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A. M. Dizhoor, E. V. Olshevskaya, I. V. Peshenko; Interactions of GCAP1 and RetGC1: The Stoichiometry of Binding in Live Transfected Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5455.
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© ARVO (1962-2015); The Authors (2016-present)
Ca2+/Mg2+ binding proteins, GCAPs, regulate photoreceptor guanylyl cyclase (retGC1 and 2) in vertebrate photoreceptors. The structure of the GCAP/retGC complexes remains poorly understood, including the stoichiometry of their binding. The purpose of this study was to evaluate the GCAP1:retGC1 complex stoichiometry using fluorescently tagged functional proteins co-expressed in cultured cells.
Recombinant GCAP1 and retGC1 fluorescently tagged with eGFP and monomeric dsRed, respectively, were co-expressed in HEK 293 cells as described previously , and the ratio between the two fluorescent tags co-localized in the membranes determined using confocal microscopy was compared against a "1:1" chimera standard, dsRedRetGC1-GCAP1eGFP, expressed as a single fusion protein in the same experiments.
When expressed in HEK293 cells, GCAP1 and retGC1 were co-localized in the membranes, thus creating a characteristic pattern strikingly different from the diffuse pattern of GCAP1 expressed in the absence of retGC1  or in the presence of retGC1 mutants insensitive to GCAP1. The co-localization required the presence of the intracellular segment of the cyclase, but not its catalytic activity: deletion of the catalytic center did not prevent binding of GCAP1. With the increased levels of GCAP1 versus retGC1 expression, the ratio of the eGFP:dsRed fluorescence for the GCAP1:retGC1 complex in the membranes approached that of the "1:1" chimera standard. The maximal GCAP1:retGC1 ratio in the complex calculated using a fractional saturation function was ~1.05.
The results indicate that each retGC1 molecule can directly bind with high affinity only one GCAP1 molecule.
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