Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding

Abstract

Abstract Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca2+-binding properties, Ca2+-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca2+-sensing, namely how similar Ca2+-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca2+-binding sites with Mg2+. All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca2+ with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg2+ either occupied the low affinity Ca2+-binding site or it shifted the affinities for Ca2+-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca2+ than by Mg2+, although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca2+-sensors.