id: Q6ZM98 gene_symbol: guca1d product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:7955 label: Danio rerio description: 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. existing_annotations: - term: id: GO:0005509 label: calcium ion binding evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - FB:FBgn0013303 - FB:FBgn0030897 - FB:FBgn0083228 - MGI:MGI:1929258 - MGI:MGI:1933131 - MGI:MGI:97883 - PANTHER:PTN000550679 - PomBase:SPAC18B11.04 - RGD:620060 - RGD:620258 - RGD:68417 - RGD:70886 - RGD:70887 - UniProtKB:P46065 - UniProtKB:P84074 - UniProtKB:Q6PIL6 - UniProtKB:Q8T893 - UniProtKB:Q9NZI2 - ZFIN:ZDB-GENE-011128-6 - ZFIN:ZDB-GENE-030829-1 - ZFIN:ZDB-GENE-040724-231 - ZFIN:ZDB-GENE-050120-1 - dictyBase:DDB_G0270210 - dictyBase:DDB_G0275931 review: summary: Calcium ion binding, propagated from the neuronal calcium sensor node of the recoverin/GCAP family. action: ACCEPT 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. supported_by: - &ca reference_id: PMID:26061947 supporting_text: 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. - reference_id: file:DANRE/guca1c/guca1c-bioinformatics/RESULTS.md supporting_text: 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 - term: id: GO:0005509 label: calcium ion binding evidence_type: IDA original_reference_id: PMID:26061947 qualifier: enables review: summary: Ca2+ titration (ITC, without Mg2+) of recombinant zGCAP4. action: ACCEPT reason: Direct binding measurement, although zGCAP4 aggregated in the presence of Mg2+. supported_by: - *ca - reference_id: PMID:26061947 supporting_text: ITC measurements with zGCAP4 were only possible in the absence of Mg2+, since the isolated protein aggregated during the time course of an experiment. - term: id: GO:0005509 label: calcium ion binding evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: enables supporting_entities: - InterPro:IPR002048 - InterPro:IPR028846 - UniRule:UR001552760 review: summary: Calcium ion binding from EF-hand and recoverin-family InterPro signatures. action: ACCEPT reason: Correct; confirmed experimentally. supported_by: - *ca - term: id: GO:0005509 label: calcium ion binding evidence_type: ISS original_reference_id: PMID:15486694 qualifier: enables supporting_entities: - UniProtKB:P51177 review: summary: Calcium ion binding by similarity, from the paper that cloned zGCAP4. action: ACCEPT reason: Correct; the Ca2+-dependent mobility shift of zGCAP4 is also shown in the same paper. supported_by: - reference_id: PMID:15486694 supporting_text: 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 ). - term: id: GO:0008048 label: calcium sensitive guanylate cyclase activator activity evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables supporting_entities: - PANTHER:PTN002746644 - ZFIN:ZDB-GENE-030829-1 - ZFIN:ZDB-GENE-040724-231 review: summary: Calcium-sensitive guanylate cyclase activator activity, from the PANTHER GCAP3 node seeded by the two zebrafish co-orthologs. action: ACCEPT reason: Core molecular function, shown directly for zGCAP4 in several assays; guca1d appearing among its own IBA sources is expected. supported_by: - &act reference_id: PMID:18777180 supporting_text: 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. - term: id: GO:0008048 label: calcium sensitive guanylate cyclase activator activity evidence_type: IDA original_reference_id: PMID:15486694 qualifier: enables review: summary: Recombinant zGCAP4 stimulated bovine rod outer segment cyclase at low Ca2+ and inhibited it at high Ca2+. action: ACCEPT reason: Direct enzymatic assay. supported_by: - reference_id: PMID:15486694 supporting_text: 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 ). - term: id: GO:0008048 label: calcium sensitive guanylate cyclase activator activity evidence_type: IDA original_reference_id: PMID:18777180 qualifier: enables review: summary: Purified myristoylated and non-myristoylated zGCAP4 activate bovine and zebrafish membrane guanylate cyclases Ca2+-dependently (abstract only in cache). action: ACCEPT reason: Direct assay; the abstract states the activation and its Ca2+ dependence. supported_by: - *act - term: id: GO:0008048 label: calcium sensitive guanylate cyclase activator activity evidence_type: IDA original_reference_id: PMID:23940527 qualifier: enables review: summary: Myristoylated zGCAP4 activated guanylate cyclases in native adult zebrafish retinal membranes. action: ACCEPT reason: The Ca2+-dependent difference in cyclase activity with zGCAP4 was highly significant in native zebrafish membranes. supported_by: - reference_id: PMID:23940527 supporting_text: '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)' - term: id: GO:0009966 label: regulation of signal transduction evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in supporting_entities: - FB:FBgn0013303 - PANTHER:PTN008588905 - RGD:68417 - SGD:S000002781 review: summary: Regulation of signal transduction, propagated from a deep neuronal calcium sensor node. action: KEEP_AS_NON_CORE 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. supported_by: - reference_id: PMID:18777180 supporting_text: 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. references: - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:15486694 title: 'Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes).' findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text. Cloning of zGCAP4; Ca2+-dependent activation of bovine ROS GC; in situ in double and long single cones, weak in short single cones, absent from rods. - id: PMID:18777180 title: The cone-specific calcium sensor guanylate cyclase activating protein 4 from the zebrafish retina. findings: [] full_text_unavailable: true reference_review: relevance: HIGH correctness: VERIFIED review_notes: Abstract only. zGCAP4 activates bovine and zebrafish cyclases, half-maximal at 520-570 nM free Ca2+; no Ca2+-myristoyl switch. - id: PMID:21829700 title: Differential calcium signaling by cone specific guanylate cyclase-activating proteins from the zebrafish retina. findings: [] reference_review: relevance: HIGH correctness: VERIFIED review_notes: Full text. Side-by-side comparison of all six zGCAPs in one assay; zGCAP4 in the low-sensitivity group (IC50 about 400 nM) with zGCAP5 and zGCAP7, zGCAP3 in the high-sensitivity group. - id: PMID:23940527 title: Zebrafish guanylate cyclase type 3 signaling in cone photoreceptors. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text. zGCAP4 activates native zebrafish retinal cyclases; proposed as the substitute for zGCAP3 in morphants. - id: PMID:26061947 title: Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text. Ca2+ binding of all zGCAPs; zGCAP4 aggregates with Mg2+. - id: PMID:30257895 title: Evolution of the calcium feedback steps of vertebrate phototransduction. findings: [] reference_review: relevance: MEDIUM correctness: VERIFIED review_notes: Full text. zGCAP3 and zGCAP4 are the 3R duplicates of GCAP3 (GCAP3a/GCAP3b). - id: file:DANRE/guca1c/guca1c-bioinformatics/RESULTS.md title: guca1c / guca1d sequence, synteny and expression analysis reference_review: relevance: HIGH correctness: VERIFIED review_notes: Manual audit verified the sidecar supports both GCAP3 duplicates retaining GCAP architecture and calcium-binding features while documenting cone expression and higher guca1c abundance. findings: [] core_functions: - description: Myristoylated neuronal Ca2+ sensor of cone photoreceptors (mainly double and long single cones) that activates the photoreceptor membrane guanylate cyclases when free Ca2+ falls; a strong activator with lower Ca2+ sensitivity (half-maximal near 0.4-0.57 uM) than its paralog zGCAP3. molecular_function: id: GO:0008048 label: calcium sensitive guanylate cyclase activator activity supported_by: - *act - reference_id: PMID:21829700 supporting_text: 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). - reference_id: PMID:15486694 supporting_text: The digoxigenin-labeled zGCAP4 antisense RNA probe hybridized specifically to the myoid region of double cones and long single cones protrude above the external limiting membrane. suggested_questions: - question: Does guca1d loss of function alter cone responses, alone or together with guca1c? - question: Which of the two copies kept the Ca2+ sensitivity of the ancestral GCAP3? suggested_experiments: - hypothesis: guca1d supports cone Ca2+ feedback at the higher Ca2+ range, complementing guca1c. description: 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. experiment_type: mutant electrophysiology