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Title: The role of the sigma subunit of adaptor protein-2, AP2σ2, in the regulation of calcium homeostasis
Author: Rogers, Angela
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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The calcium sensing receptor (CaSR), a G-protein coupled receptor (GPCR), and its downstream signalling pathways are essential for calcium homeostasis. Loss- or gain-of-function (LOF and GOF, respectively) mutations in components of these pathways result in disorders of calcium homeostasis including Familial hypocalciuric hypercalcaemia (FHH) and Autosomal dominant hypocalcaemia (ADH). FHH type 3 (FHH3) is due to LOF mutations in the s2 subunit (AP2s2) of adaptor protein-2 (AP-2). The FHH3-associated AP2s2 mutations identified to date affect the Arg15 residue and comprise heterozygous Arg15Cys, Arg15Leu, and Arg15His mutations. AP-2 is a ubiquitously expressed heterotetrameric protein, with a central role in clathrin-mediated endocytosis of transmembrane proteins, such as GPCRs. This thesis demonstrates that AP2s2 mutations account for ~7% of all FHH mutations, only affect the Arg15 residue and show evidence of mutation bias, with only AP2s2 Arg15Cys, Arg15Leu, and Arg15His mutations identified. Additionally, the study has identified de novo AP2s2 mutations and revealed that FHH3 probands may present with a broader phenotype not seen in FHH1. The heterozygous missense AP2s2 mutation was demonstrated to have no effect on the stability of the AP-2 complex in vivo. This thesis revealed that gain-of-function AP2s2 mutations are unlikely to be a cause of ADH, which is due to GOF mutations in CaSR in >70% of cases. The CaSR activates its downstream signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway, through coupling with its associated G-proteins. The AP2s2 mutations resulted in loss of activating phosphorylation events in the MAPK pathway suggesting a role for AP2s2 in CaSR signalling. Additionally, the AP2s2 Arg15Cys, Arg15His and Arg15Leu mutations were revealed to exert differential effects on the Gaq/11 and MAPK signalling pathways, suggesting mutation-bias signalling. Clathrin-mediated endocytosis is crucial for embryological development. This thesis has demonstrated that mice homozygous for an Ap2s1 splice site mutation, predicted to produce a functional knock-out, were lethal before embryonic day 12.5 (E12.5), suggesting AP2s2 is essential for cell viability in the developing embryo. Thus, the work of this thesis has further elucidated the role of AP2s2 in the biological pathways of CaSR signalling and clathrin-mediated endocytosis, and in the molecular pathology of disorders of calcium homeostasis.
Supervisor: Thakker, Rajesh V. ; Cox, Roger Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available