Localization of guanylate cyclase-activating protein 2 in mammalian retinas

Abstract

A BSTR ACTGuanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca 2؉ , a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC in bovine rod outer segment homogenates. These results suggest that although GCAP1 is involved in the Ca 2؉ -sensitive regulation of GC in rod and cone outer segments, GCAP2 may have non-phototransduction functions in photoreceptors and inner retinal neurons.In photoreceptor cells, photoactivation of rhodopsin or cone visual pigment results in a transient decrease in the concentrations of Ca 2ϩ and cGMP. These receptors and second messengers are linked through a cascade of specific activation͞ inactivation reactions in phototransduction (1, 2). The levels of Ca 2ϩ and cGMP are strictly controlled and interconnected. cGMP is a gating ligand of the plasma membrane cation channels that are permeable to Ca 2ϩ ions. After cGMP is hydrolyzed, the efflux of Ca 2ϩ exceeds the influx, resulting in decreased [Ca 2ϩ ] within the cell. The lowering of [Ca 2ϩ ] triggers production of cGMP through activation of a photoreceptor-specific particulate guanylate cyclase (GC) (3). The Ca 2ϩ sensitivity of GC (e.g., the higher activity at low levels of [Ca 2ϩ ]) is mediated by one or more Ca 2ϩ -binding proteins, termed guanylate cyclase-activating proteins (GCAPs) (4, 5).Two photoreceptor-specific GCs, GC1 and GC2, have been cloned (6-9). Although the localization of GC1 to rod and cone outer segments and synaptic terminals was established by both biochemical and immunocytochemical methods (5,(10)(11)(12)(13)(14), the localization of GC2 within photoreceptors is not known. Two GCAPs that stimulate GC1 and GC2 have also been cloned (15-17). There is abundant evidence that GCAP1 activates photoreceptor GC: (i) GCAP1 was isolated from rod outer segments (ROS) (4, 18); (ii) GCAP1 mRNA was found in the myoid region of rod and cone photoreceptor cells (15, 19); (iii) GCAP1 was localized to rod and cone outer segments and to some cone somata and synaptic terminals by immunocytochemistry (17, 18); (iv) cross-linking techniques revealed that GCAP1 was complexed with GC1 (20). In addition, the finding that GC1 is not expressed in the retina of the rd...

Guanylate cyclase-activating protein (GCAP) 1 rescues cone recovery kinetics in GCAP1/GCAP2 knockout mice

“…Alternatively, GCAP1 may play a role in unidentified pathways at the cone synapse. In wild-type mice, both GCAP1 and GC1 are found in photoreceptor terminals and could modulate cGMP channels at the synapse (8,26,33,34). G1T3 ϩ GCAP Ϫ/Ϫ mice exhibit high levels of GCAP1 in the cone somata and pedicles (Fig.…”

“…Whereas these GCAP1 mutations incur gain of function events leading to disease (41), loss of GCAP1 alters the recovery kinetics in GCAP Ϫ/Ϫ mice but does not lead to a degenerative disorder. Although GCAP2 has been identified in human cone inner segments (26) and at low levels in inner retinal cells (15), no mutations in the GCAP2 gene have been associated with retina disease (42). The high levels of GCAP1 in cone outer segments (15,43) and its ability to restore response recovery argue that GCAP1 is the predominant Ca 2ϩ -dependent GC activator in other mammalian cones as well.…”

“…For example, in situ hybridization with GCAP subclass-specific RNA in human indicated that GCAP1 and GCAP2 are present in rod and cone inner segments (14). Immunocytochemistry with GCAP2-monospecific antibodies detected GCAP2 mostly in human cone inner segments (26). However, immunocytochemistry did not detect significant levels of GCAP2 in mouse cones, whereas mouse rods expressed GCAP1 and GCAP2 at nearly the same levels (16).…”

Diversity of Guanylate Cyclase-Activating Proteins (GCAPs) in Teleost Fish: Characterization of Three Novel GCAPs (GCAP4, GCAP5, GCAP7) from Zebrafish (Danio rerio) and Prediction of Eight GCAPs (GCAP1-8) in Pufferfish (Fugu rubripes)

“…Three GCAPs (GCAP 1-3) have been cloned from vertebrate retinas (Dizhoor et al 1994Gorczyca et al 1994Gorczyca et al , 1995Haeseleer et al 1999;Imanishi et al 2002). GCAP mRNAs are abundant transcripts present in vertebrate rod and cone photoreceptors (Imanishi et al 2002;Otto-Bruc et al 1997b;Palczewski et al 1994;Subbaraya et al 1994). As judged by immunoreactivity, GCAP1 is most abundant in cone outer segments of all mammalian species tested (Cuenca et al 1998;Kachi et al 1999).…”

“…As judged by immunoreactivity, GCAP1 is most abundant in cone outer segments of all mammalian species tested (Cuenca et al 1998;Kachi et al 1999). GCAP2 was localized primarily in rod photoreceptors but was also found in cones (Kachi et al 1999;Otto-Bruc et al 1997b). GCAP3 was only identified in human and zebrafish (Danio rerio) retinas.…”