Guanylyl ayclase–activating protein

The primary processes of vertebrate visual excitation are located in the rod and cone photoreceptor cells of the retina. Illumination of the rod and cone cells triggers a biochemical cascade leading to hyperpolarization of the cell. This conversion of a light signal into an electrical signal is called phototransduction, and in the 1970s the concept of an intracellular second messenger mediating this process was developed. Two competing hypotheses were intensively discussed, the “calcium hypothesis” and the “cGMP hypothesis” to reconcile different lines of experimental results. With the identification of a cGMP-gated cation channel (cyclic nucleotide–gated channel, CNG-channel) in the plasma membrane of rod and cone cells, the second messenger of light excitation was finally identified (for a historical overview see Luo et al. 2008). Calcium on the other hand was found to be important for the sensitivity regulation (light adaptation) of photoreceptor cells (Fain et al. 2001) and was recognized as an essential part of negative feedback loops operating in photoreceptor cells (“calcium feedback”). In the current picture of phototransduction, light is absorbed by visual pigments thereby triggering a G protein–mediated signaling cascade and leading to the hydrolysis of cGMP by a phosphodiesterase. CNG-channels are open at the high concentration of cGMP in the dark and close after removal of cGMP. Synthesis of cGMP is catalyzed by retina-specific guanylate cyclases (GCs) according to the reaction scheme GTP → cGMP + pyrophosphate (PP_i). This step is under control of a calcium feedback, low Ca²⁺-concentrations increase and high Ca²⁺-concentration decrease GC activity. However, this regulatory step can only be of physiological relevance, if the cytoplasmic Ca²⁺-concentration changes over the time course of a light response. Indeed this was observed, since the cytoplasmic concentration of Ca²⁺ decreases after illumination due to the halted influx of Ca²⁺ through the CNG-channel and the continuous extrusion via a Na⁺/K⁺, Ca²⁺-exchanger. Thus a decrease in cytoplasmic Ca²⁺ accelerates cGMP synthesis. While this concept was developed in the 1980s, it became clear that the Ca²⁺-sensitive regulation of photoreceptor GC activity is mediated by a soluble Ca²⁺-binding protein (Koch and Stryer 1988). The consequent search for this protein led to the identification of a novel class of Ca²⁺-binding proteins, named guanylate cyclase–activating proteins (GCAPs) (Stephen et al. 2008; Dizhoor et al. 2010). Soon after their discovery and the determination of their amino acid sequence, it became apparent that GCAPs belong to the subfamily of neuronal calcium sensor (NCS) proteins, a group of EF-hand Ca²⁺-binding proteins mainly found in neurons and sensory cells (Burgoyne 2007).

First discovered in mammals, GCAP isoforms have been identified in many vertebrates including human, bovine, monkey, mice, chicken, fish, and amphibians. The different isoforms are classified on the basis of their amino acid sequences and are given a number like in GCAP1, GCAP2, etc. So far mammalian rod and cone cells express two or three GCAP isoforms, which are GCAP1, GCAP2, and GCAP3, but a larger variety was found in teleost fish, where the expression of eight isoforms was predicted in pufferfish (Fugu rubripes) and six isoforms were described in zebrafish (Danio rerio) and carp (Cyprinus carpio). The physiological meaning of variable GCAP expression is currently under investigation (Rätscho et al. 2010).

Tissue distribution of GCAPs had been investigated by in situ hybridization and/or immunocytochemistry. All GCAPs show a prominent transcription and/or expression in the outer vertebrate retina, presumably in the outer and inner segments of photoreceptor cells, but GCAP-specific staining was also observed in cone somata, cell bodies, axons, axon terminals, and synaptic pedicles. A comparative study on several mammalian retinas showed a species-dependent labeling pattern for GCAP1 and GCAP2. Furthermore, for some species as human and zebrafish (but not mice) GCAP3 appeared as a cone-specific isoform of GCAP. Other teleost fish–specific isoforms like for example GCAP4, GCAP5, and GCAP7 are also cone specific (Imanishi et al. 2004; Rätscho et al. 2009; Takemoto et al. 2009).

GCAPs regulate the activity of sensory GCs, in particular, the photoreceptor cell–specific forms. A triggering step of GCAP function is the light-induced decrease of the intracellular messenger cGMP and the consequent decrease of the cytoplasmic Ca²⁺-concentration in a rod or cone cell. Since GCAPs operate as Ca²⁺-sensors, they can detect changing concentrations of Ca²⁺ and thereby undergo a conformational change. GCAPs form a complex with the target GC in the Ca²⁺-free and Ca²⁺-bound state and it is assumed that the Ca²⁺-induced conformational change is transferred to the GC by protein–protein interaction. This in turn is thought to lead to an increase of catalytic activity at the cyclase catalytic domain (Fig. 2). Binding of ATP to the intracellular kinase homology domain is known to enhance the GC activity (Koch et al. 2010). The switch of a GCAP molecule from an inhibitor to an activator state does critically depend on the binding of Ca²⁺ at submicromolar and binding of Mg²⁺ at submillimolar concentrations. In the Ca²⁺-bound state (three Ca²⁺ bound) GCAPs suppress GC activity, in some cases below the basal level. Dissociation of Ca²⁺ from GCAP facilitates the exchange of Ca²⁺ for Mg²⁺ at one or two EF-hands, which is important for binding to the target GC and increase of GC activity (Dizhoor et al. 2010).

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Although GCAPs have several general properties (see above) in common, they differ in many aspects particularly concerning their regulatory properties. One characteristic distinguishing feature is the different Ca²⁺sensitivity, by which GCAP1 and GCAP2 regulate GC activity. Both proteins are present in rod and cone photoreceptor cells and overlap in their expression profile. A physiological meaning of this observation could be a so-called Ca²⁺-relay model of GC activation (Koch and Dell’Orco 2013). It is based on the observations that GCAP1 and GCAP2 are present in almost equal concentrations and that they equal in total the concentration of a GC dimer in rod cells. In the dark state of the cell, when the cytoplasmic Ca²⁺-concentration is high (Fig. 2) both GCAPs bind to a GC dimer (alternatively, one dimer could be associated with one GCAP molecule). In this state, the GC activity is very low and may just maintain the cytoplasmic dark concentration of cGMP by keeping a balance with the low dark activity of the cGMP hydrolyzing phosphodiesterase. Illumination of rod or cone cells leads to a decrease in cGMP and consequently to a fall in Ca²⁺. Depending on the light conditions and on the bleaching protocol, the cytoplasmic Ca²⁺–concentration reaches an intermediate level, which transforms only GCAP1 into an activator of the GC and keeps GCAP2 still in an inactive state (Fig. 2). This differential activating modus is in accordance with the observed differences in Ca²⁺-sensitivity. Stronger illumination then causes a further decrease of intracellular Ca²⁺, which induces the transition of GCAP2 into the activator state (Fig. 2). Changing light intensity of light flashes and background light might establish a different steady state of cytoplasmic Ca²⁺ causing a switch between different GCAP modes. By this mechanism, a photoreceptor cell could expand its dynamic range of responses to Ca²⁺.

Dysfunction or progressive visual impairment leading to blindness is often associated with inherited retinal diseases. Many proteins in photoreceptor cells with key functions for cell excitation and adaptation are known to be mutated in patients suffering from these diseases. Mutations in the GCAP gene are very often located within or near the third or fourth EF-hand and correlate with diseases like autosomal dominant cone or cone-rod dystrophies (Stephen et al. 2008; Behnen et al. 2010). Disease-related GCAP1 mutants show in almost all cases an incomplete suppression of GC activity at physiological Ca²⁺-concentration in the dark leading to a constitutive activation of the target enzyme. In turn this causes an elevated cGMP level in the cell and a distortion of the Ca²⁺-homeostasis.

The second messenger cGMP and its fine-tuned regulation of hydrolysis and synthesis are keystones of the molecular events in phototransduction. Regulation of sensory GCs by GCAPs represents a concept of cellular signaling different from the more classical case of cGMP synthesis control via hormone binding to a receptor GC (e.g., natriuretic peptide receptor GCs). Instead GCAPs are a specialized group of neuronal Ca²⁺-binding proteins that detect changes in cytoplasmic Ca²⁺ and control GC activity on the cytoplasmic part of the membrane-bound GC. The diversity of GCAPs in fishes, in particular in cones of the fish retina, could further indicate that they are an essential part of a “Ca ²⁺-relay model” operating in cone vision under changing illumination conditions. Finally, GCAPs might participate in regulatory protein–protein interactions involving other target proteins, as this has already been shown for GCAP2. The variety of GCAPs found in teleost fishes could also hint to a wider diversity of GCAP target molecules.