Diversity of GCAPs in teleost: multiple novel GCAPs in zebrafish (Danio Rerio) and pufferfish (Fugu Rubripes) retina

Abstract

Abstract: : Purpose: To explore the diversity of GCAPs and GCIP in teleost fish (Fugu rubripes and Danio rerio). Methods: In silico gene identification of 5 novel fugu and 3 novel zebrafish GCAPs, identification of GCIP genes in teleost, cloning of full–length cDNA. Bacterial expression of zGCAP 4 with his–tag and guanylate cyclase assay. In–situ hybridization of zGCAPs in retina and RT/PCR with multiple zebrafish tissue RNA to characterize the expression of zGCAPs. GCAP modeling with the HOMOLOGY module of the INSIGHTII software (Accelrys Inc., San Diego, CA), and calculation of a phylogenetic tree using Phylip (version 3.6, Felsenstein laboratory, U. of Washington). Results:We found that in addition to GCAP1–3, at least five additional GCAPs (GCAP4–8) are predicted to be present in fugu and zebrafish. We identified gene contigs encoding fuguGCAP1–8, fuguGCIP, zGCAP1–5, zGCAP7 and zGCIP. The exon/intron arrangements of the zGCAP1–5/GCAP7 and fGCAP1–8 genes are identical to those of known vertebrate GCAP genes suggesting that these genes arose by gene duplications from a common ancestor. In contrast to mammalian, the GCAP1 and GCAP2 genes apparently are not arranged in a tail–to–tail array in the pufferfish genome while the GCAP7,8 genes are in a head–to–tail array separated by less than 2 kb intergenic region. The positions of three of the four introns in GCIP genes are identical to those in GCAP genes indicating a close evolutionary relationship of GCAPs and GCIP. Sequence analysis of the predicted fugu and zebrafish GCAPs identified a myristoylation site at position 2 and three functional EF–hand Ca²⁺–binding motifs, EF2–4. Recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca²⁺–dependent manner. RT–PCR with GCAP specific primers showed specific expression of GCAPs and GCIP in the retina, while GCAP1, GCAP7, and GCIP mRNA is also present in the brain. In situ hybridization with anti–sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. Conclusions: The presence of at least eight GCAP genes suggests an unexpected complexity of regulation of photoreceptor GC in the teleost retina, and possibly additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4–8 or GCIP genes.

Keywords: photoreceptors • calcium • protein structure/function