphorylation and arrestin binding to activated Rhodopsin, reduction of photolyzed chromophore and regeneration processes with 11-cis-retinal probably consti- tute the sole pathway for recovery of rod sensitivity. The role of RK in rods would thus be to accelerate inactivation of activated Rhodopsin molecules that in concert with regeneration leads to the normal rate of recovery of sensitivity. Cones may rely mainly on regeneration for the inactivation of photolyzed visual pigment, but RK also contributes to cone recovery.

  • Molecular cloning and localization of Rhodopsin Kinase in the mammalian pineal
    Visual neuroscience, 1997
    Co-Authors: Xinyu Zhao, Jing Huang, Françoise Haeseleer, Robert N. Fariss, Wolfgang Baehr, Ann H. Milam, Krzysztof Palczewski
    Abstract:

    Several retinal photoreceptor proteins involved in phototransduction have also been found in the mammalian pineal. This study demonstrates that rat and human pineals express protein Kinases that are identical to the corresponding rod photoreceptor Rhodopsin Kinases. The deduced amino acid sequence of rat and human Rhodopsin Kinases have 84% sequence similarity to the earlier reported sequence of the bovine retinal enzyme, with complete conservation of the topological regions containing the position of the catalytic domain and sites of posttranslational modifications. Rat pineal also expresses rod opsin and putative blue cone opsin. Using immunocytochemistry, rod opsin and Rhodopsin Kinase were found to be co-localized in pinealocytes in the human tissue. These data demonstrate that the mammalian pineal contains light-sensitive opsins and a Kinase involved in their inactivation. These findings correlate with an earlier report that neonatal rats show extraretinal light sensitivity, and suggest that a functional photoreceptive system may be present in the adult mammalian pineal.

  • Mechanism of Rhodopsin phosphorylation.
    Biophysical Chemistry, 1995
    Co-Authors: Xinyu Zhao, Krzysztof Palczewski, Hiroshi Ohguro
    Abstract:

    A key reaction in the inactivation of Rhodopsin is its phosphorylation by Rhodopsin Kinase. In recent years, extensive studies related to Rhodopsin Kinase function and enzymatic properties were carried out. Rhodopsin Kinase is a Ser/Thr protein Kinase and a member of the G protein-coupled receptor Kinases sub-family (GRKs) which consists of six recently identified members. Photolyzed Rhodopsin is phosphorylated by Rhodopsin Kinase sequentially, with the first phosphate transferred preferentially to Ser-338, and subsequent phosphates transferred to Ser-343 and Thr-336. The binding of arrestin to the receptor, and reduction of the photolyzed chromophore all-trans-retinal to all-trans-retinol limits physiologically significant phosphorylation at no more than three sites (H. Ohguro, R.S. Johnson, L.H. Ericsson, K.A. Walsh and K. Palczewski, Biochemistry, 33 (1994) 1023). A similar phosphorylation reaction is implicated in most, if not all, G protein-coupled receptors during their desensitization.

Ivan I. Senin - One of the best experts on this subject based on the ideXlab platform.

  • Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase
    Frontiers in molecular neuroscience, 2012
    Co-Authors: I. I. Grigoriev, Karl-wilhelm Koch, Sergei E. Permyakov, Ivan I. Senin, Evgeni Yu. Zernii, Konstantin E. Komolov, N.k. Tikhomirova, Pavel P. Philippov
    Abstract:

    Phosphorylation of photoactivated Rhodopsin by Rhodopsin Kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.

  • Involvement of the recoverin C-terminal segment in recognition of the target enzyme Rhodopsin Kinase.
    The Biochemical journal, 2011
    Co-Authors: Evgeni Yu. Zernii, Sergei E. Permyakov, Eugene A. Permyakov, Konstantin E. Komolov, Tatiana V. Kolpakova, Daniele Dell'orco, Annika Poetzsch, Ekaterina L. Knyazeva, I. I. Grigoriev, Ivan I. Senin
    Abstract:

    -dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and Rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for Rhodopsin Kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-Kinase complex provided insight into the protein-protein interface between the Kinase and the C-terminus of recoverin. Based on these results we suggest that Phe 3 from the N-terminal helix of Rhodopsin Kinase and Lys 192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognitionbyrecoverin.Takentogether,theresultsofthepresent study reveal a novel Rhodopsin-Kinase-binding site within the C- terminal region of recoverin, and highlights its significance for target recognition and regulation.

  • Involvement of recoverin C-terminal segment in recognition of the target enzyme Rhodopsin Kinase
    Biochemical Journal, 2011
    Co-Authors: Evgeni Yu. Zernii, Sergei E. Permyakov, Eugene A. Permyakov, Konstantin E. Komolov, Tatiana V. Kolpakova, Daniele Dell'orco, Annika Poetzsch, Ekaterina Knyazeva, Ilya Grigoriev, Ivan I. Senin
    Abstract:

    Neuronal calcium sensor (NCS) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini that could therefore serve as structural control elements providing specific functions like target recognition or Ca2+-sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of Rhodopsin Kinase, GRK1, in a Ca2+-dependent manner. We investigated a series of recoverin forms that were mutated at the C-terminus. Using pull down assay, surface plasmon resonance spectroscopy and Rhodopsin phosphorylation assay, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for Rhodopsin Kinase. Site directed mutagenesis of single amino acids in combination with structural analysis and computational modeling of recoverin-Kinase complex provided insight in the protein-protein interface between Kinase and the C-terminus of recoverin. Based on these data we suggest that F3 from the N-terminal helix of Rhodopsin Kinase and K192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, our study revealed a novel Rhodopsin Kinase binding site within C-terminal region of recoverin and highlights its significance for target recognition and regulation.

  • Mechanism of Rhodopsin Kinase regulation by recoverin.
    Journal of neurochemistry, 2009
    Co-Authors: Konstantin E. Komolov, Muhammad Akhtar, Pavel P. Philippov, Ivan I. Senin, Valeriya A. Churumova, I. I. Grigoriev, Nadezda A. Kovaleva, Mathias P. Christoph, Karl-wilhelm Koch
    Abstract:

    Recoverin is suggested to inhibit Rhodopsin Kinase (GRK1) at high [Ca(2+)] in the dark state of the photoreceptor cell. Decreasing [Ca(2+)] terminates inhibition and facilitates phosphorylation of illuminated Rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of Rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with Rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca(2+)-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with Rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic Kinase groove. Ca(2+)-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.

  • One of the Ca2+ binding sites of recoverin exclusively controls interaction with Rhodopsin Kinase.
    Biological chemistry, 2005
    Co-Authors: Konstantin E. Komolov, Pavel P. Philippov, Ivan I. Senin, Valeriya A. Churumova, Dimitri V Zinchenko, Svetlana A Vaganova, Oliver H. Weiergräber, Karl-wilhelm Koch
    Abstract:

    Recoverin is a neuronal calcium sensor protein that controls the activity of Rhodopsin Kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target Rhodopsin Kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.

Joyce E Young - One of the best experts on this subject based on the ideXlab platform.

  • variation in Rhodopsin Kinase expression alters the dim flash response shut off and the light adaptation in rod photoreceptors
    Investigative Ophthalmology & Visual Science, 2011
    Co-Authors: Keisuke Sakurai, Shahrokh C. Khani, Joyce E Young, Vladimir J Kefalov
    Abstract:

    Rod photoreceptors are exquisitely sensitive light detectors that are perfectly suited for function in dim light.1 The detection of light and the conversion of its energy into an electric signal take place at the membrane discs in the outer segments of rod photoreceptors. Phototransduction is initiated by the absorption of a photon by a molecule of visual pigment, Rhodopsin.2 In its active state, Rhodopsin (R*) binds to a G protein, transducin, triggering the exchange of guanosine-5′-triphosphate (GTP) for guanosine-5′-diphosphate (GDP) on its α-subunit (Tα). In turn, the activated Tα-GTP binds to an effector enzyme, cGMP phosphodiesterate (PDE) which eventually results in closure of cGMP-gated channels in the outer segment. The amplification of the rod signal is produced by two phototransduction components: a single R* activating multiple Tα subunits, and a single Tα/PDE complex hydrolyzing multiple cGMP molecules. Response termination requires the timely inactivation of both R* and Tα/PDE. First, R* is partially inactivated on phosphorylation by Rhodopsin Kinase (GRK1),3,4 a reaction inhibited in darkness by recoverin.5 Phosphorylated Rhodopsin is then completely inactivated on binding to arrestin.6 Transducin is inactivated in a GTP hydrolysis reaction catalyzed by regulator of G protein signalling 9 (RGS9)7 which returns transducin into its inactive GDP-bound state. The molecular mechanisms that rate-limit the inactivation of the transduction cascade and dominate the light response shut off have been an active area of research but are still subjects of debate. While early studies indicated that shut off of the transduction cascade is controlled by the inactivation of R*8, a recent study demonstrated that the inactivation of the Tα/PDE complex is the rate-limiting step in the shut off of the light response in mouse rods.9 The same study stated that overexpression of Rhodopsin Kinase does not affect the termination of the light response and concluded that the inactivation of R* is very rapid (≤80 ms) and substantially faster than that of Tα/PDE (see also Ref. 10). However, whether the inactivation of R* by Rhodopsin Kinase is slow enough to modulate the overall response kinetics in rods remains controversial.11 More importantly, it is not known whether Rhodopsin phosphorylation affects the function of rods during light adaptation. We recently generated transgenic mice with rods and cones overexpressing GRK1 driven by the full length Rhodopsin Kinase promoter12 in preparation for studying how GRK1 expression modulates cone function. We performed initial recordings from the rods of these mice to confirm that, as previously suggested, overexpression of GRK1 in mouse rods does not affect the kinetics of their responses.9 Surprisingly, we observed a notable acceleration of rod response shut off in rods overexpressing GRK1. We proceeded to characterize in detail the effect of GRK1 expression level on the function of mouse rods in darkness and during background adaptation. Our results demonstrate that R* inactivation by Rhodopsin Kinase affects the kinetics of the single-photon response and plays a role in the background adaptation of mammalian rods.

  • conserved interactions of a compact highly active enhancer promoter upstream of the Rhodopsin Kinase grk1 gene
    Genomics, 2007
    Co-Authors: Joyce E Young, Eileen M. Kasperek, Agnieszka Lis, Todd Vogt, Shahrokh C. Khani
    Abstract:

    Rhodopsin Kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  • Conserved interactions of a compact highly active enhancer/promoter upstream of the Rhodopsin Kinase (GRK1) gene.
    Genomics, 2007
    Co-Authors: Joyce E Young, Todd M. Vogt, Eileen M. Kasperek, Agnieszka Lis, Shahrokh C. Khani
    Abstract:

    Rhodopsin Kinase (RK) is a conserved component of the light adaptation and recovery pathways shared among rod and cone photoreceptors of a variety of species. To gain insight into transcriptional mechanisms driving RK and potentially other genes of similar spatial profile, the components and the interactions of the highly compact enhancer/promoter region (E/P) upstream of the human RK gene were examined. Cross-species comparison outlined an active 49-bp widely shared E/P core as the major site of conservation in the entire 5' flanking sequence. The area consisted of a bicoid-type homeodomain recognition cassette and a unique T-rich module interacting with TATA-binding proteins. Homeodomain interactions involved primarily Crx and secondarily Otx2. Both strongly stimulated the E/P. In the absence of Crx, persistent E/P activity shifted from the outer retina to the inner to follow the Otx2 pattern. The spatial patterns were largely unaffected by the absence of rod transcription factors, Nrl and Nr2e3, and the RK transcriptional activity preceded the surge in rod-specific transcription. Conserved bicoid homeodomain factors thus appear to be the key factors governing localization of RK E/P activity in retina and photoreceptors.

  • Conserved structure and spatiotemporal function of the compact Rhodopsin Kinase (GRK1) enhancer/promoter.
    Molecular vision, 2005
    Co-Authors: Joyce E Young, Kenneth W. Gross, Shahrokh C. Khani
    Abstract:

    Purpose: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of Rhodopsin Kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites. Methods: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development. Results: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening. Conclusions: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.

  • conserved structure and spatiotemporal function of the compact Rhodopsin Kinase grk1 enhancer promoter
    Molecular Vision, 2005
    Co-Authors: Joyce E Young, Kenneth W. Gross, Shahrokh C. Khani
    Abstract:

    Purpose: To demonstrate that the crucial elements responsible for the spatial and temporal expression patterns of Rhodopsin Kinase (Rk) are contained within a narrow conserved segment immediately flanking the Rk transcription start sites. Methods: Sequences upstream of the mouse Rk gene were compared to the human sequence to identify areas of conservation. Transgenic mice carrying a segment of the conserved human DNA sequence linked upstream of the green fluorescent protein (GFP) gene were examined by fluorescence microscopy and RT-PCR to localize GFP expression in retina and pineal gland. Rk and GFP temporal expression patterns were further compared by immunostaining and real-time RT-PCR in transgenic eyes during development. Results: Comparison of the mouse and human 5' flanking sequence revealed only a small island of conserved sequence upstream of the respective Rk start sites. Uniform GFP expression was supported by a 0.2 kb fragment of the conserved human sequence in the transgenic mouse rods, cones, and pinealocytes. Developmental studies revealed an exponential rise in Rk and GFP transcripts in the first ten day postnatal period followed by a plateau later extending to adulthood. Rk and GFP proteins were first detected after postnatal day 10 and rose in parallel afterwards, overlapping in time with the maturation of photoreceptor outer segments and eye opening. Conclusions: The conserved short enhancer/promoter immediately upstream of the Rk gene contains the key elements required for appropriate response to spatial and temporal cues during photoreceptor cell differentiation and fate determination. The above studies narrow the core sequences that govern gene expression in photoreceptors in vivo.

Karl-wilhelm Koch - One of the best experts on this subject based on the ideXlab platform.

  • molecular recognition of Rhodopsin Kinase grk1 and recoverin is tuned by switching intra and intermolecular electrostatic interactions
    Biochemistry, 2019
    Co-Authors: Seher Abbas, Valerio Marino, Daniele Dellorco, Karl-wilhelm Koch
    Abstract:

    G protein-coupled receptor Kinase 1 (GRK1) or Rhodopsin Kinase is under specific control of the neuronal Ca2+-sensor protein recoverin, which is a critical feedback mechanism responsible for the modulation of the shape and sensitivity of the rod cell photoresponse. This process requires the precise matching of interacting protein surfaces and the dynamic changes in protein conformations. Here we study the molecular recognition process of recoverin and GRK1 by testing the hypothesis of a cation−π interaction pair in the recoverin–GRK1 complex. The critical role of residue K192 in recoverin was investigated by site-directed mutagenesis and subsequent structural and functional analysis. The following methods were used: isothermal titration calorimetry, fluorescence and circular dichroism spectroscopy, Ca2+-dependent membrane binding, and protein–protein interaction analysis by back scattering interferometry and surface plasmon resonance. While neutralizing the charge at K in the mutant K192L did not prevent ...

  • Synergetic Effect of Recoverin and Calmodulin on Regulation of Rhodopsin Kinase
    Frontiers in molecular neuroscience, 2012
    Co-Authors: I. I. Grigoriev, Karl-wilhelm Koch, Sergei E. Permyakov, Ivan I. Senin, Evgeni Yu. Zernii, Konstantin E. Komolov, N.k. Tikhomirova, Pavel P. Philippov
    Abstract:

    Phosphorylation of photoactivated Rhodopsin by Rhodopsin Kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.

  • Mechanism of Rhodopsin Kinase regulation by recoverin.
    Journal of neurochemistry, 2009
    Co-Authors: Konstantin E. Komolov, Muhammad Akhtar, Pavel P. Philippov, Ivan I. Senin, Valeriya A. Churumova, I. I. Grigoriev, Nadezda A. Kovaleva, Mathias P. Christoph, Karl-wilhelm Koch
    Abstract:

    Recoverin is suggested to inhibit Rhodopsin Kinase (GRK1) at high [Ca(2+)] in the dark state of the photoreceptor cell. Decreasing [Ca(2+)] terminates inhibition and facilitates phosphorylation of illuminated Rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of Rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with Rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca(2+)-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with Rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic Kinase groove. Ca(2+)-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.

  • One of the Ca2+ binding sites of recoverin exclusively controls interaction with Rhodopsin Kinase.
    Biological chemistry, 2005
    Co-Authors: Konstantin E. Komolov, Pavel P. Philippov, Ivan I. Senin, Valeriya A. Churumova, Dimitri V Zinchenko, Svetlana A Vaganova, Oliver H. Weiergräber, Karl-wilhelm Koch
    Abstract:

    Recoverin is a neuronal calcium sensor protein that controls the activity of Rhodopsin Kinase in a Ca(2+)-dependent manner. Mutations in the EF-hand Ca2+ binding sites are valuable tools for investigating the functional properties of recoverin. In the recoverin mutant E121Q (Rec E121Q ) the high-affinity Ca2+ binding site is disabled. The non-myristoylated form of Rec E121Q binds one Ca2+ via its second Ca(2+)-binding site (EF-hand 2), whereas the myristoylated variant does not bind Ca2+ at all. Binding of Ca2+ to non-myristoylated Rec E121Q apparently triggers exposure of apolar side chains, allowing for association with hydrophobic matrices. Likewise, an interaction surface for the recoverin target Rhodopsin Kinase is constituted upon Ca2+ binding to the non-acylated mutant. Structural changes resulting from Ca(2+)-occupation of EF-hand 2 in myristoylated and non-myristoylated recoverin variants are discussed in terms of critical conditions required for biological activity.

  • recoverin and Rhodopsin Kinase activity in detergent resistant membrane rafts from rod outer segments
    Journal of Biological Chemistry, 2004
    Co-Authors: Ivan I. Senin, Pavel P. Philippov, Valeriya A. Churumova, N.k. Tikhomirova, Doris Hoppnerheitmann, Olga O Polkovnikova, Karl-wilhelm Koch
    Abstract:

    Abstract Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of Rhodopsin Kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca2+ concentrations. Inhibition of Rhodopsin Kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca2+ sensitivity of Rhodopsin Kinase inhibition in DRMs was shifted to lower free Ca2+ concentration in comparison with the initial rod outer segment membranes (IC50 = 0.76 μm in DRMs and 1.91 μm in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-β-cyclodextrin yielded a similar shift of the Ca2+-dependent dose-response curve of Rhodopsin Kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca2+-dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.