Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein

Igor V. Peshenko; Elena V. Olshevskaya; Alexander M. Dizhoor

doi:10.1074/jbc.M116.742288

Functional Study and Mapping Sites for Interaction with the Target Enzyme in Retinal Degeneration 3 (RD3) Protein *

From the Pennsylvania College of Optometry, Department of Research, Salus University, Elkins Park, Pennsylvania 19027

↵1 The Martin and Florence Hafter Chair Professor of Pharmacology. To whom correspondence should be addressed: Research S416, Salus University, 8360 Old York Rd., Elkins Park, PA 19027; Tel.: 215-780-1468; Fax: 215-780-1464; E-mail: adizhoor{at}salus.edu.

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, ⁸⁷KIHP⁹⁰, ⁹³CGPAI⁹⁷, ⁹⁹RFRQ¹⁰², and ¹¹⁹RSVL¹²², reduced the RD3 apparent affinity for the cyclase 180–700-fold. Changes of amino acid sequences outside the Lys⁸⁷–Leu¹²² central portion of the molecule either failed to prevent RD3 binding to the cyclase or had a much smaller effect. Mutations in the ⁹³CGPAI⁹⁷ 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 Cys⁹³ 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 Lys⁸⁷–Leu¹²² region.

Footnotes

↵* This work was supported by National Institutes of Health Grant EY11522 from the NEI and a Pennsylvania Department of Health CURE Formula grant. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.