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Vertebrate photoreceptor cells are exquisite light detectors operating under very dim and bright illumination. The photoexcitation and adaptation machinery in photoreceptor cells consists of protein complexes that can form highly ordered supramolecular structures and control the homeostasis and mutual dependence of the secondary messengers cGMP and Ca2+. The visual pigment in rod photoreceptors, the G protein-coupled receptor rhodopsin is organized in tracks of dimers thereby providing a signaling platform for the dynamic scaffolding of the G protein transducin. Illuminated rhodopsin is turned off by phosphorylation catalyzed by rhodopsin kinase GRK1 under control of Ca2+-recoverin. The GRK1 protein complex partly assembles in lipid raft structures, where shutting off rhodopsin seems to be more effective. Re-synthesis of cGMP is another crucial step in the recovery of the photoresponse after illumination. It is catalyzed by membrane bound sensory guanylate cyclases and is regulated by specific neuronal Ca2+-sensor proteins called GCAPs. At least one guanylate cyclase (ROS-GC1) was shown to be part of a multiprotein complex having strong interactions with the cytoskeleton and being controlled in a multimodal Ca2+-dependent fashion. The final target of the cGMP signaling cascade is a cyclic nucleotide-gated channel that is a hetero-oligomeric protein located in the plasma membrane and interacting with accessory proteins in highly organized microdomains. We summarize results and interpretations of findings related to the inhomogeneous organization of signaling units in photoreceptor outer segments.

Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations.In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g. Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a two-fold shift in the Ca2+-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca2+-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca2+-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases.

The membrane guanylate cyclase family has been branched into three subfamilies: natriuretic peptide hormone surface receptors, Ca2+-modulated neuronal ROS-GC, and Ca2+-modulated odorant surface receptor ONE-GC. The first subfamily is solely modulated by the extracellularly-generated hormonal signals; the second, by the intracellularly-generated sensory and sensory-linked signals; and the third, by combination of these two. The present study defines (1) a new paradigm and (2) a new mechanism of Ca2+ signaling. (1) ANF-RGC, the prototype member of the surface receptor subfamily, is stimulated by free [Ca2+]i. The stimulation occurs via myristoylated form of neurocalcin , and both the guanylate cyclase and the calcium sensor neurocalcin are present in the glomerulosa region of the adrenal gland. (2) The EF-2, EF-3 and EF-4 hands of GCAP1 sense the progressive increment of [Ca2+]i and with a K1/2 of 100 nM turn ROS-GC1 OFF. In total reversal, the same EF hands upon sensing the progressive increment of [Ca2+]i with K1/2 turn ONE-GC ON. The findings suggest a universal Ca2+-modulated signal transduction theme of the membrane guanylate cyclase family; demonstrate that signaling of ANF-RGC occurs by the peptide hormones and also by [Ca2+]i signals; that for the Ca2+ signal transduction, ANF-RGC functions as a two-component transduction system consisting of the Ca2+ sensor neurocalcin  and the transducer ANF-RGC; and that the neurocalcin  in this case expands beyond its NCS family. Furthermore, the study shows a novel mechanism of the [Ca2+]i sensor GCAP1 where it acts as an antithetical NCS for the signaling mechanisms of ROS-GC1 and ONE-GC.

Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca2+/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca2+-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance (SPR) technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca2+-sensing protein. The synergetic inhibition of RK by both Ca2+-sensors occurs over a broader range of Ca2+-concentration than by recoverin alone, indicating increased Ca2+-sensitivity of RK regulation in the presence of both Ca2+-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK.