Identificación de nuevas Dianas funcionales de las Quinasas de receptores acoplados a proteinas G (GRKs)

• Autores: María Helena Holguín Asensio
• Directores de la Tesis: Ana Ruiz Gómez (dir. tes.), Federico Mayor Menéndez (dir. tes.)
• Lectura: En la Universidad Autónoma de Madrid ( España ) en 2009
• Idioma: español
• Tribunal Calificador de la Tesis: Jorgina Satrústegui Gil-Delgado (presid.), Sonsoles Campuzano Corrales (secret.), Miguel Medina Padilla (voc.), Francisco Javier Vitorica Ferrández (voc.), Anna M. Aragay i Combas (voc.)
• Materias:
  • Ciencias de la vida
    • Biología molecular
• Enlaces
  • Tesis en acceso abierto en: Biblos-e Archivo
• Resumen

  • G protein-coupled receptor kinases (GRKs) represent an important point of convergence of different signalling routes. GRKs were first reported to participate together with arrestins in the regulation of multiple G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological importance.

    More recent evidence indicates that GRKs (and in particular the ubiquitous GRK2 isoform) can also phosphorylate certain tyrosine kinase receptors and a growing number of non-GPCR substrates and interact with a variety of proteins involved in cell signaling, acting as agonist-regulated adaptor scaffolds, thus suggesting that these proteins could have diverse ¿effector¿ functions.

    This work has been focused in the identification of new functional interactions of GRK proteins. First, we have caracterized that DREAM/calsenilin/KChIP3 protein interacts and is phosphorylated by GRK2 at Ser- 95. This phosphorylation did not modify the repressor activity of DREAM, but mimicking phosphorylation at this site impairs DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. In addition, inhibition of calcineurin, a phosphatase able to dephosphorylate DREAM protein that has been phosphorylated by GRK2 ¿in vitro¿, also blocked DREAM-mediated Kv4.2 channel trafficking. Our results thus suggest that these dynamic posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

    We have also identified Presenilin 1 (PS1), a DREAM/calsenilin/KChIP3-interacting protein itself, as a novel GRK2 functional partner. PS1 interacts and is phosphorylated by GRK2 in a ß 2 -adrenergic receptor activation and internalization-dependent manner. GRK2/PS1 association is increased upon phosphorylation of GRK2 by cSrc and ERK. Phosphorylation of full-length PS1 by GRK2 promotes PS1 degradation by the proteasome pathway, whereas phosphorylation of the processed PS1 (NTF/CTF) enhances ¿-secretase activity towards APP in a GRK2- activity and phosphorylated-state-dependent manner.

    Finally, we have shown that Gprk2, a Drosophila member of the GRKs family, plays a key role in the Smo signal transduction pathway. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. Using Drosophila melanogaster as an experimental model, we have shown that lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. We have found that Gprk2 function is required for transducing the Smo signal and that when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary.