Characterization of retinal guanylate cyclase-activating protein 3 (GCAP3) from zebra®sh to man

Calmodulin-like neuronal Ca-binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF-hand Ca-binding motifs. The guanylate cyclase-activating proteins 1±3 (GCAP1±3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1±2 (GC1±2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modi®cation, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebra®sh) retina and localized them to rod cells, short single cones (GCAP1±2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to de®ne class-speci®c residues for the NCBP subfamilies. Introduction Ca-binding proteins from the calmodulin (CaM) superfamily, termed GCAPs, are involved in the regulation of photoreceptor GC (Polans et al., 1996). GCAPs stimulate GC1 and/or GC2 in low [Ca]free, and this regulation is responsible, in part, for modulating the sensitivity of photoreceptor cells and extending their operation through a broad range of light intensities (Mendez et al., 2001). Two proteins, GCAP1 and GCAP2, which share only »50% homology were discovered initially (reviewed in Polans et al., 1996). More recently, we reported the presence of a new retina-speci®c GCAP, termed GCAP3 (Haeseleer et al., 1999), and GCIP (Li et al., 1998). For detailed understanding of phototransduction, the pairing of GCAPs with GCs is of great importance, however, it remains unclear. In situ hybridization with short GCAP1 RNA probes showed the transcript abundantly present in cone myoid regions and, to a lesser degree, in rod inner segments in human and bovine retina (Palczewski et al., 1994; Subbaraya et al., 1994). The intensity of GCAP1's immunoreactivity was strong in cone outer segments for all mammalian species tested but weaker in rod outer segments (ROS) (Gorczyca et al., 1995), particularly in primates (Kachi et al., 1999), cats (Cuenca et al., 1998), and mouse retina (Cuenca et al., 1998; Howes et al, 1998). Initially, GCAP2 was localized in rod photoreceptors (Dizhoor et al, 1995). However, in subsequent experiments, we and colleagues localized GCAP2 to the cone inner segments, somata, and synaptic terminals and, less signi®cantly, to the rod inner segments and inner retinal neurons. A number of interesting differences were observed between species (Otto-Bruc et al., 1997; Cuenca et al., 1998; Kachi et al., 1999). In mouse retina, GCAP2 was nearly undetectable in cones (Howes et al., 1998). All of these studies were carried out initially without knowledge of the existence of GCAP3 (Haeseleer et al., 1999), whose expression pattern and localization had not yet been reported. Mammalian photoreceptors express two retina-speci®c membraneassociated types of GCs. GC1 immunoreactivity in human retina was detected in the photoreceptor outer segments, primarily in cones (Dizhoor et al, 1994; Liu et al., 1994). Although radioisotopic in situ hybridization localized human GC2 mRNA to photoreceptor inner segments and outer nuclear layer (Lowe et al., 1995), a higher resolution technique is required to determine the expression in rods and cones. Electron microscopy of rat retina showed that both Correspondence: Dr Krzysztof Palczewski, Department of Ophthalmology or Dr Wolfgang Baehr, Department of Ophthalmology, as above. E-mail: [email protected] *Present address: Novasite Pharmaceuticals, Inc., 3520 Dunhill Street, San Diego, CA 92121, USA Received 30 September 2001, revised 15 November 2001, accepted 19 November 2001 European Journal of Neuroscience, Vol. 15, pp. 63±78, 2002 ã Federation of European Neuroscience Societies