Subcellular localization of blindness-causative protein RD3 assessed by a genetic approach

Identifier:  TFG:2119

Handle: https://hdl.handle.net/20.500.11797/TFG2119

Authors:  Soler Boronat, Alba

Abstract:
Light transduction to an electrical signal at photoreceptor cells of the retina constitutes the first step in vision. Gene mutations causing functional defects at this level lead to blindness. Mutations in RD3, a protein required for the stability and trafficking of retinal guanylate cyclase that synthesizes cGMP, the key signaling molecule in phototransduction, are linked to severe forms of blindness. It is not yet clear how mutations in RD3 compromise the protein function and photoreceptor cell physiology, leading to retinal degeneration. Controversial results from different laboratories have made the subcellular localization of RD3 a key unresolved question. We here have assessed RD3 subcellular localization in rods by making transient transgenic mice that express RD3.V5 in rod photoreceptors of rd3 mice, a naturally-occurring mouse model of RD3 loss of function. By using antibodies to the V5 tag, we show that RD3 localizes mainly to the inner segment compartment of rods, and is largely excluded from the outer segment. Importantly, its expression restores normal RetGC expression and trafficking to the outer segment. Expression of transgenic K87E/RD3.V5, a mutant that leads to retinitis pigmentosa, reveals that mutant RD3 distributes as the control protein, but fails to fully rescue RetGC trafficking to the outer segment. In K87E/RD3.V5 transfected cells, a fraction of RetGC miss localizes at the cell proximal compartments. Therefore we conclude that the retinitis pigmentosa-causative mutation K87E leads to an intermediate phenotype between complete loss-of-function mutations linked to LCA12, and the wild type protein in a physiological context. Our results explain the mild phenotype of K87E mutation, linked to RP and not to LCA, at the time that emphasize the toxicity of RetGC miss localization to proximal compartments.