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Rhodopsin Interacts With Retinal Guanylate Cyclase; Involvement In Phototransduction
Author(s) -
Bondarenko Vladimir
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb150
Subject(s) - colocalization , biology , membrane , rhodopsin , recombinant dna , microbiology and biotechnology , retinal , biophysics , biochemistry , gene
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 in cell. 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 rhodopsin is required for its full activation by GCAP. To study interaction between Rho and retGC we used pull down assay and colocalization experiments. Fluorescent protein‐tagged bovine Rho and intradiscal domain of retGC were expressed in HEK293 cells and membrane fraction containing Rho and soluble fraction containing intracellular domain of retGC were prepared. Membrane and soluble fractions were mixed together and upon incubation, membranes were separated from soluble fraction by centrifugation and the presence of both recombinant proteins in washed membranes has been analyzed by western blot. Intradiscal domain of retGC was present only in cell membranes containing Rho suggesting direct interaction between these proteins, which was also supported by colocalization experiments.