Role of Guanylate Cyclase Activating Proteins in photoreceptor cells of the retina in health and disease
In the last two decades, it has been done a thoroughly research about the role of Guanylate Cyclase Activating Proteins (GCAPs) in photoreceptor cells of the retina as activity regulators of Retinal Guanylate Cyclase (RetGC), which allow to restore cGMP levels to darkness ones when intracellular Ca2...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2014 |
| País: | España |
| Recursos: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/283566 |
| Acesso em linha: | http://hdl.handle.net/10803/283566 |
| Access Level: | acceso abierto |
| Palavra-chave: | Ciències de la salut Ciencias biomédicas Medical sciences Malalties de la retina Enfermedades de la retina Retinal diseases Sinapsi Sinapsis Synapses Fotoreceptors Fotorreceptores Photoreceptors Ciències de la Salut 616 |
| Resumo: | In the last two decades, it has been done a thoroughly research about the role of Guanylate Cyclase Activating Proteins (GCAPs) in photoreceptor cells of the retina as activity regulators of Retinal Guanylate Cyclase (RetGC), which allow to restore cGMP levels to darkness ones when intracellular Ca2+ falls. However, little is known about: a) ¿What determines GCAPs distribution within the cell?, b) ¿Which other functions GCAP proteins, GCAP1 and GCAP2, carry out at other cellular compartments different from the sensory one? and c) ¿How they cause cell death when they are mutated? In this study we want address these questions. 1. First of all, we own a mouse model that expresses a GCAP2 mutated form unable to bind Ca2+ (bEF-GCAP2). Other mutations described for GCAP1 and present in some autosomic dominant Cone Rod Dystrophies (adCORD), prevent Ca2+ binding to some of its EF-hand domains which produces the constitutive activation of RetGC, and consequently, high cGMP levels that result in toxicity for the cell. However, we observe that our model causes the death by other mechanism, as RetGC is not activated by GCAP2, because GCAP2 is retained in the inner segment and does not translocate to the sensory compartment. We want to identify interactions that GCAP2 establish differentially in this compartment and could be retaining it. We find out 14-3-3 family of proteins by mass-spectrometry and liquid chromatography. Furthermore, bEF-GCAP2 is abnormally phosphorylated in vivo and GCAP2 phosphorylation promotes its binding to 14-3-3 binding. We demonstrate that GCAP2 phosphorylation in residue serine 201 is the cause of its retention in the inner segment, avoiding its translocation to the outer segment, and when we mutate serine 201 into a glycine, this retention is reverted in vivo. Finally, we propose that GCAP2 phosphorylation and its binding to 14-3-3 is what retains GCAP2 in the inner segment, and this happens in a balance way during dark/light day cycles. When this system overloads will cause retinal degeneration by the formation of aggregates. We believe that mutations in GCAP2 or light conditions promoting GCAP2 accumulation in its Ca2+-free form in the inner segment of the cell, bring to cell death by GCAP2 conformational instability. Most important, we propose that this will also apply for genetic scenarios mimicking the effects to constant light exposure, the so called “equivalent-light” scenarios. 2. Secondly, as a result of the identification of GCAP2 interaction to RIBEYE (Venkatesan et al. 2010) the major component of synaptic ribbons in the photoreceptor cell terminal, we developed an ultrastructural study of the role that GCAP2 may play in this compartment. Through confocal and electronic microscopy we have demonstrated the presence of GCAP1 and GCAP2 in rod synaptic ribbons. However, GCAP1 and GCAP2 are not necessary during synaptic ribbons assembling and basic maintenance. As GCAP2 overexpression in the wildtype background (which means a higher GCAP2:GCAP1 ratio) promotes ribbons disassembling, we propose that GCAP2 may play a role mediating the morphological changes that take place in the synaptic ribbons in response to variations in [Ca2+]. |
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