Abstract
Purpose: :
The purpose of this study is to demonstrate communication between mammalian rhodopsin (Rho) and retinal guanylate cyclase (retGC) and to understand the role of their communication in phototransduction.
Methods: :
We expressed bovine Rho and intradiscal domain of retGC in HEK293 and used membrane and soluble fractions pull down assay to demonstrate interaction between these proteins. We also co-expressed fluorescently-tagged proteins in cultured cells to analyze their co-localization.
Results: :
It has been shown that retGC activity in isolated ROS membranes was much higher than activity of the same membranes isolated under dark conditions suggesting that communication of retGC with activated rhodopsin is required for its full activation. To test this hypothesis we used pull down assay. Fluorescent protein-tagged bovine Rho and intradiscal domain of retGC were transiently expressed in HEK293 cells. Membrane fraction containing Rho and soluble fraction containing intracellular domain of retGC were prepared from HEK293 cells transfected with plasmid vector expressing one of the proteins. Membrane and soluble fractions were mixed together with the ratio corresponding to their content in photoreceptors. Upon incubation, membranes were separated from soluble fraction by centrifugation and have been washed several times in isotonic buffer. The presence of both recombinant proteins in washed membranes has been analyzed by western blot. We found that intradiscal domain of retGC was present only in cell membranes containing Rho suggesting direct interaction between these proteins. Moreover, co-expression of Rho and retGC intradiscal domain, attached to different fluorescent proteins, demonstrated that they colocalize in mammalian cell. This is additional evidence suggesting their interaction in vivo.
Conclusions: :
Based on our observations, we propose that communication between Rho and retGC occurs in intradiscal space and is necessary for more efficient recovery of photoreceptors to the dark state.
Keywords: photoreceptors • protein structure/function • signal transduction