Detailed Localization of Photoreceptor Guanylate Cyclase Activating Protein-1 and -2 in Mammalian Retinas using Light and Electron Microscopy

Abstract

Guanylate cyclase activating proteins, GCAP-1 and GCAP-2, have a pivotal role in the activation of guanylate cyclase in phototransduction. Previous studies on the localization of GCAP-1 and GCAP-2 are contradictory. In this study, we tried to avoid possible artifacts accompanied by immunocytochemistry. Immunolabeling of a GCAP was carried out using antibodies pre-adsorbed with a different type of GCAP. In addition, immunolabeling was performed using three different animal species under different fixation and embedding. Electron microscopic immunocytochemistry was also performed to reveal subcellular localization of GCAPs as well as confirming data obtained by light microscopy. All data indicate that anti-GCAP-1 antibody binding sites were found predominantly in cone outer segments, in particular, in disk membrane regions. Sparse labeling was observed in rod outer segments, but the labeling was much lower than that seen in cone outer segments. Less labeling is also found in synaptic regions and inner segments of cones. No labeling was detected in connecting cilia and its cytoplasmic extensions. Such labeling patterns were similar among human, monkey and bovine retinas. The localization of GCAP-1 is consistent with the pattern of a recently reported human cone-specific degeneration. Anti-GCAP-2 antibody binding sites were detected in both inner and outer segments of rods and cones of all three animals although the labeling density was slightly different among species. Cryo-immuno-labeling of GCAP-2 in bovine retinas revealed that labeling sites were more concentrated in rods than those of cones, and that synaptic regions were also labeled. The different localization of GCAPs suggest that roles of GCAP-1 and GCAP-2 may be different.