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.

The R838S Mutation in Retinal Guanylyl Cyclase 1 (RetGC1) Alters Calcium Sensitivity of cGMP Synthesis in the Retina and Causes Blindness in Transgenic Mice

“…The lack of RD3 causes retinal degeneration in the affected humans and in the rd3/rd3 mouse strain (20). Evidently, both proposed functions of RD3 contribute to photoreceptor survival, because photoreceptors in mice lacking RD3 degenerate even faster than in mice completely devoid of both RetGC1 and RetGC2 (23). We find that R838S RetGC1 reaches the outer segment in a seemingly normal fashion (Fig.…”

“…GCAP1 and RD3-Myristoylated bovine GCAP1 D6S was expressed from a Novagen pET11d vector in BLR(DE3) Escherichia coli strain harboring N-myristoyl transferase and purified to ϳ95% electrophoretic homogeneity from inclusion bodies using urea extraction, hydrophobic, and size exclusion chromatography as described previously in detail (75,76). Human RD3 was expressed from pET11d vector in BL21(DE3) CodonPlus E. coli strain (Stratagene/Agilent Technologies), extracted from the inclusion bodies and purified to ϳ95% electrophoretic homogeneity as previously described in detail (22,23).…”

“…Two RetGC isozymes expressed in photoreceptors, RetGC1 and RetGC2 (6 -8), bind calcium-sensor proteins, GCAPs (7)(8)(9)(10)(11)(12), which decelerate the cyclase activity at high intracellular calcium concentrations in the dark and accelerate it in the light, when the influx of calcium through the cGMP-gated channels is shut off (13)(14)(15)(16)(17)(18)(19). RetGC also binds retinal degeneration 3 (RD3) protein (20 -21), which prevents cyclase activation by GCAPs (22)(23) and promotes accumulation of RetGC1 in the outer segments (21,24,25). RetGC1 (human gene GUCY2D) accounts for most of the cGMP synthesis in mammalian photoreceptors (26,27), therefore mutations in RetGC1 that disable the cyclase activity (28 -33) cause recessive blindness at birth, Leber congenital amaurosis type 1 (LCA1) (33), mostly a non-degenerative or partially degenerative (31,32) loss-of-function hereditary retinal disease.…”

Retinal degeneration 3 (RD3) protein, a retinal guanylyl cyclase regulator, forms a monomeric and elongated four-helix bundle

“…Lack of RD3 strongly reduces RetGC content in photoreceptors, likely because of the RD3 involvement in a process of RetGC delivery to the outer segment (11,12). RD3 also acts as a potent negative regulator of RetGC activity that binds to the cyclase with high affinity and blocks its activation by GCAPs (13,14). The ability of RD3 to bind and suppress RetGC is likely required for preventing aberrant activation of the cyclase by GCAPs and is critical for the survival of photoreceptors (14).…”

“…RD3 also acts as a potent negative regulator of RetGC activity that binds to the cyclase with high affinity and blocks its activation by GCAPs (13,14). The ability of RD3 to bind and suppress RetGC is likely required for preventing aberrant activation of the cyclase by GCAPs and is critical for the survival of photoreceptors (14). The RetGC binding domain in RD3 was previously hypothesized to occupy the central region of the RD3 primary structure (14); however, the three-dimensional structure of RD3 is currently unknown and could not be reliably predicted because of a lack of sequence homology with other known protein structures.…”