guca1d (guanylate cyclase activator 1d; protein name zGCAP4) is one of two zebrafish co-orthologs of vertebrate GUCA1C (GCAP3), retained from the teleost-specific genome duplication; its paralog is guca1c (zGCAP3). It encodes a 185 aa N-myristoylated neuronal calcium sensor of the recoverin/GCAP family with four EF-hands, of which EF2-EF4 carry intact Ca2+-binding loops. Recombinant zGCAP4 is a strong activator of photoreceptor membrane guanylate cyclases from bovine and zebrafish retina, stimulating them at low free Ca2+ and inhibiting them at high Ca2+, with half-maximal activation near 0.4-0.57 uM free Ca2+, a lower Ca2+ sensitivity than zGCAP3. Myristoylation does not change its Ca2+-sensor properties and it shows no Ca2+-myristoyl switch. guca1d is expressed only in cone photoreceptors, most strongly in double cones and long single cones and weakly in short single (UV) cones, from about 3-4 dpf; in adult cones its transcript is about a tenth as abundant as guca1c. No loss-of-function data exist.
| 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 measured directly for zGCAP4. The EF2-EF4 loops keep the Ca2+-coordinating residues annotated on human GUCA1C (bioinformatics RESULTS.md in the guca1c folder). guca1d (ZFIN:ZDB-GENE-040724-231) is among the 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:26061947 Retina specific GCAPs in zebrafish acquire functional select... | ACCEPT | Summary: Ca2+ titration (ITC, without Mg2+) of recombinant zGCAP4. Reason: Direct binding measurement, although zGCAP4 aggregated in the presence of Mg2+. 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. PMID:26061947 ITC measurements with zGCAP4 were only possible in the absence of Mg2+, since the isolated protein aggregated during the time course of an experiment. |
| 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. 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 similarity, from the paper that cloned zGCAP4. Reason: Correct; the Ca2+-dependent mobility shift of zGCAP4 is also shown in the same paper. Supporting Evidence: PMID:15486694 zGCAP4-His 6 displayed a minor mobility change in the presence and absence of Ca 2+ ( Fig. 3B , inset), as observed for other GCAPs ( Palczewski et al. 1994 ). |
| 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, shown directly for zGCAP4 in several assays; guca1d appearing among its own IBA sources is expected. Supporting Evidence: PMID:18777180 zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520-570 nM free Ca(2+) concentration. |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:15486694 Diversity of guanylate cyclase-activating proteins (GCAPs) i... | ACCEPT | Summary: Recombinant zGCAP4 stimulated bovine rod outer segment cyclase at low Ca2+ and inhibited it at high Ca2+. Reason: Direct enzymatic assay. Supporting Evidence: PMID:15486694 When assayed in the presence of bovine rod outer segment GC, zGCAP4 modulated cyclase activity as expected in a dependent manner, stimulating at low and inhibiting at high [Ca 2+ ] ( Fig. 3B ). |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:18777180 The cone-specific calcium sensor guanylate cyclase activatin... | ACCEPT | Summary: Purified myristoylated and non-myristoylated zGCAP4 activate bovine and zebrafish membrane guanylate cyclases Ca2+-dependently (abstract only in cache). Reason: Direct assay; the abstract states the activation and its Ca2+ dependence. Supporting Evidence: PMID:18777180 zGCAP4 was a strong activator of membrane-bound guanylate cyclases from bovine and zebrafish retina, showing half-maximal activation at 520-570 nM free Ca(2+) concentration. |
| GO:0008048 calcium sensitive guanylate cyclase activator activity | IDA PMID:23940527 Zebrafish guanylate cyclase type 3 signaling in cone photore... | ACCEPT | Summary: Myristoylated zGCAP4 activated guanylate cyclases in native adult zebrafish retinal membranes. Reason: The Ca2+-dependent difference in cyclase activity with zGCAP4 was highly significant in native zebrafish membranes. Supporting Evidence: PMID:23940527 Differences of activities in the presence of zGCAPs were highly significant: zGCAP3 (t = 9.53, ***P≤0.001); zGCAP4 (t = 10.26, ***P≤0.001) |
| 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 for a GCAP that feeds back on cone phototransduction, but very general, and no in vivo data exist for guca1d. The specific activity is recorded in core_functions. Supporting Evidence: PMID:18777180 Thus, expression and biochemical properties of zGCAP4 are in agreement with its function as an efficient Ca(2+)-sensitive regulator of guanylate cyclase activity in cone vision. |
Loading supporting content…
Download this section (compressed HTML)Q: Does guca1d loss of function alter cone responses, alone or together with guca1c?
Q: Which of the two copies kept the Ca2+ sensitivity of the ancestral GCAP3?
Experiment: Make guca1d mutants (promoter-less alleles to avoid transcriptional adaptation) and record cone photoresponse recovery and light adaptation by ERG, alone and with guca1c mutants.
Hypothesis: guca1d supports cone Ca2+ feedback at the higher Ca2+ range, complementing guca1c.
Type: mutant electrophysiology
Loading supporting content…
Download this section (compressed HTML)Loading supporting content…
Download this section (compressed HTML)