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  • GCIPs are highly conserved Ca binding proteins first

    2021-09-15

    GCIPs are highly conserved Ca2+-binding proteins first discovered in the frog [42] and later in teleost [9]. With regard to gene structure, sequence conservation, and function, GCIPs are evolutionarily related to the GCAPs. The GCIP genes have acquired an additional intron in the N-terminal region while preserving the positions of the three C-terminal introns at exactly the same junctions as in GCAP genes. GCIP genes/ESTs could not be retrieved from human, mouse, rat, or other mammalian genomic databases, suggesting that the GCIP genes were lost in mammals or arose in teleost/amphibia after mammalian and teleost divergence.
    The function of GCAP1 as revealed through animal models The tail-to-tail GCAP gene array facilitates a GCAP double knockout with one construct eliminating C-terminal exons of both genes (Fig. 3A). Mouse retinas lacking GCAPs can be evaluated for their contribution to the recovery in rods and cones. As shown previously [43], Ca2+ regulation of GCs in GCAPs−/− retinas is abolished as expected from biochemical characterization of GCAP function. Single cell flash responses from dark-adapted GCAPs−/− rods were larger and slower than responses from wild-type rods (Fig. 3D). Transgenic bovine GCAP2 expressed in GCAPs−/− rods restored Ca2+-sensitive GC activity, but did not restore normal flash response kinetics [43]. In transgenic mice expressing mouse GCAP1 on a GCAP knockout background, the levels of expression in rod photoreceptors varied, presumably due to limited capacity of the partial GCAP1 promoter used to generate these mice (Fig. 3E). However, the majority of the rods generated flash responses that were indistinguishable from those of wild-type [39] (Fig. 3C). Under paired flash ERG conditions stimulating Senexin A exclusively, a delayed recovery of both the cone driven b-wave and a-wave was observed in GCAP knockout retinas. As shown for rods, transgenic GCAP1 could restore normal cone response recovery. Paired flash ERGs clearly demonstrated that the recovery of the cone-driven a-wave was restored to normal [44]. It appears from these experiments that transgenic mouse GCAP1 expressed at normal levels on a GCAP knockout background supports the generation of wild-type flash responses in rods and cones in the absence of GCAP2. To determine the precise role of GCAP2 in recovery, Ca2+ dependent regulation of GC1 and/or GC2, or a potential role in adaptation, current experiments are aiming at expression of mouse GCAP2 on the GCAP knockout background, and expression of GCAP1 and GCAP2 on GC1/GC2 null backgrounds.
    GCAP genes and human retina disease Deletion of GCAP1 and GCAP2 genes in mice does not affect morphology of the retina, indicating that functional GCAP genes are not essential for the development or the survival of photoreceptor cells. The GCAP1 gene, however, has been linked to autosomal dominant cone dystrophy (adCD) [45]. A mutation (Y99C) results in a Tyr to Cys change found in humans afflicted with dominant cone dystrophy [46], [47]. The cone-specific degeneration is consistent with high expression levels of GCAP1 in the outer segment of this cell type, which also expresses GCAP3 and GCAP2. The residue Y99 is flanking the 12 amino acid EF-hand 3 loop and is conserved in most GCAPs (zebrafish and fugu GCAPs carry an F instead of Y, Fig. 2). The Y99C mutation has been shown to alter the Ca2+ sensitivity of GCAP1, leading to the constitutive stimulation of GC1 at high [Ca2+]i, limiting its ability to fully inactivate GC1 under physiological dark conditions (a similar mutation in GCAP2, Y104C, also affects Ca2+ sensitivity [46]). An increase in the concentration of cGMP is expected to ensue, and such alterations have been linked in other studies to the degeneration of photoreceptor cells in animal models such as the rd1 mouse [48] and the rcd1 Irish setter [49]. The E155G mutation [50] is predicted to affect Ca2+ coordination at EF-hand 4 and exert similar effects on GC1 as does GCAP1(Y99C). The residue E155 of GCAP1 is 100% conserved in all GCAPs sequenced so far (Fig. 2). We expressed a similar mutant, GCAP1(E155D), in vitro and showed that it was biologically active but less Ca2+-sensitive than normal GCAP1, very similar to E111D (Fig. 6A in [30]) and GCAP1(Y99C) (Fig. 4 in [47]). A third mutation, GCAP (I143NT), was recently identified and shown to cause adCD [63]. The fourth mutation (GCAP1(P50L)) [51] most likely causes disease by an entirely different mechanism. In contrast to Y99 and E155, the residue P50 is not present in other GCAPs and is not consistently conserved in all GCAP1 sequences (Fig. 2). Recombinant GCAP1(P50L) was biologically active, indistinguishable from GCAP1 [52], but the mutant was less stable and more susceptible to proteolysis [51]. Thus, GCAP1(P50L) may exert a dominant negative effect causative of the human disease. No disease-causing mutations have been identified in the human GCAP2 gene [53].