guca1c (guanylate cyclase activator 1C; protein name zGCAP3) is one of two zebrafish co-orthologs of vertebrate GUCA1C (GCAP3), retained from the teleost-specific genome duplication; its paralog is guca1d (zGCAP4). It encodes a small (188 aa) N-myristoylated neuronal calcium sensor of the recoverin/GCAP family with four EF-hands, of which EF2-EF4 bind Ca2+. Like other GCAPs it activates the photoreceptor membrane guanylate cyclases at low free Ca2+ and inhibits them at high Ca2+, so it mediates the Ca2+ feedback that restores cGMP after light exposure. Among the zebrafish GCAPs it activates the cyclases with high Ca2+ sensitivity (half-maximal near 30 nM free Ca2+, similar to zGCAP1 and zGCAP2 and more than tenfold lower than zGCAP4). guca1c is expressed in all four cone types and not in rods, from about 3-4 dpf; it is the most abundant GCAP transcript in adult cones and is present in large molar excess over the cone cyclase zGC3. Morpholino knockdown did not change larval optokinetic or optomotor responses, and a CRISPR knockout reported Muller-cell gliosis and some ganglion-cell apoptosis.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0005509 calcium ion binding | IBA GO_REF:0000033 | ACCEPT | Summary: Calcium ion binding, propagated from the neuronal calcium sensor node of the recoverin/GCAP family. Reason: Correct and directly confirmed for this protein. zGCAP3 binds three Ca2+ ions with high affinity, and its EF2-EF4 loops keep the Ca2+-coordinating residues annotated on human GUCA1C (bioinformatics RESULTS.md). guca1c (ZFIN:ZDB-GENE-030829-1) appears among the IBA sources because it has its own experimental annotation. Supporting Evidence: PMID:26061947 In all zGCAPs at least two binding sites exhibited high affinities for Ca2+ with KD values in the submicromolar range (KD1 and KD2 in Table 2), for zGCAP3 also the third site (KD3) showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range. file:DANRE/guca1c/guca1c-bioinformatics/RESULTS.md All 14 Ca2+-coordinating positions annotated on human GUCA1C (EF2, EF3 and EF4 loops) align to residues compatible with Ca2+ binding in both zebrafish copies |
| GO:0005509 calcium ion binding | IDA PMID:11860507 Characterization of retinal guanylate cyclase-activating pro... | ACCEPT | Summary: Calcium ion binding shown by the Ca2+-dependent electrophoretic mobility shift of recombinant zGCAP3. Reason: The mobility shift is a standard indication of Ca2+ binding by GCAPs, and later titrations measured the binding directly. Supporting Evidence: PMID:11860507 zGCAP3 displayed a minor mobility change in the presence and absence of Ca 2+ , as observed for other GCAPs |
| GO:0005509 calcium ion binding | IDA PMID:21829700 Differential calcium signaling by cone specific guanylate cy... | ACCEPT | Summary: Direct Ca2+ titration of recombinant zGCAP3. Reason: Direct binding measurement; all six zebrafish GCAPs bind at least three Ca2+ ions. Supporting Evidence: PMID:21829700 Direct Ca(2+)-binding revealed that all zGCAPs bound at least three Ca(2+). |
| GO:0005509 calcium ion binding | IDA PMID:26061947 Retina specific GCAPs in zebrafish acquire functional select... | ACCEPT | Summary: Isothermal titration calorimetry and SPR of zGCAP3 Ca2+ binding. Reason: Direct measurement; zGCAP3 is the only zebrafish GCAP in which all three sites bind Ca2+ with high affinity. Supporting Evidence: PMID:26061947 In all zGCAPs at least two binding sites exhibited high affinities for Ca2+ with KD values in the submicromolar range (KD1 and KD2 in Table 2), for zGCAP3 also the third site (KD3) showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range. |
| GO:0005509 calcium ion binding | IEA GO_REF:0000120 | ACCEPT | Summary: Calcium ion binding from EF-hand and recoverin-family InterPro signatures. Reason: Correct; confirmed experimentally for this protein. Supporting Evidence: PMID:26061947 In all zGCAPs at least two binding sites exhibited high affinities for Ca2+ with KD values in the submicromolar range (KD1 and KD2 in Table 2), for zGCAP3 also the third site (KD3) showed high affinity for Ca2+, whereas for other zGCAPs the affinity of KD3 was in the micromolar range. |
| GO:0005509 calcium ion binding | ISS PMID:15486694 Diversity of guanylate cyclase-activating proteins (GCAPs) i... | ACCEPT | Summary: Calcium ion binding by sequence similarity, from the comparative analysis of teleost GCAPs. Reason: Correct; the paper notes three functional EF-hands in all GCAPs, and binding was later measured directly. Supporting Evidence: PMID:15486694 Sequence analysis of the predicted polypeptides identified a myristoylation site at position 2 and three functional EF-hand Ca 2+ -binding motifs, EF2-4, characteristic of all GCAPs. |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IBA GO_REF:0000033 | ACCEPT | Summary: Calcium-sensitive guanylate cyclase activator activity, from the PANTHER GCAP3 node seeded by the two zebrafish co-orthologs. Reason: Core molecular function. The node's donors are guca1c itself and its paralog guca1d, both of which have experimental annotations for this activity; the target appearing among its own sources is expected. Supporting Evidence: PMID:21829700 The IC50 values of zGCAPs could be separated in two groups; one group consisting of zGCAP1, 2, and 3 had IC50 values around 30 nM free [Ca2+], the second group of zGCAP4, 5 and 7 had IC50 values between 180 and 520 nM centered around 400 nM (Table 2 and Figure 3). |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:11860507 Characterization of retinal guanylate cyclase-activating pro... | ACCEPT | Summary: Recombinant zGCAP3 stimulated bovine rod outer segment guanylate cyclase at low Ca2+ and inhibited it at high Ca2+. Reason: Direct enzymatic assay showing the defining Ca2+-dependent activation. Supporting Evidence: PMID:11860507 In the GC assay, GCAPs display the expected properties, modulating ROS GCs in a Ca 2+ -dependent manner stimulating at low and inhibiting at high [Ca 2+ ] ( Fig. 9B ). |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:21829700 Differential calcium signaling by cone specific guanylate cy... | ACCEPT | Summary: Ca2+-dependent activation of membrane guanylate cyclase by purified zGCAP3, with IC50 near 30 nM free Ca2+. Reason: Direct assay comparing all six zebrafish GCAPs; zGCAP3 is a strong activator with high Ca2+ sensitivity. Supporting Evidence: PMID:21829700 Isoforms 1-4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. PMID:21829700 The IC50 values of zGCAPs could be separated in two groups; one group consisting of zGCAP1, 2, and 3 had IC50 values around 30 nM free [Ca2+], the second group of zGCAP4, 5 and 7 had IC50 values between 180 and 520 nM centered around 400 nM (Table 2 and Figure 3). |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:22212098 Operation profile of zebrafish guanylate cyclase-activating ... | ACCEPT | Summary: Myristoylated and non-myristoylated zGCAP3 regulate a membrane guanylate cyclase (abstract only in cache). Reason: The abstract reports that both forms modulate the cyclase, with different kinetic parameters; consistent with the other assays. Supporting Evidence: PMID:22212098 Differences between myristoylated and non-myristoylated zGCAP3 were also observed in modulating the kinetic and catalytic parameters of the GCAP-target, a membrane bound guanylate cyclase. |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:23940527 Zebrafish guanylate cyclase type 3 signaling in cone photore... | ACCEPT | Summary: Myristoylated zGCAP3 activated guanylate cyclases in native adult zebrafish retinal membranes in a Ca2+-dependent way. Reason: Assay with the native zebrafish cyclases; zGCAP3 gave the highest activities of the cone GCAPs tested. Supporting Evidence: PMID:23940527 The highest activities were measured with zGCAP3 indicating that zGCAP3 is indeed a major activator of zGCs in native zebrafish retinae. |
| GO:0009966 regulation of signal transduction | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Regulation of signal transduction, propagated from a deep neuronal calcium sensor node. Reason: Broadly true, since GCAP-mediated cyclase activation feeds back on cone phototransduction, but the term is very general and was placed for the whole NCS family. The specific process is the Ca2+-dependent positive regulation of guanylate cyclase activity, recorded in core_functions. No in vivo phototransduction measurement exists for guca1c. Supporting Evidence: PMID:21829700 Light causes a drop of intracellular Ca2+, which triggers activation of membrane bound guanylate cyclases (GC) in rod and cone cells. |
| GO:0030250 guanylate cyclase activator activity | ISS PMID:15486694 Diversity of guanylate cyclase-activating proteins (GCAPs) i... | ACCEPT | Summary: Guanylate cyclase activator activity by similarity to human GCAP1. Reason: Correct parent of the experimentally supported calcium-sensitive term; zGCAP3 activates the cyclase in direct assays. Supporting Evidence: PMID:15486694 Recombinant zGCAP3 was previously shown to stimulate bovine GC, suggesting conservation of residues critically important for interaction between partners of distantly related species. |
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Download this section (compressed HTML)Q: Is the tenfold difference in Ca2+ sensitivity between zGCAP3 (guca1c) and zGCAP4 (guca1d) derived in one copy, and which copy resembles gar GCAP3 when assayed in the same system?
Q: Does guca1d compensate in guca1c mutants (transcriptional adaptation after the NMD-type allele), and does a guca1c; guca1d double mutant alter cone photoresponse recovery?
Q: Are the plexiform-layer and ganglion-cell-layer immunosignals for zGCAP3 real, or cross-reactivity, given that single-cell and in situ data restrict the transcript to cones?
Experiment: Generate guca1c and guca1d single and double mutants (ideally promoter-less or RNA-less alleles to avoid transcriptional adaptation) and record cone ERG a-wave recovery and paired-flash recovery in larvae and adults.
Hypothesis: guca1c and guca1d are partly redundant in cone Ca2+ feedback.
Type: double mutant electrophysiology
Experiment: Express spotted gar GCAP3 alongside zGCAP3 and zGCAP4 and measure GC activation versus free Ca2+ with the same native zebrafish retinal membranes.
Hypothesis: The Ca2+ sensitivity difference between the copies is a protein-level divergence from the ancestral GCAP3.
Type: in vitro enzyme assay
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