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Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3


Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein–coupled receptors (GPCRs)1,2,3. AP2, a heterotetramer of α, β, μ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins1,4. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins4, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone5,6,7. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR)8,9,10,11,12, which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.

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Figure 1: Evolutionary conservation of AP2σ2 Arg15 and structural analysis of mutants.
Figure 2: Schematic model for CaSR signaling and its endocytosis mediated by β-arrestin and AP2.
Figure 3: AP2S1 Arg15 mutants increase the EC50 value of CaSR-expressing cells.

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This work was supported by the UK Medical Research Council (MRC) programme grants G9825289 and G1000467 (to M.A.N., F.M.H., A.A.C.R., C.E.T. and R.V.T.); the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme (to M.A.N. and R.V.T.); the High-Throughput Genomics Group, Wellcome Trust Centre for Human Genetics (Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070); the Research and Development Office, Northern Ireland (to U.G., S.J.H. and P.J.M.); and the Shriners Hospitals for Children (grant 15958) (to M.P.W.). S.A.H. is a Wellcome Trust Clinical Research Training Fellow.

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Authors and Affiliations



M.A.N. and R.V.T. designed the experiments. M.A.N., F.M.H., S.A.H., A.A.C.R. and C.E.T. performed experiments and analyzed data. M.A.N., S.A.H., A.A.C.R., T.C., C.E.T., L.G. and A.J.R. carried out sequencing and information technology. G.M. and D.B. directed the information technology and the exome capture DNA sequencing infrastructure. T.C., U.G., P.J.M., S.J.H., M.P.W. and R.V.T. recruited subjects and families with FHH. M.A.N., F.M.H., S.A.H. and N.R. performed the AP2S1 functional calcium assays. M.A.N., F.M.H. and R.V.T. wrote the manuscript. All authors checked the manuscript for scientific content and contributed to the final drafting of the manuscript.

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Correspondence to Rajesh V Thakker.

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Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Note (PDF 321 kb)

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Nesbit, M., Hannan, F., Howles, S. et al. Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3. Nat Genet 45, 93–97 (2013).

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