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    • br Biochemical properties and post

    2022-01-19


    Biochemical properties and post-translational modification of GCAPs GCAPs are hydrophobic proteins that are likely permanently bound to their GC target under physiological conditions [5]. They interact with the photoreceptor GCs through the intracellular domains (reviewed in [17]). GCAP1 has been shown to be myristoylated at the N-terminal Gly residue [4], and this post-translational modification is predicted to be common to all GCAPs. In bovine retina, GCAP1 is heterogeneously acylated with different lipids (saturated and unsaturated) as has been observed for other acylated proteins in the retina [26]. The functional role of this modification is not fully understood and explored, outside of obvious roles in interaction with membranes as a hydrophobic anchor (reviewed in [15]). Unlike recoverin, valdecoxib mg which features a so-called Ca2+/myristoyl switch [27] that depends on the free [Ca2+], the myristoyl group in GCAP2 and most likely other GCAPs is exposed at all Ca2+ concentrations [28]. A number of studies, based on mutagenesis and biophysical approaches, were carried out which enhanced our understanding of the structure and function of GCAPs. Among these were endogenous Trp fluorescence and engineered Trp reporter residues in GCAP1 [29], limited proteolysis [30], and SH group titration with EPR spectroscopy [31], [32], mutagenesis of GCAP1 and GCAP2 [28], [33], [34], [35] (reviewed in [13]), NMR [36], and peptide valdecoxib mg studies for the activation of GCs [37]. Inactivation of two EF-hand motifs (EF-hand 3 and EF-hand 4) of GCAP1 converts this protein into a Ca2+-insensitive activator at all physiological concentrations of [Ca2+] (reviewed in [15]). Based on these studies, two mechanisms emerged describing the stimulation of GC in the recovery phase of phototransduction: one model assumes changes within the flexible central helix of GCAP1 upon Ca2+ dissociation, causing relative reorientation of two structural domains containing a pair of EF-hand motifs and thus switching its target, GCs, from a low active to a more active enzyme. We favor this model. The second model proposes that dimerization of GCAPs is part of the mechanism by which this protein regulates GCs [38].
    Gene structures of GCAPs and GCIPs The vertebrate GCAP structural genes (translation start to stop) show significant variation in length, from 876bp (fugu GCAP8) to 45,732bp (human GCAP3). All GCAP genes sequenced so far or identified as contigs in the GenBank have three introns. With regard to the polypeptide sequences, the positions of introns in GCAP genes have been precisely conserved over several hundred millions of years of evolution, from pufferfish to chicken and human GCAPs. Introns 1 and 3 interrupt the coding sequences of EF-hand 2 and EF-hand 4 precisely between residues 4 and 5 of the 12-amino acid Ca2+-binding loop. Intron 3 is invariably positioned between the sixth and seventh amino acid after EF-hand 3. Thus, the known GCAP gene structures are identical throughout vertebrate species. The human and mouse GCAP1 and GCAP2 genes are organized in a tail-to-tail array (genes arranged on opposite strands) in vertebrates [39], [40] separated by relatively short intergenic regions containing polyadenylation sites for transcript termination. The GCAP1/2 gene array, most likely arising by a gene duplication/inversion event, is also present in chicken, while in teleost the distance between GCAP1 and GCAP2 genes is much larger (>80kb in zebrafish); thus, it seems unlikely that the tail-to-tail array has been preserved in these species. In humans, the GCAP gene array is located on 6p21, and the GCAP3 gene is located on 3q13.1 [20], suggesting a gene duplication and translocation event in this species. In mice, the GCAP3 gene was shown to be inactive and disabled by multiple stop codons in the coding region (potentially a pseudogene). It is reasonable to assume that GCAP4-8 in teleost were generated by additional gene duplications since the teleost and mammalian lineages diverged about 450 million years ago [41]. For example, the fugu GCAP7 and fugu GCAP8 genes are arranged in a head-to-tail gene array (both genes on the same strand in the same direction), separated by only 2kb of genomic DNA (stop codon of fugu GCAP7 to ATG of fugu GCAP8). The conservation of GCAP gene structures over several hundred millions of years of evolution and multiple rounds of gene duplications is remarkable. Based on genome searches and EST analyses, the mouse, rat, and human genomes do not harbor GCAP4-8 genes.