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I. V. Peshenko, E. V. Olshevskaya, S. Yao, H. H. Ezzeldin, S. J. Pittler, A. M. Dizhoor; The Study of GCAP1 Binding to RetGC1: Disruption by LCA-Associated Mutations. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5454.
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Ca2+/Mg2+ binding proteins, GCAPs, regulate photoreceptor guanylyl cyclase (retGC1 and 2). Mutations in GCAPs and retGC1 have been linked to various hereditary retinal degenerations. Two new mutations, D639Y and R768W, in retGC1 were detected in patients with Leber congenital amaurosis. The purpose of this study was to evaluate the functional consequences of these two mutations for retGC1 regulation and interaction with GCAP1.
We expressed recombinant retGC1 and GCAP1 in HEK 293 cells and measured retGC1 activation by GCAP1 in vitro and the association between fluorescently tagged retGC1 and GCAP1 in cyto using confocal microscopy as described in .
When expressed in HEK293 cells, both mutants demonstrated basal activity in the presence of Mn2+, but in contrast to wild type, were completely inactive in the presence of Mg2+ and saturating concentrations of GCAP1. When co-expressed with wild type retGC1, untagged or tagged with monomeric dsRed, eGFP-tagged GCAP1 effectively associated with the cyclase in the membranes of HEK293 cells. However, it completely failed to associate with D639Y or R768W retGC1 under the same conditions. Both mutations, D639Y and R768W, were originally found in heterozygous patients, therefore, we tested whether or not either mutant had a dominant negative effect by co-expressing them with wild type retGC1 at equal proportion. We deleted a short 15-kDa fragment from the long extracellular domain of the wild type cyclase (ΔretGC1), which did not inactivate retGC1 or affect its sensitivity to Ca2+/GCAP1 regulation, but made it possible to quantify the ratio between the ΔretGC1 and each mutant by immunoblot when co-expressed in HEK cells co-transfected with either D639Y or R768W. We found no evidence for a strong dominant negative effect for either mutant.
Point mutations, D639Y or R768W, in the KHD domain of retGC1 completely disrupt interaction of the retGC1 with GCAP1. Ref.:  Peshenko et al., J Biol Chem 283, 21747 (2008)
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