Abstract
Purpose::
Guanylyl cyclase activating proteins (GCAPs) are Ca(2+)/Mg(2+)-sensor proteins that impart Ca(2+) sensitivity to retinal guanylyl cyclase (RetGC) [1,2]. The purpose of this study was to investigate the role of Mg(2+) binding by GCAP-1 in the activation of RetGC-1 in the light.
Methods::
Site-directed mutagenesis was used to disable Ca(2+) or Ca(2+)/Mg(2+) binding in individual EF-hands of GCAP-1. RetGC-1 was produced in transfected HEK 293-F cells and reconstituted with GCAP-1 mutants produced in E. coli.
Results::
Mutations in all three metal-binding EF-hand loops of GCAP-1 that prevented binding of both Ca(2+) and Mg(2+) resulted in the loss of the ability of GCAP-1 to activate RetGC-1 while the mutations that prevented binding of only Ca(2+), but not of Mg(2+) produced constitutive activator form of GCAP-1. We have found that of the three metal binding EF-hands, Mg(2+)-bound EF-hands 2 and 3 were critical for the ability of GCAP-1 to interact with and activate RetGC-1 at low free Ca(2+). Binding of Mg(2+) in EF-hand 2 affected the conformation of EF-1, the N-terminal EF-hand-like domain implicated in RetGC recognition by GCAPs. The replacement of Mg(2+) in EF-hands 2 and 3 by Ca(2+) maintained the ability of GCAP-1 to activate RetGC-1, but only when Ca(2+) binding in the EF-hand 4 was disabled. Mg(2+) binding in EF-hand 2 was critical for high-affinity binding to RetGC-1 at low free Ca(2+). Mg(2+) binding in EF-hand 3 enhanced the level of RetGC-1 activation. Mg(2+) binding in EF-hand 4 was not required for RetGC-1 activation, but Ca(2+) binding in this EF-hand was essential for the RetGC inhibition.
Conclusions::
The cation-free form of GCAP-1 is inactive as RetGC-1 regulator, and the physiological RetGC activator form of GCAP-1 is Mg(2+)-bound. Replacement of Ca(2+) by Mg(2+) in EF-hands 2 and 3 (but not of EF-hand 4) in light-adapted photoreceptors is essential for creating the activator confromation of GCAP-1. References: [1] Peshenko & Dizhoor (2004) J. Biol. Chem. 279, 16903-6; [2] Peshenko & Dizhoor (2006) J. Biol. Chem. 281, 23830-41
Keywords: calcium • photoreceptors • protein structure/function