Mutations Found in Patients Leber's Congenital Amaurosis Disrupt Retinal Guanylate Cyclase Interaction with Rhodopsin In Vitro; Involvement In Phototransduction

Abstract

We use bovine rod outer membranes membrane and recombinant intradiscal domain of retinal guanylate cyclase (retGC) expressed in HEK293 in pull down assay to demonstrate interaction between rhodopsin (Rho) and retGC.It has been previously shown that retGC activity in light‐exposed rod outer segment membranes was much higher than activity of the same membranes isolated under dark conditions suggesting that communication of retGC with activated Rho is required for its full activation by GCAP. To study the effect of the mutations found in Leber's congenital amaurosis (LCA) in intradiscal domain of retGC on its interaction with Rho we used pull down assay and colocalization experiments. Fluorescent protein‐tagged intradiscal domain of bovine retGC was expressed in HEK293 cells and soluble fraction containing recombinant wild type or mutant proteins were prepared. We used photobleached and hypotonically washed rod outer segment (ROS) membrane as a source of photoactivated Rho. Soluble fractions containing recombinant proteins were incubated with ROS membranes. After washing several times with isotonic buffer presence of recombinant proteins in membranes and soluble fraction were analyzed by western blot using retGC‐specific antibodies. Only wild type intradiscal domain of retGC was detected in ROS membrane fraction suggesting that mutation causing LCA disrupt the interaction between retGC and Rho.