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Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R.

ABSTRACT: In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca²⁺ levels, thus contributing to the Ca²⁺/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca²⁺ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca²⁺/Mg²⁺-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg²⁺ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca²⁺. Conversely, nonmyristoylated GCAP2 does not bind Mg²⁺ over the physiological range and remains as a monomer in the absence of Ca²⁺. Both myristoylated and nonmyristoylated GCAP2 bind Ca²⁺ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca²⁺-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.

SUBMITTER: Avesani A

PROVIDER: S-EPMC8113879 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeTo identify pathogenic mutations in the guanylate cyclase-activating protein 1 (GCAP1) and GCAP2 genes and to characterize the biochemical effect of mutation on guanylate cyclase (GC) stimulation.MethodsThe GCAP1 and GCAP2 genes were screened by direct sequencing for mutations in 216 patients and 421 patients, respectively, with various hereditary retinal diseases. A mutation in GCAP1 segregating with autosomal dominant cone degeneration was further evaluated biochemically by employing recombinant proteins, immunoblotting, Ca2+-dependent stimulation of GC, fluorescence emission spectra, and limited proteolysis in the absence and presence of Ca2+.ResultsA novel GCAP1 mutation, I143NT (substitution of Ile at codon 143 by Asn and Thr), affecting the EF4 Ca2+-binding loop, was identified in a heterozygote father and son with autosomal dominant cone degeneration. Both patients had much greater loss of cone function versus rod function; previous histopathologic evaluation of the father's eyes at autopsy (age 75 years) showed no foveal cones but a few, scattered cones remaining in the peripheral retina. Biochemical analysis showed that the GCAP1-I143NT mutant adopted a conformation susceptible to proteolysis, and the mutant inhibited GC only partially at high Ca2+ concentrations. Individual patients with atypical or recessive retinitis pigmentosa (RP) had additional heterozygous GCAP1-T114I and GCAP2 gene changes (V85M and F150C) of unknown pathogenicity.ConclusionsA novel GCAP1 mutation, I143NT, caused a form of autosomal dominant cone degeneration that destroys foveal cones by mid-life but spares some cones in the peripheral retina up to 75 years. Properties of the GCAP1-I143NT mutant protein suggested that it is incompletely inactivated by high Ca2+ concentrations as should occur with dark adaptation. The continued activity of the mutant GCAP1 likely results in higher-than-normal scotopic cGMP levels which may, in turn, account for the progressive loss of cones.

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Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R.

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