Focus on molecules: guanylate cyclase-activating proteins (GCAPs).
Ontology highlight
ABSTRACT:
SUBMITTER: Baehr W
PROVIDER: S-EPMC2739054 | biostudies-literature | 2009 Jun
REPOSITORIES: biostudies-literature
Publications
Focus on molecules: guanylate cyclase-activating proteins (GCAPs).
Baehr Wolfgang W Palczewski Krzysztof K
Experimental eye research 20090108 1
Similar Datasets
Project description:Photoreceptor ROS-GC1 (rod outer segment membrane guanylate cyclase) is a vital component of phototransduction. It is a bimodal Ca(2+) signal transduction switch, operating between 20 and ∼1000 nM. Modulated by Ca(2+) sensors guanylate cyclase activating proteins 1 and 2 (GCAP1 and GCAP2, respectively), decreasing [Ca(2+)](i) from 200 to 20 nM progressively turns it "on", as does the modulation by the Ca(2+) sensor S100B, increasing [Ca(2+)](i) from 100 to 1000 nM. The GCAP mode plays a vital role in phototransduction in both rods and cones and the S100B mode in the transmission of neural signals to cone ON-bipolar cells. Through a programmed domain deletion, expression, in vivo fluorescence spectroscopy, and in vitro reconstitution experiments, this study demonstrates that the biochemical mechanisms modulated by two GCAPs in Ca(2+) signaling of ROS-GC1 activity are totally different. (1) They involve different structural domains of ROS-GC1. (2) Their signal migratory pathways are opposite: GCAP1 downstream and GCAP2 upstream. (3) Importantly, the isolated catalytic domain, translating the GCAP-modulated Ca(2+) signal into the generation of cyclic GMP, in vivo, exists as a homodimer, the two subunits existing in an antiparallel conformation. Furthermore, the findings demonstrate that the N-terminally placed signaling helix domain is not required for the catalytic domain's dimeric state. The upstream GCAP2-modulated pathway is the first of its kind to be observed for any member of the membrane guanylate cyclase family. It defines a new model of Ca(2+) signal transduction.
| S-EPMC3431457 | biostudies-literature
Project description:Calmodulin-like neuronal Ca2+-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca2+-binding motifs. The guanylate cyclase-activating proteins 1-3 (GCAP1-3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1-2 (GC1-2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class-specific residues for the NCBP subfamilies.
| S-EPMC1363676 | biostudies-literature
Project description:Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage.
| S-EPMC3376486 | biostudies-literature
Project description:Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.
| S-EPMC5811540 | biostudies-literature
Project description:ImportanceAs genetic and genomic screening is becoming more widely accessed, correctly distinguishing pathogenic from nonpathogenic variants is of increasing relevance.ObjectiveTo reevaluate a previously reported family in whom the p.(Pro50Leu) variant in the gene GUCA1A was associated with a dominant retinal dystrophy, in light of new examination findings in the proband's daughter.Design, setting, and participantsA genetic study relating to a family with an inherited retinal dystrophy was performed at the retinal genetics service of Moorfields Eye Hospital from October 27, 2009, to May 23, 2019, after the proband's daughter underwent fundus examination.Main outcomes and measuresResults of sequencing of X chromosome-linked retinitis pigmentosa genes in the proband and specific analysis of the repetitive ORF15 region of the RPGR gene.ResultsA frame-shifting single-nucleotide deletion was found in the ORF15 exon of RPGR (GRCh37 [hg19] x:38145160delT; NM_001034853.1: c.3092delA p.[Glu1031Glyfs*58]), which may be associated with the loss of 121 amino acid residues at the carboxyl terminus of the protein. The p.(Pro50Leu) variant in GUCA1A was also found to be too common in a publicly available genome database to be a fully penetrant cause of a dominant retinal dystrophy.Conclusions and relevanceThe phenotype in the family is now associated with the variant in RPGR. The findings suggest that the p.(Pro50Leu) variant in GUCA1A should not be regarded as pathogenic. This report also highlights the relevance of examining relatives, of reevaluating diagnoses in light of new data, and of considering X chromosome-linked inheritance in apparently autosomal dominant pedigrees unless there is clear male-to-male transmission.
| S-EPMC6902164 | biostudies-literature
Project description:The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway plays a key role in the pathogenesis of pulmonary hypertension (PH). Targeted treatments include phosphodiesterase type 5 inhibitors (PDE5i) and sGC stimulators. The sGC stimulator riociguat is approved for the treatment of pulmonary arterial hypertension (PAH) and chronic thromboembolic pulmonary hypertension (CTEPH). sGC stimulators have a dual mechanism of action, enhancing the sGC response to endogenous NO and directly stimulating sGC, independent of NO. This increase in cGMP production via a dual mechanism differs from PDE5i, which protects cGMP from degradation by PDE5, rather than increasing its production. sGC stimulators may therefore have the potential to increase cGMP levels under conditions of NO depletion that could limit the effectiveness of PDE5i. Such differences in mode of action between sGC stimulators and PDE5i could lead to differences in treatment efficacy between the classes. In addition to vascular effects, sGC stimulators have the potential to reduce inflammation, angiogenesis, fibrosis and right ventricular hypertrophy and remodelling. In this review we describe the evolution of treatments targeting the NO-sGC-cGMP pathway, with a focus on PH.
| S-EPMC10957071 | biostudies-literature
Project description: Not available
| S-EPMC8502073 | biostudies-literature
Project description: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.
| S-EPMC8113879 | biostudies-literature
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.
| S-EPMC1475955 | biostudies-literature
Project description:Neuronal Ca(2+) sensors (NCS) are high-affinity Ca(2+)-binding proteins critical for regulating a vast range of physiological processes. Guanylate cyclase-activating proteins (GCAPs) are members of the NCS family responsible for activating retinal guanylate cyclases (GCs) at low Ca(2+) concentrations, triggering synthesis of cGMP and recovery of photoreceptor cells to the dark-adapted state. Here we use amide hydrogen-deuterium exchange and radiolytic labeling, and molecular dynamics simulations to study conformational changes induced by Ca(2+) and modulated by the N-terminal myristoyl group. Our data on the conformational dynamics of GCAP1 in solution suggest that Ca(2+) stabilizes the protein but induces relatively small changes in the domain structure; however, loss of Ca(+2) mediates a significant global relaxation and movement of N- and C-terminal domains. This model and the previously described "calcium-myristoyl switch" proposed for recoverin indicate significant diversity in conformational changes among these highly homologous NCS proteins with distinct functions.
| S-EPMC2822722 | biostudies-literature