Structural diversity of neuronal calcium sensor proteins and insights for activation of retinal guanylyl cyclase by GCAP1.

Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca(2) (+)-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca(2) (+)-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca(2) (+)-binding to both recoverin and NCS-1 cause large protein conformational changes that ejects the covalently attached myristoyl group into the solvent exterior and promotes membrane targeting (Ca(2) (+)-myristoyl switch). The GCAP proteins undergo much smaller Ca(2) (+)-induced conformational changes and do not possess a Ca(2) (+)-myristoyl switch. Recent structures of GCAP1 in both its activator and Ca(2) (+)-bound inhibitory states will be discussed to understand structural determinants that control their Ca(2) (+)-dependent activation of retinal guanylyl cyclases

Role of guanylyl cyclase modulation in mouse cone phototransduction

A negative phototransduction feedback in rods and cones is critical for the timely termination of their light responses and for extending their function to a wide range of light intensities. The calcium feedback mechanisms that modulate phototransduction in rods have been studied extensively. However, the corresponding modulation mechanisms that enable cones to terminate rapidly their light responses and to adapt in bright light, properties critical for our daytime vision, are still not understood. In cones, calcium feedback to guanylyl cyclase is potentially a key step in phototransduction modulation. The guanylyl cyclase activity is modulated by the calcium-binding guanylyl cyclase activating proteins (GCAP1 and GCAP2). Here, we used single-cell and transretinal recordings from mouse to determine how GCAPs modulate dark-adapted responses as well as light adaptation in mammalian cones. Deletion of GCAPs increased threefold the amplitude and dramatically prolonged the light responses in dark-adapted mouse cones. It also reduced the operating range of mouse cones in background illumination and severely impaired their light adaptation. Thus, GCAPs exert powerful modulation on the mammalian cone phototransduction cascade and play an important role in setting the functional properties of cones in darkness and during light adaptation. Surprisingly, despite their better adaptation capacity and wider calcium dynamic range, mammalian cones were modulated by GCAPs to a lesser extent than mammalian rods. We conclude that a disparity in the strength of GCAP modulation cannot explain the differences in the dark-adapted properties or in the operating ranges of mammalian rods and cones

Impaired Ca2+ sensitivity of a novel GCAP1 variant causes cone dystrophy and leads to abnormal synaptic transmission between photoreceptors and bipolar cells

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1-cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers' accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified

Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2

Journal ArticlePURPOSE: To identify gene arrangement, chromosomal localization, and expression pattern of mouse guanylate cyclase activating proteins GCAP1 and GCAP2, retina-specific Ca2+-binding proteins, and photoreceptor guanylate cyclase activators. METHODS: The GCAP1 and GCAP2 genes were cloned from genomic libraries and sequenced. The chromosomal localization of the GCAP array was determined using fluorescent in situ hybridization. The expression of GCAP1 and GCAP2 in mouse retinal tissue was determined by immunocytochemistry. RESULTS: In this study, the mouse GCAP1 and GCAP2 gene array, its chromosomal localization, RNA transcripts, and immunolocalization of the gene products were fully characterized. The GCAP tail-to-tail array is located at the D band of chromosome 17. Each gene is transcribed into a single transcript of 0.8 kb (GCAP1) and 2 kb (GCAP2). Immunocytochemistry showed that both GCAP genes are expressed in retinal photoreceptor cells, but GCAP2 was nearly undetectable in cones. GCAP2 was also found in amacrine and ganglion cells of the inner retina. Light-adapted and dark-adapted retinas showed no significant difference in the distribution of the most intense GCAP2 staining within the outer segment and outer plexiform layers. CONCLUSIONS: Identical GCAP gene structures and the existence of the tail-to-tail gene array in mouse and human suggest an ancient gene duplication-inversion event preceding mammalian diversification. Identification of both GCAPs in synaptic regions, and of GCAP2 in the inner retina suggest roles of these Ca-binding proteins in addition to regulation of phototransduction

Enzymatic Relay Mechanism Stimulates Cyclic GMP Synthesis in Rod Photoresponse: Biochemical and Physiological Study in Guanylyl Cyclase Activating Protein 1 Knockout Mice

Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1−/− retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1−/− retinas became more sensitive to Ca2+ and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1−/−, as well as in RetGC1−/−GCAP1−/−, and RetGC2−/−GCAP1−/− hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1−/− rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1−/− was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca2+ and specificities toward RetGC isozymes. GCAP1 is the ‘first-response’ sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed-up photoreceptor recovery

Unveiling novel components of the protein complex responsible for cGMP synthesis in retinal photoreceptors: role in cell physiology and disease

In photoreceptor cells of the retina, light triggers a protein G-mediated enzymatic cascade that leads to hydrolysis of cGMP. The drop in cGMP levels causes the closure of cGMP-channel at the plasma membrane, decreasing the influx of cations, mainly Na+ and Ca2+, hyperpolarizing the cell. This hyperpolarization decreases the rate of neurotransmitter release at the synaptic terminal. Photoreceptor cells must recover the darkness equilibrium and adapt their light sensitivity to the wide range of light intensities present in the natural world. Genetic defects at both activation and termination cascades leads to inherited retinal dystrophies. The cGMP levels are restored to darkness equilibrium by new synthesis by the complex formed by a membrane form of guanylate cyclase (RetGC) which is bound to a couple of proteins that confers it calcium sensitivity, Guanylate Cyclase Activating Proteins (GCAP1 and GCAP2). RetGC activity is stimulated by the drop of Ca2+ concentration because of close of cGMP-channels. There is a feedback loop between cGMP and Ca2+ that has a fundamental role in the processes of termination of light response and light adaptation. RetGC1 is responsible for cGMP synthesis in rods and cones. Mutations of genes involved in the cGMP synthesis complex have been linked to autosomal dominant inherited retinal dystrophies, both retinitis pigmentosa (adRP) as Leber’s congenital amaurosis (LCA) The regulation of RetGC by GCAPs proteins has been extensively studied in vitro, at biochemical and structural levels. However, many relevant aspects of regulation and trafficking of this complex in vivo remains unknown. By subretinal electroporation, we have analyzed the molecular determinants of subcellular distribution of GCAPs. We have determined that the complex between RetGC1 and GCAP1 is assembled in the inner segment and then transported to the outer segment, playing a determinant role the myristoylation of GCAP1 and the binding of GCAP1 to the cyclase. On the other hand, phosphorylation plays an essential role in subcellular distribution of GCAP2, and failures in subcellular localization of GCAP2 could contribute to explain the pathophysiology of the human G157R mutation linked to adRP. We here report a proteomic approach to identify novel interactors of Guanylate Cyclase Activating Protein 1 (GCAP1) that led to the unexpected discovery of inosine monophosphate dehydrogenase 1 (IMPDH1) interaction with retinal guanylate cyclase 1 (RetGC1). IMPDH1 is the rate-limiting step in de novo GTP synthesis, and mutations in impdh1 gene have been associated to adRP and LCA. We reveal an unanticipated direct interaction between IMPDH1 and RetGC1 at photoreceptor outer segments where phototransduction takes place. The interaction involves the dimerization and catalytic domains of RetGC1, and is significantly affected by IMPDH1 mutations associated to blindness. This finding links de novo GTP synthesis to GTP conversion to cGMP, bridging blindness-causative genes so far considered unrelated and creating a new scenario for the development of therapeutic strategies. By bridging distinct blindness-causative genes in a common biochemical pathway, we here contribute to reduce the apparent complexity of inherited retinal dystrophies grouping them on base common metabolic pathways. The main aim of this strategy of grouping genes on base of their function is to identify “hubs” of cell damage. We also have characterized the interaction between RetGC1 and Creatine kinase B, which could be supplying locally the ATP needed to maintain the catalytic activity in cones. This work aid to understanding about regulation and trafficking of RetGC/GCAPs complex, as well as the interplaying between the cGMP synthesis complex and de novo GTP synthesis, opening a new conceptual framework for pharmacological treatment of diseases that trigger changes in intracellular levels of cGMP, which in a prolonged way affect to cell survival, leading to inherited blindness.En fotorreceptores de retina, la respuesta a luz desencadena la hidrólisis del cGMP. La síntesis de cGMP recae en el complejo formado por una forma de membrana de la guanilato ciclasa (RetGC1 y RetGC2) y unas proteínas que le confieren sensibilidad a calcio (Guanylate Cyclase Activating Proteins GCAP1 y GCAP2). Mutaciones en los genes que codifican para las proteínas integrantes de este complejo han sido ligadas distrofias hereditarias de retina autosómicas dominantes. La regulación de este complejo ha sido extensamente estudiada in vitro, sin embargo, muchos aspectos relacionados con este complejo en el entorno de la célula viva se desconocen. Determinamos mediante electroporación subretinal que el ensamblaje del complejo formado por RetGC1 y GCAP1 precede a su transporte hacia el segmento externo y tanto la miristoilación como la unión a la ciclasa por parte de GCAP1 son necesarias para su transporte. Por otro lado, la fosforilación juega un papel clave en la distribución celular de GCAP2, y fallos en la localización de GCAP2 podrían contribuir a explicar la patofisiología de la mutación hG157R ligada a retinosis pigmentaria autosómica dominante. Mediante una aproximación proteómica para identificar nuevos interactores de GCAP1, hemos caracterizado la interacción directa entre la guanilato ciclasa y la inosina monofosfato deshidrogenasa (IMPDH1), la enzima responsable del paso limitante en la síntesis de novo de GTP. Mutaciones en el gen impdh1 se han asociado a distrofias hereditarias de retina autosómicas dominantes. Ambas proteínas se localizan en el compartimento sensorial, interaccionan en el orden micromolar, involucrando a los dominios de dimerización y catalítico de RetGC1 y la interacción se afecta significativamente por los mutantes asociados a ceguera en IMPDH1. Además también se ha caracterizado la interacción de RetGC con la Creatina quinasa B (CKB), la cual podría está proporcionando el ATP local necesario para mantener la actividad catalítica específicamente en conos. Este trabajo arroja luz sobre la regulación y transporte del complejo RetGC/GCAPs, así como la interconexión entre los complejos de síntesis de cGMP y síntesis de novo de GTP, integrando genes asociados a enfermedad en base a su implicación en procesos metabólicos comunes, abriendo un nuevo escenario para el tratamiento farmacológico de enfermedades que provoquen cambios en los niveles de cGMP intracelulares

Constitutive activation of guanylate cyclase by the G86R GCAP1 variant is due to "locking" cation-\u3c0 interactions that impair the activator-to-inhibitor structural transition

Guanylate Cyclase activating protein 1 (GCAP1) mediates the Ca2+-dependent regulation of the retinal Guanylate Cyclase (GC) in photoreceptors, acting as a target inhibitor at high [Ca2+] and as an activator at low [Ca2+]. Recently, a novel missense mutation (G86R) was found in GUCA1A, the gene encoding for GCAP1, in patients diagnosed with cone-rod dystrophy. The G86R substitution was found to affect the flexibility of the hinge region connecting the N- and C-domains of GCAP1, resulting in decreased Ca2+-sensitivity and abnormally enhanced affinity for GC. Based on a structural model of GCAP1, here, we tested the hypothesis of a cation-\u3c0 interaction between the positively charged R86 and the aromatic W94 as the main mechanism underlying the impaired activator-to-inhibitor conformational change. W94 was mutated to F or L, thus, resulting in the double mutants G86R+W94L/F. The double mutants showed minor structural and stability changes with respect to the single G86R mutant, as well as lower affinity for both Mg2+ and Ca2+, moreover, substitutions of W94 abolished "phase II" in Ca2+-titrations followed by intrinsic fluorescence. Interestingly, the presence of an aromatic residue in position 94 significantly increased the aggregation propensity of Ca2+-loaded GCAP1 variants. Finally, atomistic simulations of all GCAP1 variants in the presence of Ca2+ supported the presence of two cation-\u3c0 interactions involving R86, which was found to act as a bridge between W94 and W21, thus, locking the hinge region in an activator-like conformation and resulting in the constitutive activation of the target under physiological conditions