β-Arrestins are multifunctional signalling adaptors, facilitating and regulating many signalling pathways in a variety of sub-cellular locales. This thesis work describes a novel role for β-arrestin 1 at the Golgi where it acts as a homeostatic regulator of Golgi morphology and function. β-Arrestin 1 knock-out MEFs have a more dispersed Golgi morphology when compared to WT MEFs, a difference in Golgi morphology that cannot be explained by differences in cell size or the cytoskeleton. Consistent with this observation, the overexpression of β-arrestin 1 in MEFs and HT29 cells causes Golgi compaction, suggesting a regulatory role for β-arrestin 1 at the Golgi. Furthermore, immunofluorescence experiments confirm that endogenous β-arrestin 1 is localised to this organelle. Mutants of β-arrestin 1 with characterised disruptions in protein binding abilities were expressed in HT29 cells and Golgi morphology was assessed by immunofluorescence. A mutant of β-arrestin 1 previously shown to lack the ability to bind AP2 failed to induce the compact Golgi phenotype associated with wild-type β-arrestin 1 expression. We confirmed by co-immunoprecipitation that β-arrestin 1 binds to AP1 suggesting that the association of β-arrestin 1 with AP1 is required for maintenance of a tightly packed Golgi structure. Conversely, the expression of a previously reported non-Src binding mutant produced an excessively compact Golgi. Inhibition of ERK, but not Src, activity recapitulated this phenotype suggesting this mutant may be defective in both Src and ERK binding, and implicating ERK in β-arrestin 1-mediated Golgi dispersal. Trafficking of the ts045-VSV-G protein is also perturbed when these β-arrestin 1 mutants are expressed, suggesting a functional role for β-arrestin 1 at the Golgi. β-Arrestin 1/AP1 complex formation is potentiated by active Src and serves to negatively regulate anterograde trafficking in addition to preventing Golgi dispersal. Conversely, β-arrestin 1 mediates ERK activation to facilitate anterograde trafficking and promote Golgi dispersal. β-Arrestin 1 thus likely plays a homeostatic role at the Golgi maintaining Golgi structure in the face of highly variable membrane flux through this organelle.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:626677 |
| Date | January 2014 |
| Creators | Webb, K. F. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1418064/ |
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