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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyCritical role of central 139 loop in stability and binding selectivityof arrestin-1Sergey A. Vishnivetskiy, Faiza Baameur, Kristen R. Findley,Vsevolod V. Gurevich. Pharmacology, Vanderbilt University,Nashville, TN.Purpose: To elucidate the functional role of central 139-loop on thereceptor-binding surface of arrestin-1Methods: Deletions of 139-loop and mutations that disrupt itsinteractions with other elements of the molecule were introduced inwild type arrestin-1 and its enhanced 3A mutant with high binding tounphosphorylated light-activated rhodopsin (Rh*). We tested bindingselectivity and thermal stability of these mutantsResults: Using intra-molecular distance measurements by DEER wedetected large movement of arrestin-1 139-loop to the side, awayfrom the incoming receptor, upon its binding to activephosphorhodopsin (P-Rh*). Therefore, we hypothesized thatelimination of this loop would promote rhodopsin binding. Deletionsof increasing length in 139-loop progressively enhanced arrestin-1binding to non-preferred forms of rhodopsin: Rh*, dark P-Rh, andphospho-opsin. The interactions of 139-loop with the neighboringfinger loop involved in receptor binding were disrupted by chargeeliminations and reversals on both sides. These milder perturbationsincreased arrestin-1 binding to non-preferred forms of rhodopsin, butto a lesser extent. In all cases an increase in binding to Rh*, dark P-Rh, and phospho-opsin correlated with a decrease in thermal stabilityof the mutants. Similar effects of deletions and mutations in 139-loopon both binding selectivity and thermal stability were observed in thecontext of WT arrestin-1 and an enhanced 3A mutant. whichdemonstrates higher binding to non-preferred forms of rhodopsinConclusions: Central 139 loop is an earlier unappreciated elementstabilizing basal arrestin-1 conformation and precluding its binding tonon-preferred forms of rhodopsin. Thermal stability of the basal stateof arrestin-1 directly correlates with its selectivity for P-Rh*. NIHgrants EY011500, GM077561, and GM081756 (VVG).Commercial Relationships: Sergey A. Vishnivetskiy, None; FaizaBaameur, None; Kristen R. Findley, None; Vsevolod V. Gurevich,NoneSupport: NIH grants EY011500, GM077561, and GM081756(VVG).Program Number: 2459 Poster Board Number: D0064Presentation Time: 2:45 PM - 4:30 PMModulation of Mouse Rod cGMP-Gated Channels by Grb14Raju V. Rajala 1 , Michael L. Woodruff 2 , Gordon L. Fain 2, 3 .1 Ophthal/Dean McGee Eye Inst, Univ of Oklahoma Hlth Sci Ctr,Oklahoma City, OK; 2 Integrative Biology and Physiology, UCLA,Los Angeles, CA; 3 Jules Stein Eye Inst, UCLA, Los Angeles, CA.Purpose: Previous experiments indicated that growth factor receptorboundprotein 14 (Grb14) may modulate rod cyclic guanosinenucleotide (cGMP) gated channels by decreasing channel affinity forcGMP. We tested this hypothesis by recording electrical responsesfrom rods in which the gene for the Grb14 protein had been deleted.Methods: Grb14 -/- mice were obtained from Dr. Roger Daly, GarvanInstitute of Medical Research, Australia. Suction-electrode recordingswere made from single mouse rods by methods previously described(e.g. Chen et al., J. Neurosci. 30:16232-40, 2010).Results: Rod responses of dark-adapted Grb14 -/- mice decayed morerapidly than strain-controlled wild-type (WT) rods (Figure 1) withdecreased values of τ REC at all light intensities; integration timedecreased from 292 ± 20 ms (SE, n=15) in WT to 215 ± 12 ms (17)in Grb14 -/- mice. This result is consistent with an increase in channelaffinity for cGMP produced by deletion of Grb14. Grb14 -/- mouserods also showed a large and significant decrease in the limiting timeconstant τ D from 171 ± 15 ms (15) in WT to 118 ± 5 ms (17) inGrb14 -/- rods. Similar though smaller decreases in both τ REC and τ Dwere previously reported from mice whose channels lack thecalmodulin binding site (Chen et al., 2010), which should also resultin increased channel affinity for cGMP. Although Grb14 wasreported to translocate from inner to outer segment in the light(Rajala et al. Biochemistry 48:5563-5572, 2009), we saw no apparentdifference in the effect of Grb14 gene deletion on response decay indark-adapted and bleached or background adapted rods, and lightadaption in Grb14 -/- rods was essentially the same as in WT.Conclusions: Our results confirm a role of Grb14 as a modulator ofmouse rod cGMP-gated channels in both dark-adapted and lightadaptedrods. The physiological role of this modulation remains to beelucidated.Figure 1. Responses of WT and Grb14 -/- rods to 10 ms flashes ofintensities from 2.4 to 2600 photons μm -2 .Commercial Relationships: Raju V. Rajala, None; Michael L.Woodruff, None; Gordon L. Fain, NoneSupport: NIH/NEI grant (EY01844, EY016507, EY00871)Program Number: 2460 Poster Board Number: D0065Presentation Time: 2:45 PM - 4:30 PMIndependent manipulation of binding selectivity and selfassociationof arrestin-1Vsevolod V. Gurevich 1 , Sergey A. Vishnivetskiy 1 , Qiuyan Chen 1 ,Maria C. Palazzo 1 , Evan K. Brooks 2 , Christian Altenbach 2 , Tina M.Iverson 1 , Wayne L. Hubbell 2 . 1 Pharmacology, Vanderbilt University,Nashville, TN; 2 JSEI, UCLA, Los Angeles, TN.Purpose: To test whether binding selectivity and self-association ofarrestin-1 can be independently manipulated for research andtherapeutic useMethods: Versions of arrestin-1 with activating mutations enhancingthe binding to unphosphorylated light-activated rhodopsin (Rh*) andsubstitutions suppressing self-association were constructed,expressed, and their binding selectivity, stability, and oligomerizationwere testedResults: Arrestin-1 preferentially binds active phosphorylatedrhodopsin (P-Rh*). An enhanced mutant with increased binding toRh* partially compensates for the lack of rhodopsin phosphorylationin vivo. We show that reengineering of the receptor-binding surfaceof arrestin-1 further improves the binding to Rh* while preservingprotein stability. Mammalian arrestin-1 readily self-associates atphysiological concentrations. To elucidate the biological role of thisphenomenon, wild type arrestin-1 in living animals must be replacedwith a non-oligomerizing mutant retaining all other functions.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyConstitutively monomeric forms of arrestin-1 with wild typeselectivity for P-Rh* are sufficiently stable for in vivo expression.The same mutations eliminate self-association of enhanced forms,while preserving high Rh* binding and stabilityConclusions: Individual functions of arrestin-1 can be independentlymanipulated to generate mutants with the desired combinations offunctional characteristics. Stable forms of arrestin-1 with high Rh*binding can be constructed with or without the ability to selfassociate.These results pave the way for testing of the biological roleof arrestin-1 self-association and elucidation of full potential ofcompensational gene therapy of gain-of-function GPCR mutations.NIH grants EY011500, GM077561, GM081756 (VVG), EY05216and the Jules Stein Professorship Endowment (WLH), GM079419,GM095633 (TII)Commercial Relationships: Vsevolod V. Gurevich, None; SergeyA. Vishnivetskiy, None; Qiuyan Chen, None; Maria C. Palazzo,None; Evan K. Brooks, None; Christian Altenbach, None; TinaM. Iverson, None; Wayne L. Hubbell, NoneSupport: NIH grants EY011500, GM077561, GM081756 (VVG),EY05216 and the Jules Stein Professorship Endowment (WLH),GM079419, GM095633 (TII)Program Number: 2461 Poster Board Number: D0066Presentation Time: 2:45 PM - 4:30 PMA New Form of Photoreceptor Light Adaptation: EnhancedCurrent Following Sustained IlluminationAlex S. McKeown, Timothy W. Kraft. Vision Sciences, Univ ofAlabama at Birmingham, Birmingham, AL.Purpose: Photoreceptors have been extensively studied for the pasthalf century, but important details remain undiscovered in the realmof biochemical adaptation. Certain stimulus conditions can elicitprotein translocation, but these occur well outside the working rangeof rods. More relevant to fast adaptation are the biochemical changesthat occur in response to changing calcium levels, which result inaltered phototransduction response kinetics. Here we present a uniqueform of photoreceptor adaptation that results in increased totalcirculating dark current following sustained illumination. We alsoexamine the idea that the adaptation is caused by the dynamicinteraction of calmodulin with the cyclic nucleotide gated (CNG)channels of the rod outer segment.Methods: Using single cell suction electrode recordings of mousephotoreceptors, we have discovered a form of adaptation that occursin response to a steady illumination capable of bleaching only 1-4%of the photopigment in mouse rod cells. Following illumination of 1-3 minutes (5000 photons µm-2 s-1) there is a transient 35% increasein response amplitude that returns to baseline with a time constant of10 seconds. We also investigated this unique form of adaptation in amutant mouse lacking the calmodulin binding site on the beta subunitof the rod outer segment CNG channel (CaM-del mouse).Results: Following exposures of sustained illumination, rods exhibita 35% increase in circulating current upon return to darkness (n=11WT). Following identical duration and intensity of sustainedillumination, calmodulin mutant rods exhibit a 65% (vs. 35% in WT)increase in circulating current upon returning to darkness (p = 0.006)(n=14 CaM-del).Conclusions: These findings demonstrate both a unique form of lightadaptation in photoreceptors that we call dark potentiation, andsuggest a role for calmodulin in attenuating this form of adaptation.The excess channel opening following the light step is likely due tofluctuations in the concentration of cGMP, as determined byguanylate cyclase and phosphodiesterase. The role of calcium andGCAPs in this form of adaptation is being investigated. Additionalpsychophysical studies may reveal how such photoreceptor changescan impact human perception. The adaptation may enhancesensitivity in dim environments like those found in a deep forest, orduring specific times of day, such as dawn or dusk.Commercial Relationships: Alex S. McKeown, None; Timothy W.Kraft, NoneProgram Number: 2462 Poster Board Number: D0067Presentation Time: 2:45 PM - 4:30 PMEM-structure and functional properties of the rhodopsintransducincomplexBeata Jastrzebska, Tivadar Orban, Marcin Golczak, Andreas Engel,Krzysztof Palczewski. Pharmacology, Case Western ReserveUniversity, Cleveland, OH.Purpose: To determine the molecular assembly, structure andfunctional properties of the visual G protein-coupled receptor(GPCR), rhodopsin (Rho), in the complex with its cognitiveheterotrimeric G protein, transducin (Gt).Methods: By photoactivation of Rho (Meta II or Rho*) andnucleotide depletion, we trapped and purified the nucleotide-freeRho*-Gt complex and calculated its molecular envelope fromprojections of negatively stained Rho*-Gt particles. Using sConA asa probe, we identified the quaternary organization of Rho moleculesin this complex. Functional properties of Rho molecules bound to Gtwithin the stable Rho*-Gt complex was investigated by UV-visiblespectroscopy and HPLC retinoid analysis.Results: The 3-D envelope determined for the Rho*-Gt complexaccommodated two Rho molecules and one Gt heterotrimer(pentameric assembly). Within the complex, the photoactivated Rhodimer serves as a platform for binding the Gt heterotimer. We foundthat binding of Gt stabilizes one Rho in its active Meta II state,whereas the second progresses toward the opsin conformation.Therefore, each monomer contributes unevenly to the pentamericcomplex, indicating an asymmetry of the Rho dimer.Conclusions: This study unequivocally demonstrates dimericassembly of Rho* in the active complex with transducin and providesa clear functional distinction between each monomer, indicating thatoligomeric assembly and activation of family A GPCRs areintimately associated.Commercial Relationships: Beata Jastrzebska, None; TivadarOrban, None; Marcin Golczak, None; Andreas Engel, None;Krzysztof Palczewski, QLT Inc (F), Polgenix Inc (E), Visum Inc(P), Amegen Inc (F)Support: RES506768Program Number: 2463 Poster Board Number: D0068Presentation Time: 2:45 PM - 4:30 PMMechanisms of Retinal Vascular Alteration inHyperhomocysteinemiaAmany M. Tawfik 1, 2 , Shanu Markand 1, 2 , Sylvia Magyerdi 2, 3 ,Mohamed A. Al-Shabrawey 2, 3 , Sylvia B. Smith 1, 2 . 1 Cellular Biology& Anatomy, Georgia Health Sciences Univ, Augusta, GA; 2 VisionDiscovery Institute, GHSU, Augusta, GA; 3 Oral Biology/Anatomy,GHSU, Augusta, GA.Purpose: Excess homocysteine (Hcy), a sulfur containing nonproteinogenicamino acid, is implicated in human vascular disorders.Recently, we described the retinal phenotype inhyperhomocysteinemic mice lacking/deficient in the gene encodingcystathionine β−synthase (CBS). These mice have marked disruptionof retinal neuronal layers, decreased ERG function & altered retinalvasculature suggestive of ischemic retinopathy. In this study weexplored the mechanism of Hcy-induced retinopathy specificallyscreening markers of ER stress, oxidative stress, inflammation &NMDA receptor (NMDAr) activation.©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.
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