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, 87KIHP90 ,93 CGPAI97 ,99 RFRQ102 , and119 RSVL122 , 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.