title: Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein.
abstract: Retinal degeneration 3 (RD3) protein, essential for normal expression of retinal membrane guanylyl cyclase (RetGC) in photoreceptor cells, blocks RetGC catalytic activity and stimulation by guanylyl cyclase-activating proteins (GCAPs). In a mouse retina, RD3 inhibited both RetGC1 and RetGC2 isozymes. Photoreceptors in the rd3/rd3 mouse retinas lacking functional RD3 degenerated more severely than in the retinas lacking both RetGC isozymes, consistent with a hypothesis that the inhibitory activity of RD3 has a functional role in photoreceptors. To map the potential target-binding site(s) on RD3, short evolutionary conserved regions of its primary structure were scrambled and the mutations were tested for the RD3 ability to inhibit RetGC1 and co-localize with the cyclase in co-transfected cells. Substitutions in 4 out of 22 tested regions, (87)KIHP(90), (93)CGPAI(97), (99)RFRQ(102), and (119)RSVL(122), reduced the RD3 apparent affinity for the cyclase 180-700-fold. Changes of amino acid sequences outside the Lys(87)-Leu(122) central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the (93)CGPAI(97) portion of a predicted central α-helix most drastically suppressed the inhibitory activity of RD3 and disrupted RD3 co-localization with RetGC1 in HEK293 cells. Different side chains replacing Cys(93) profoundly reduced RD3 affinity for the cyclase, irrespective of their relative helix propensities. We conclude that the main RetGC-binding interface on RD3 required for the negative regulation of the cyclase localizes to the Lys(87)-Leu(122) region.
authors.affiliation: From the Pennsylvania College of Optometry, Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Peshenko
authors.affiliation: From the Pennsylvania College of Optometry, Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Olshevskaya
authors.affiliation: From the Pennsylvania College of Optometry, Department of Research, Salus University, Elkins Park, Pennsylvania 19027 adizhoor@salus.edu.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Dizhoor
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D011956:Receptors, Cell Surface; C516799:retinal degeneration 3 protein, mouse; D006162:Guanylate Cyclase; C484774:Gucy2d protein, mouse
keywords: calcium-binding proteins; cyclic GMP (cGMP); eye; guanylate cyclase (guanylyl cyclase); neurobiology; photoreceptor; phototransduction; retina; signal transduction