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I. V. Peshenko, E. V. Olshevskaya, A. M. Dizhoor; Binding of Mg(2+) in EF-Hand 2 of GCAP-1 Controls Its Binding to RetGC-1. Invest. Ophthalmol. Vis. Sci. 2008;49(13):148.
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Guanylyl cyclase activating proteins (GCAPs) are Ca(2+)/Mg(2+)-sensor proteins that impart Ca(2+) sensitivity to retinal guanylyl cyclase (RetGC) [1,2]. We previously demonstrated that blocking Mg(2+) binding to EF-hand 2 of GCAP-1 prevented activation of RetGC-1[1,2]. The purpose of this study was to elucidate the role of EF-hands in binding of GCAP-1 to RetGC-1.
GFP-tagged GCAP-1 mutants with disabled Ca(2+) or Ca(2+)/Mg(2+) binding in individual EF-hands were expressed in E. coli and in HEK293 cells, with or without co-transfection with wild-type or RFP-tagged RetGC-1 (as DsRed-Monomer, Clontech). Activity of the tagged proteins was assayed in vitro and their co-localization was studied in vivo, using fluorescent microscopy of live or fixed cultured cells.
Both RFP-RetGC-1 and GFP-GCAP-1 were functionally active in vitro. When expressed in HEK293 cells, RFP-RetGC-1 showed membrane localization while GFP-GCAP-1 was localized to the cytoplasm. However, co-transfection of GFP-GCAP-1 with a non-tagged RetGC-1 resulted in primarily membrane localization of GFP-GCAP-1 and its co-localization with RetGC1, detected using anti-RetGC-1 antibody. Co-transfection with RFP-RetGC-1 also resulted in membrane localization of GFP-GCAP-1, coinciding with that of RFP-RetGC-1. Consistent with the previous findings using non-tagged GCAP-1 mutant, disabling Ca(2+), but not Mg(2+), binding in all EF-hands preserved RetGC-1 activation by GFP-labeled GCAP-1. When co-expressed with RetGC-1, the tagged mutant retained its co-localization with RetGC-1 in HEK293 cells. Inactivation of Mg(2+) binding in EF-hand 2, but not in the EF-hands 3 or 4, not only drastically reduced the affinity of GFP-GCAP-1 for RetGC-1 in cyclase activation assay, but the GFP-tagged EF-hand 2 mutant co-expressed with RetGC-1 was unable to maintain its membrane co-localization with the cyclase and was found primarily in cytoplasm of the co-transfected cells.
We optimized a system of co-expression of functionally active fluorescently tagged GCAP-1 and RetGC-1 that allows monitoring of co-localization of the two proteins and its alteration by mutations. Binding of Mg(2+) to EF-hand 2 is crucial for GCAP-1 binding to RetGC-1 and anchoring it to the membrane as a part of RetGC/GCAP complex.References.  Peshenko & Dizhoor (2006) J. Biol. Chem. 281, 23830-41;  Peshenko & Dizhoor (2007) J. Biol. Chem. 282, 21645-52
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