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Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3

Abstract

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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References

  1. Collins, B.M., McCoy, A.J., Kent, H.M., Evans, P.R. & Owen, D.J. Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523–535 (2002).

    Article  CAS  Google Scholar 

  2. Edeling, M.A. et al. Molecular switches involving the AP-2β2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev. Cell 10, 329–342 (2006).

    Article  CAS  Google Scholar 

  3. Ohno, H. Physiological roles of clathrin adaptor AP complexes: lessons from mutant animals. J. Biochem. 139, 943–948 (2006).

    Article  CAS  Google Scholar 

  4. Kelly, B.T. et al. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456, 976–979 (2008).

    Article  CAS  Google Scholar 

  5. McMurtry, C.T. et al. Significant developmental elevation in serum parathyroid hormone levels in a large kindred with familial benign (hypocalciuric) hypercalcemia. Am. J. Med. 93, 247–258 (1992).

    Article  CAS  Google Scholar 

  6. Lloyd, S.E., Pannett, A.A., Dixon, P.H., Whyte, M.P. & Thakker, R.V. Localization of familial benign hypercalcemia, Oklahoma variant (FBHOk), to chromosome 19q13. Am. J. Hum. Genet. 64, 189–195 (1999).

    Article  CAS  Google Scholar 

  7. Nesbit, M.A. et al. Identification of a second kindred with familial hypocalciuric hypercalcemia type 3 (FHH3) narrows localization to a <3.5 megabase pair region on chromosome 19q13.3. J. Clin. Endocrinol. Metab. 95, 1947–1954 (2010).

    Article  CAS  Google Scholar 

  8. Pollak, M.R. et al. Mutations in the human Ca2+-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75, 1297–1303 (1993).

    Article  CAS  Google Scholar 

  9. Hannan, F.M. et al. Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites. Hum. Mol. Genet. 21, 2768–2778 (2012).

    Article  CAS  Google Scholar 

  10. Brown, E.M. & MacLeod, R.J. Extracellular calcium sensing and extracellular calcium signaling. Physiol. Rev. 81, 239–297 (2001).

    Article  CAS  Google Scholar 

  11. Pearce, S.H. et al. Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J. Clin. Invest. 96, 2683–2692 (1995).

    Article  CAS  Google Scholar 

  12. Pearce, S.H. et al. Functional characterization of calcium-sensing receptor mutations expressed in human embryonic kidney cells. J. Clin. Invest. 98, 1860–1866 (1996).

    Article  CAS  Google Scholar 

  13. Kirchhausen, T. et al. AP17 and AP19, the mammalian small chains of the clathrin-associated protein complexes show homology to Yap17p, their putative homolog in yeast. J. Biol. Chem. 266, 11153–11157 (1991).

    CAS  PubMed  Google Scholar 

  14. Heath, H. III, Jackson, C.E., Otterud, B. & Leppert, M.F. Genetic linkage analysis in familial benign (hypocalciuric) hypercalcemia: evidence for locus heterogeneity. Am. J. Hum. Genet. 53, 193–200 (1993).

    PubMed  PubMed Central  Google Scholar 

  15. Wagener, B.M., Marjon, N.A., Revankar, C.M. & Prossnitz, E.R. Adaptor protein-2 interaction with arrestin regulates GPCR recycling and apoptosis. Traffic 10, 1286–1300 (2009).

    Article  CAS  Google Scholar 

  16. Brown, E.M. et al. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366, 575–580 (1993).

    Article  CAS  Google Scholar 

  17. Hofer, A.M. & Brown, E.M. Extracellular calcium sensing and signalling. Nat. Rev. Mol. Cell Biol. 4, 530–538 (2003).

    Article  CAS  Google Scholar 

  18. Grant, M.P., Stepanchick, A., Cavanaugh, A. & Breitwieser, G.E. Agonist-driven maturation and plasma membrane insertion of calcium-sensing receptors dynamically control signal amplitude. Sci. Signal. 4, ra78 (2011).

    Article  Google Scholar 

  19. Hough, T.A. et al. Activating calcium-sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification. Proc. Natl. Acad. Sci. USA 101, 13566–13571 (2004).

    Article  CAS  Google Scholar 

  20. Reyes-Ibarra, A.P. et al. Calcium-sensing receptor endocytosis links extracellular calcium signaling to parathyroid hormone-related peptide secretion via a Rab11a-dependent and AMSH-sensitive mechanism. Mol. Endocrinol. 21, 1394–1407 (2007).

    Article  CAS  Google Scholar 

  21. Leach, K. et al. Identification of molecular phenotypes and biased signaling induced by naturally occurring mutations of the human calcium-sensing receptor. Endocrinology 153, 4304–4316 (2012).

    Article  CAS  Google Scholar 

  22. Guarnieri, V. et al. Calcium-sensing receptor (CASR) mutations in hypercalcemic states: studies from a single endocrine clinic over three years. J. Clin. Endocrinol. Metab. 95, 1819–1829 (2010).

    Article  CAS  Google Scholar 

  23. Fang, Y., Huang, C.C., Kain, S.R. & Li, X. Use of coexpressed enhanced green fluorescent protein as a marker for identifying transfected cells. Methods Enzymol. 302, 207–212 (1999).

    Article  CAS  Google Scholar 

  24. Nesbit, M.A. et al. Characterization of GATA3 mutations in the hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome. J. Biol. Chem. 279, 22624–22634 (2004).

    Article  CAS  Google Scholar 

  25. Gaynor, K.U. et al. A missense GATA3 mutation, Thr272Ile, causes the hypoparathyroidism, deafness, and renal dysplasia syndrome. J. Clin. Endocrinol. Metab. 94, 3897–3904 (2009).

    Article  CAS  Google Scholar 

  26. Goldsmith, P.K., Fan, G., Miller, J.L., Rogers, K.V. & Spiegel, A.M. Monoclonal antibodies against synthetic peptides corresponding to the extracellular domain of the human Ca2+ receptor: characterization and use in studying concanavalin A inhibition. J. Bone Miner. Res. 12, 1780–1788 (1997).

    Article  CAS  Google Scholar 

  27. Mounkes, L., Kozlov, S., Burke, B. & Stewart, C.L. The laminopathies: nuclear structure meets disease. Curr. Opin. Genet. Dev. 13, 223–230 (2003).

    Article  CAS  Google Scholar 

  28. Kim, Y.M. & Benovic, J.L. Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein–coupled receptor trafficking. J. Biol. Chem. 277, 30760–30768 (2002).

    Article  CAS  Google Scholar 

  29. Nesterov, A., Carter, R.E., Sorkina, T., Gill, G.N. & Sorkin, A. Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis. EMBO J. 18, 2489–2499 (1999).

    Article  CAS  Google Scholar 

  30. Hannan, S. et al. γ-aminobutyric acid type B (GABA(B)) receptor internalization is regulated by the R2 subunit. J. Biol. Chem. 286, 24324–24335 (2011).

    Article  CAS  Google Scholar 

  31. Clark, M.J. et al. Performance comparison of exome DNA sequencing technologies. Nat. Biotechnol. 29, 908–914 (2011).

    Article  CAS  Google Scholar 

  32. Boyden, L.M. et al. Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482, 98–102 (2012).

    Article  CAS  Google Scholar 

  33. Lunter, G. & Goodson, M. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 21, 936–939 (2011).

    Article  CAS  Google Scholar 

  34. Li, H. et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

    Article  Google Scholar 

  35. Robinson, J.T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  36. Bataille, S., Berland, Y., Fontes, M. & Burtey, S. High Resolution Melt analysis for mutation screening in PKD1 and PKD2. BMC Nephrol. 12, 57 (2011).

    Article  CAS  Google Scholar 

  37. Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

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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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). https://doi.org/10.1038/ng.2492

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