Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase Activating Protein 1 (GCAP1) [Neurobiology]

February 24th, 2014 by Peshenko, I. V., Olshevskaya, E. V., Lim, S., Ames, J. B., Dizhoor, A. M.

Retinal guanylyl cyclase (RetGC) activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affect activation of RetGC1 localize to a distinct patch formed by the surface of non-metal binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch can completely block activation of the cyclase without affecting Ca2+ binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1 including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1 are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the 'binding patch' prevent co-localization with RetGC1, except for Met26, Lys85, and Trp94. With these residues mutated, GCAP1 completely fails to stimulate cyclase activity but still binds RetGC1 and competes with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the 'binding patch' with additional activation step involving Met26, Lys85 and Trp94.