Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyformation. Rho-paGFP-1D4 mice were bred to mice expressingrhodopsin (WT) or the rhodopsin C-terminal truncation autosomaldominant retinitis pigmentosa mutant, Q344ter. We havecharacterized the fusion protein, monitored localization underdifferent environments, and performed real time ex vivo traffickingstudies.Methods: To test for proper folding, rho-paGFP-1D4 was expressedin COS cells, reconstituted, solubilized, and subjected to UV/Visspectroscopy. Activation of the G-protein transducin was tested byuptake of GTPγ 35 S whereas rhodopsin phosphorylation was tested forby using ATPγ 32 P. Polarized mouse Inner Medullary Collecting Duct(IMCD) cells were transfected with rho-paGFP-1D4 cDNA toobserve in vitro localization relative to a primary cilium. TransgenicXenopus laevis were created expressing rho-paGFP-1D4 under theXenopus opsin promoter to examine in vivo localization in transgenicanimals and knock-in mice were generated expressing rho-paGFP-1D4 to monitor in vivo localization and real time rhodopsin transport.Results: Rho-paGFP-1D4 folds, activates transducin, and isphosphorylated similarly to WT rhodopsin. Rho-paGFP-1D4localizes similarly to WT rhodopsin in vitro and in vivo unless therhodopsin trafficking mutant Q344ter is expressed in the samesystem. This has allowed us to monitor the differences in proper andimproper transport of rhodopsin in live mouse retinas.Conclusions: Rho-paGFP-1D4 functions and localizes similarly toWT, despite the addition of 258 amino acids to the C-terminus. Assuch, it is being used as a real time probe of rhodopsin transport whenexpressed in retinas containing wild type properly-localizingrhodopsin and the mislocalizing mutant Q344ter.Commercial Relationships: Joshua Sammons, None; Alecia K.Gross, NoneSupport: EY019311Program Number: 702 Poster Board Number: D0259Presentation Time: 10:30 AM - 12:15 PMCharacterisation of a novel antibody against the loop betweenTMD 3 and 4 of human Bestrophin1Caroline Pasquay, Annabella Janise, Birgit Lorenz, Markus N.Preising. Dept. of Ophthalmology, Justus-Liebig-University,Giessen, Germany.Purpose: Purpose: Bestrophin1 is part of an integral membraneprotein complex located in the basolateral membrane of the retinalpigment epithelium. The gene is mutated in Best vitelliform maculardystrophy (VMD), an autosomal dominant macular degeneration withhighly variable expressivity and reduced penetrance. We want toinvestigate whether mutant bestrophin1 subunits form complexeswith wildtype protein subunits to get further information about thefunction of mutant protein complexes which is not well understoodup to now. For this purpose we constructed a novel antibody againstthe loop between transmembrane domains (TMD) three and four andconfirmed its applicability in Western blotting.Methods: Methods: An alignment with the four isoforms of humanbestrophin (1-4) was performed. A human bestrophin1 specificsequence stretch (AA204-220: PILLQSLLNEMNTLRTQ) within theloop between TMD 3 and 4 was chosen as antigen and anoligopeptide was synthesized. The antibody was raised against theoligopeptide in chicken and affinity purified by a commercialsupplier. The antibody specificity was confirmed by an ELISAagainst the oligopeptideMDCK II cells were seeded on transwell filters and cultured in highglucose DMEM supplemented with 10% FCS and 5 timespenicillin/streptomycin at 37°C and 5% CO 2 . His-tagged BEST1cloned into a pcDNA3(-) vector was transfected into MDCKII cellsusing RotiFect® according to the manufacturers’ instructions atsubconfluency. Cells were harvested 24 h after transfection andlysed. After lysis proteins were extracted and protein preparation tookplace after a modified protocol according to Bordier (JBC 1981).SDS-PAGE and Western blotting were performed following astandard protocol.Results: Results: The antibody showed a single specific bandcorresponding to a molecular weight of approx. 67 kD on SDS-PAGE and Western blotting. This corresponds well with themonomer size of human bestrophin1. Counterstaining by acommercially available anti-His antibody labelled the identical band.Conclusions: Conclusion: A novel antibody (cab-BEST1) was raisedin chicken against the intracellular loop between TMD 3 and 4 ofhuman bestrophin1. The antibody was specific for humanbestrophin1 in Western blots and will be useful in testing the functionof the N-terminal half of bestrophin1. Further data on its applicabilityin immunohistochemistry will be available at the meeting.Commercial Relationships: Caroline Pasquay, None; AnnabellaJanise, None; Birgit Lorenz, Optos (F); Markus N. Preising, NoneSupport: Dr. Gertrud und Franz-Karl Portmann-Stiftung, GiessenerLichtblicke e.V., Marie-Louise Geissler StiftungProgram Number: 703 Poster Board Number: D0260Presentation Time: 10:30 AM - 12:15 PMThe presynaptic glutamate transporter in rods binds to a distinctsubset of PDZ domain containing proteinsSarah Hengel 1 , Joseph G. Laird 1 , Will Watkins 2 , Randy A. Hall 2 ,Sheila A. Baker 1 . 1 Department of Biochemistry, Carver College ofMedicine, University of Iowa, Iowa City, IA; 2 Department ofPharmacology, Emory University School of Medicine, Atlanta, GA.Purpose: Visual signaling depends on removal of glutamate from thesynaptic cleft between photoreceptors and bipolar cells. Excitatoryamino acid transporter 5 (EAAT5) is the glutamate transporter foundin rod inner segments and synapses. The location and activity ofEAAT5 is likely regulated by interacting proteins, however bindingpartners have yet to be identified. The C-terminus of EAAT5contains a PDZ binding motif which prompted us to screen for PDZdomain interactions.Methods: We generated a GST fusion protein containing the 30 C-terminal amino acids of EAAT5 and probed an array of 96 PDZdomains. GST pull-downs and fluorescence anisotropy assays wereused to confirm interactions. A membrane-associated GFP reporterfused to EAAT5’s C-terminus was expressed in the rods of transgenicfrogs and subcellular location was visualized with confocalmicroscopy.Results: The C-terminus of EAAT5 bound to 13 of the 96 PDZdomains on the array. Only three of these positive hits, β2-syntrophin, SAP97, and MAGI-1a, are known to be expressed in theouter plexiform layer of the retina along with EAAT5. All three ofthese interactions were validated by independent GST pull-downassays. The β2-syntrophin PDZ domain interacts with the EAAT5 C-terminal peptide with a calculated KD=4.3 +/-0.3 µM. In transgenictadpoles the membrane reporter accumulates in the outer segmentregardless of the presence of EAAT5’s C-terminus.Conclusions: We identified multiple novel interaction partners forthe glutamate transporter EAAT5. These interactions are unlikely tobe necessary for the trafficking of EAAT5, suggesting they maymodulate its activity.Commercial Relationships: Sarah Hengel, None; Joseph G.Laird, None; Will Watkins, None; Randy A. Hall, None; Sheila A.Baker, NoneSupport: EY020542-04Program Number: 704 Poster Board Number: D0261©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.