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    • br For some time it

    2022-06-20


    For some time, it was known that membrane-bound guanylate cyclases (GCs) of photoreceptor bimatoprost mg in the retina respond to changes in the [Ca]. Lolley and Racz observed that synthesis of cyclic GMP (cGMP) was stimulated at only low [Ca], concluding that Ca has an inhibitory effect on the regulation of GCs. These authors suggested that in photoreceptors, changes in intracellular [Ca] may regulate the synthesis of cyclic GMP. Next, Koch and Stryer showed that stimulation of GC at low [Ca] is mediated by a protein that can be released from the outer segment membranes in low salt buffer, and that Ca sensitivity is partially restored by adding back the soluble extract to the depleted membranes. Several years of efforts to isolate the Ca-dependent mediator finally succeeded when Gorczyca et al. isolated a CaM-like protein that had the required properties. This protein was named guanylate cyclase-activating protein, or GCAP. The molecular cloning of GCAP from several species soon followed and an important monoclonal antibody against GCAP was generated . Immunoaffinity chromatography with this antibody led to the identification of a second modulator, termed GCAP2 , which also was independently purified and cloned by Dizhoor and colleagues. Subsequently, GCAP1 was also purified, expressed, and functionally reconstituted with GCs by Koch’s group . Recently, the GCAP family expanded significantly by the discovery of additional GCAP genes (GCAP4-8) present in the zebrafish and pufferfish genomes . Phylogenetic analysis of these genes suggests that teleost species underwent a series of gene duplications not observed in mammals. In addition to GCAPs, Li et al. cloned a related protein termed guanylate cyclase-inhibitory protein (GCIP) from frog retina, also present in teleost . GCIP is unable to stimulate GC but inhibits this enzyme at high free [Ca], and competes with GCAP1 and GCAP2 for GC regulatory sites. The major structural difference between GCIP and GCAPs was observed in the fourth EF-hand Ca-binding motif, which in GCIP is disabled for Ca binding. In frog retina, GCIP localized in the inner segments, somata, and synaptic terminals of cone photoreceptors. Several extensive reviews summarize the regulation of GCs by GCAPs , , , , , , , . In this contribution, we will discuss some more recent findings and address questions that will need to be answered by new experimental approaches. Cloning GCAPs from multiple species and their localization in the retina and pineal gland Initially, GCAP was cloned from mammalian retina and only two forms were identified, GCAP1 and GCAP2 [4], [5], [7], [19]. Haeseleer et al. [20] identified a human EST encoding a novel GCAP, GCAP3. GCAP1 and GCAP2 were expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones [21]. The GCAP1 and GCAP2 expression levels differ between species and direct comparison of the expression between species is not justified [22]. As a consequence of extensive modification in the GCAP3 gene, it is not expressed in mouse retina. In contrast, all three GCAPs were present in teleost (zebrafish) retina and were localized to rod cells, short single cones (GCAP1-2), and all subtypes of cones (GCAP3) [21]. More recently, GCAP4-8 were cloned or predicted from pufferfish (Fugu rubripes) and zebrafish (Danio rerio) [9]. In situ hybridization with anti-sense zGCAP4, zGCAP5, and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. In some species, GCAPs are also expressed in the pineal gland [23], likely due to the evolutionary relationship between these two organs. ESTs encoding human GCAP1 have been cloned from other sources, e.g., testes (BE937608) and brain (BM564184). GCAP2 ESTs were identified in chicken ovaries (BU455923) and Xenopus brain (CD328160), and a human GCAP3 EST was found in the thalamus (AV732101). These findings suggest the presence of GCAPs at low levels in these tissues. The sequence comparisons and evolutionary trace analysis allow one to identify the key conserved residues that are critical for GCAP structure and function, as well as to define class-specific residues for the NCBP subfamilies. A phylogenetic tree of all known GCAPs, GCIP, and recoverins in relation to CaM is shown in Fig. 1. The Ca2+-binding loops (EF-hand motifs) and surrounding helices are the most strictly conserved regions among GCAPs (Fig. 2). Thus, EF-hand motifs including adjacent regions could be responsible for binding to photoreceptor GCs as proposed recently [9]. GCAPs have only three active Ca2+ loops (EF-hand 2 and EF-hand 4); the first loop (EF-hand 1) is disabled for Ca2+ coordination. This is in contrast to CaM in which all four loops are active, or CaBPs [24], [25] in which EF-hand 2 is disabled, or recoverins, which have only two active loops (EF-hand 2 and EF-hand 3) [15].