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)(1-3). AP2, a heterotetramer of alpha, beta, mu and sigma subunits, links clathrin to vesicle membranes and binds to tyrosine-and dileucine-based motifs of membrane-associated cargo proteins(1,4). Here we show that missense mutations of AP2 s subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins(4), result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone(5-7). We found AP2S1 mutations in > 20% of cases of FHH without mutations in calcium-sensing GPCR (CASR)(8-12), 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.