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Alexander M Dizhoor, Elena V Olshevskaya, Igor V Peshenko; Mutually exclusive binding of guanylyl cyclase activating proteins (GCAP) to retinal guanylyl cyclase 1 (RetGC1): implications for regulation of photoresponse and GUCY2D-linked Leber’s congenital amaurosis.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4662.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase activating proteins (GCAPs) controls photoreceptor recovery and causes blinding disorders when affected by mutations. We tested the principal models for GCAP1 and GCAP2 binding to RetGC1 - by using a common primary docking interface versus independent binding sites formed by distant parts of the cyclase intracellular domain.
We tested co-localization of GFP-tagged GCAP1 and GCAP2 with mOrange-tagged RetGC1 mutants in HEK293 cells, as well as the ability of GCAPs to activate and regulate in a Ca2+-sensitive manner RetGC1 variants expressed in HEK293 cells and native RetGC1 in ROS of transgenic mouse retinas.
At near-saturating concentrations, GCAP1 and GCAP2 activate the recombinant cyclase from HEK293 cells or the native RetGC1 isozyme from RetGC2-/-GCAPs1,2-/- retinas in a non-additive fashion. The Met26Arg GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 also displaced both GCAP1-GFP and GCAP2-GFP from the complex with mOrange-RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor guanylyl cyclase (NPRA) failed to bind GCAPs, but replacing its kinase-homology (KHD) and dimerization (DD) domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr1016-Ser1103 fragment in a human RetGC1 (numbered from Met1 of the leader peptide) did not block GCAP2 binding to the cyclase. In contrast, substitutions in KHD, Trp708Arg and Ile734Thr, linked to Leber’s congenital amaurosis (LCA) inactivated binding of both GCAP1-GFP and GCAP2-GFP.
Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg488-Arg851 portion of RetGC1, and mutations in that region causing congenital LCA blindness disrupt activation of the cyclase by both GCAP1 and GCAP2.
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