Normal GCAPs partly compensate for altered cGMP signaling in retinal dystrophies associated with mutations in GUCA1A

Abstract

Missense mutations in the GUCA1A gene encoding guanylate cyclase-activating protein 1 (GCAP1) are associated with autosomal dominant cone/cone-rod (CORD) dystrophies. The nature of the inheritance pattern implies that a pool of normal GCAP proteins is present in photoreceptors together with the mutated variant. To assess whether human GCAP1 and GCAP2 may similarly regulate the activity of the retinal membrane guanylate cyclase GC-1 (GC-E) in the presence of the recently discovered E111V-GCAP1 CORD-variant, we combined biochemical and in silico assays. Surprisingly, human GCAP2 does not activate GC1 over the physiological range of Ca2+ whereas wild-type GCAP1 significantly attenuates the dysregulation of GC1 induced by E111V-GCAP1. Simulation of the phototransduction cascade in a well-characterized murine system, where GCAP2 is able to activate the GC1, suggests that both GCAPs can act in a synergic manner to mitigate the effects of the CORD-mutation. We propose the existence of a species-dependent compensatory mechanism. In murine photoreceptors, slight increases of wild-type GCAPs levels may significantly attenuate the increase in intracellular Ca2+ and cGMP induced by E111V-GCAP1 in heterozygous conditions. In humans, however, the excess of wild-type GCAP1 may only partly attenuate the mutant-induced dysregulation of cGMP signaling due to the lack of GC1-regulation by GCAP2.

Impaired Ca2+ Sensitivity of a Novel GCAP1 Variant Causes Cone Dystrophy and Leads to Abnormal Synaptic Transmission Between Photoreceptors and Bipolar Cells

“…Although the reason behind such change in electrophoretic mobility has not been completely elucidated, in the absence of ions NCS proteins migrate at their theoretical molecular mass (MM), whereas they exhibit a shift upon Ca 2+ -binding proportional to their affinity [ 34 ]. This is also the case for WT GCAP1 [ 26 ], which shifts from ~23 kDa in the absence to ~17 kDa in the presence of Ca 2+ ( Figure 4 B), and for its variant N104K-G105R, which shows a less prominent shift upon Ca 2+ -binding, suggesting a reduced affinity for Ca 2+ .…”

“…Recombinant human guanylate cyclases 1 and 2 (GC1 and GC2, respectively) were expressed in permanent HEK293 cells after transfection with PEI and 10-day selection with geneticin [ 69 ]. After cell lysis, cell membranes containing GCs were resuspended in 50 mM HEPES pH 7.4, 50 mM KCl, 20 mM NaCl, 1 mM DTT buffer and incubated with 5 µM GCAP1 variants for 10 min at 30 °C in the presence of 1 mM Mg 2+ and either 2 mM EGTA (free Ca 2+ < 19 nM) or ~30 µM Ca 2+ [ 26 ]. Enzymatic reactions were carried out in 30 mM MOPS/KOH pH 7.2, 60 mM KCl, 4 mM NaCl, 1 mM GTP, 3.5 mM MgCl 2 , 0.3 mM ATP, 0.16 mM Zaprinast buffer and blocked with the addition of 50 mM EDTA and boiling.…”

“…Clinically similar phenotypes may indeed result from very different molecular features of the affected GCAP1 variant and, conversely, similar molecular properties may result in heterogenous phenotypes [ 13 ]. The dominant nature of the transmission in COD associated with GUCA1A poses tremendous challenges to possible therapeutic options, because the compensation of the detrimental effects of the mutant allele, which in other species may arise from other GCAP isoforms, does not seem to occur in humans [ 26 ].…”

Regulation of retinal membrane guanylyl cyclase (RetGC) by negative calcium feedback and RD3 protein

“…Considering the specificity of GCAPs in the regulation of RetGC isozymes, it is important to emphasize that the physiological data assessing the interactions between the endogenous RetGC1 isozyme and GCAP isoforms in vivo using gene-knockout and transgenic mouse models unambiguously demonstrate that GCAP2 can effectively stimulate RetGC1, whereas GCAP1 (either endogenous murine or transgenically expressed bovine) does not significantly stimulate RetGC2 in vivo [80]. A controversy in the field remains about how effectively GCAP2 can activate human RetGC1 [21,98]. It appears that GCAPs of different species expressed in Escherichia coli can be sensitive to the process of renaturation after extraction from the inclusion bodies, because a human GCAP2 refolded from guanidine chloride used as denaturing agent is unable to stimulate human RetGC1 in vitro [21], whereas the same isoform purified using urea-extraction activates human RetGC1, albeit with an apparent affinity lower than GCAP1 [98].…”

“…A controversy in the field remains about how effectively GCAP2 can activate human RetGC1 [21,98]. It appears that GCAPs of different species expressed in Escherichia coli can be sensitive to the process of renaturation after extraction from the inclusion bodies, because a human GCAP2 refolded from guanidine chloride used as denaturing agent is unable to stimulate human RetGC1 in vitro [21], whereas the same isoform purified using urea-extraction activates human RetGC1, albeit with an apparent affinity lower than GCAP1 [98]. Obviously, it is the physiology that should ultimately elucidate the role of GCAPs.…”