Abstract
Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism.
Highlights
Retinal guanylyl cyclase activating proteins (GCAPs) form a subfamily within the neuronal calcium sensor (NCS) proteins group of the EF-hand superfamily
With the previously reported observations using native photoreceptor membranes (Otto-Bruc et al, 1997; Hwang and Koch, 2002), the G2A GCAP1 stimulated the activity of the recombinant RetGC1 expressed in HEK293 cells much less efficiently (Figure 1B)
It needs to be emphasized that GCAP binding to retinal guanylyl cyclase (RetGC) cannot be measured directly, because detergents required for extraction of RetGC from the membranes inactivate GCAP/RetGC interaction (Koch, 1991; Lambrecht and Koch, 1992)
Summary
Retinal guanylyl cyclase activating proteins (GCAPs) form a subfamily within the neuronal calcium sensor (NCS) proteins group of the EF-hand superfamily (reviewed in: Burgoyne, 2007). Binding in EF-hand domains of GCAPs (Peshenko and Dizhoor, 2004), and at the low free Ca2+ levels in the light it converts GCAPs into Mg2+-bound, RetGC activator state.
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