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

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.

Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors

“…S3). The Ca 2+ -induced broadening of NMR peaks was also observed for unmyristoylated GCAP5, which argues against a possible Ca 2+ -myristoyl switch for GCAP5 16 .…”

“…4D), consistent with possible Fe 2+ binding at EF2. In the presence of physiological Mg 2+ levels (1 mM), Mg 2+ interferes with Ca 2+ -binding to GCAP5 16 and is known to bind to EF2 in GCAP1 17,29 . To test whether Fe 2+ can bind to EF2 in place of Mg 2+ , an HSQC spectrum of Mg 2+ -bound GCAP5 was recorded in the presence and absence of saturating Fe 2+ (Fig.…”

“…Acylation of the GCAP5 D3N mutant was originally confirmed in living cells by a click-chemistry approach 16 . Cloning of the C15A/C17A double mutant was similar as described for other point mutants of GCAPs 11, 13 .…”

CaF2nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies

“…Binding stoichiometry of GCAP1 interacting with NP was tested by isothermal titration calorimetry (ITC) as described earlier for other binding processes involving NCS proteins. 18,29,30 Briefly, ITC was performed using a VP-ITC instrument from MicroCal (Northampton, MA, USA) at T = 25°C. Purified GCAP1 or the mutant D100E was present in the recording cell in a decalcified buffer composed of 5 mM Tris-HCl pH 7.5 and 150 mM KCl.…”

Protein and Signaling Networks in Vertebrate Photoreceptor Cells

“…The Ca 2+ -myristoyl switch of recoverin (Zozulya and Stryer, 1992 ) is prototypical for a variety of myristoylated proteins and has served as a benchmark for studying related switching mechanisms in other Ca 2+ -sensors (O‘Callaghan et al, 2003 ; Li et al, 2011 ; Lim et al, 2011 , 2014 ; Burgoyne and Haynes, 2015 ; Marino et al, 2015 ; Sulmann et al, 2015 ) or by characterizing the biocompatibility of nanoparticles (Marino et al, 2014 ). The Ca 2+ -binding and switching process in recoverin had been investigated in more detail by comparing WT recoverin with mutants containing disabled EF-hands or a truncated C-terminus (Senin et al, 2002a , 2003 ; Weiergräber et al, 2003 , 2006 ).…”