| الملخص: | <p>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 σ2 subunit (AP2σ2) of adaptor protein-2 (AP-2). The FHH3-associated AP2σ2 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.</p> <p>This thesis demonstrates that AP2σ2 mutations account for &Tilde;7% of all FHH mutations, only affect the Arg15 residue and show evidence of mutation bias, with only AP2σ2 Arg15Cys, Arg15Leu, and Arg15His mutations identified. Additionally, the study has identified <em>de novo</em> AP2σ2 mutations and revealed that FHH3 probands may present with a broader phenotype not seen in FHH1. The heterozygous missense AP2σ2 mutation was demonstrated to have no effect on the stability of the AP-2 complex in vivo. This thesis revealed that gain-of-function AP2σ2 mutations are unlikely to be a cause of ADH, which is due to GOF mutations in CaSR in &GT;70% of cases.</p> <p>The CaSR activates its downstream signalling pathways, including the mitogen-activated protein kinase (MAPK) pathway, through coupling with its associated G-proteins. The AP2σ2 mutations resulted in loss of activating phosphorylation events in the MAPK pathway suggesting a role for AP2σ2 in CaSR signalling. Additionally, the AP2σ2 Arg15Cys, Arg15His and Arg15Leu mutations were revealed to exert differential effects on the Gα<sub>q/11</sub> and MAPK signalling pathways, suggesting mutation-bias signalling.</p> <p>Clathrin-mediated endocytosis is crucial for embryological development. This thesis has demonstrated that mice homozygous for an <em>Ap2s1</em> splice site mutation, predicted to produce a functional knock-out, were lethal before embryonic day 12.5 (E12.5), suggesting AP2σ2 is essential for cell viability in the developing embryo.</p> <p>Thus, the work of this thesis has further elucidated the role of AP2σ2 in the biological pathways of CaSR signalling and clathrin-mediated endocytosis, and in the molecular pathology of disorders of calcium homeostasis.</p>
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