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

Abstract

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.