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 internalises 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-based and dileucine-based motifs of membrane-associated cargo proteins1,4. Here, we show that AP2 sigma subunit (AP2S1) missense mutations, which all involved the Arg15 residue (Arg15Cys, Arg15His and Arg15Leu) that 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 parathyroids, kidneys and bone5-7 These AP2S1 mutations occurred in >20% of FHH patients without calcium-sensing GPCR (CaSR) mutations which cause FHH18-12. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular-calcium and reduced CaSR endocytosis, likely through a loss of interaction with a C-terminus CaSR dileucine-based motif whose disruption also decreased intracellular signalling. Thus, our results reveal a new role for AP2 in extracellular-calcium homeostasis.

A G-protein Subunit-α11 Loss-of-Function Mutation, Thr54Met, Causes Familial Hypocalciuric Hypercalcemia Type 2 (FHH2)

“…1) was performed using leucocyte DNA and gene‐specific primers (Sigma‐Aldrich, St. Louis, MO, USA), as previously reported 11. Publicly accessible databases, including dbSNP (http://www.ncbi.nlm.nih.gov/projects/SNP/), 1000 genomes (http://browser.1000genomes.org), the National Heart, Lung and Blood Institute (NHLBI) Exome Sequencing Project (http://evs.gs.washington.edu/EVS/, EVS data release ESP6500SI) representing the exomes of approximately 6500 individuals, and the Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org) representing exomes of 60,706 unrelated individuals, were examined for the presence of sequence variants, and any potential pathogenic sequence abnormality identified within the patient DNA was confirmed by restriction endonuclease analyses, as described 12…”

“…The bidirectional pBI‐CMV2 cloning vector was used because it facilitated the co‐expression of Gα 11 and GFP,11, 12 and site‐directed mutagenesis was used to generate the mutant GNA11 construct using the Quikchange Lightning Site‐directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and gene‐specific primers (Sigma‐Aldrich), as described 19. Cells were maintained in DMEM‐Glutamax media (Thermo‐Fisher, Waltham, MA, USA) with 10% fetal bovine serum (Gibco, Thermo‐Fisher) and 400 μg/mL geneticin (Thermo‐Fisher) at 37°C, 5% CO 2 .…”

“…Cells were maintained in DMEM‐Glutamax media (Thermo‐Fisher, Waltham, MA, USA) with 10% fetal bovine serum (Gibco, Thermo‐Fisher) and 400 μg/mL geneticin (Thermo‐Fisher) at 37°C, 5% CO 2 . Successful transfection was confirmed by visualizing GFP fluorescence using an Eclipse E400 fluorescence microscope with a Y‐FL Epifluorescence attachment and a triband 4,6‐diamidino‐2‐phenylindole‐FITC‐Rhodamine filter, and images captured using a DXM1200C digital camera and NIS Elements software (Nikon, Tokyo, Japan) 11, 12, 13. The expression of Gα 11 and CaSR proteins was also determined by Western blot analyses using anti‐Gα 11 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐GFP (Santa Cruz), anti‐calnexin (Millipore, Billerica, MA, USA) or anti‐CaSR (AbCam, Cambridge, UK) antibodies.…”

“…The Ca 2+ i responses of HEK293‐CaSR cells expressing wild‐type or mutant Gα 11 proteins were assessed by a flow cytometry‐based assay, as reported 11, 12, 13. In brief, HEK293‐CaSR cells were cultured in T75 flasks and transiently transfected 24 hours later with 16 μg DNA 11.…”

“…Transfected cells, in suspension, were stimulated by sequentially adding Ca 2+ to the Ca 2+ ‐ and Mg 2+ ‐free HBSS to increase the [Ca 2+ ] o in a stepwise manner from 0 to 15 mM and then analyzed on a MoFlo modular flow cytometer (Beckman Coulter, Indianapolis, IN, USA) by simultaneous measurements of GFP expression (at 525 nm), Ca 2+ i ‐bound Indo‐1AM (at 410 nm), and free Indo‐1AM (ie, not bound to Ca 2+ i ) (at 485 nm), using a JDSU Xcyte UV laser (Coherent Radiation, Santa Clara, CA, USA), on each cell at each [Ca 2+ ] o , as described11, 12. The peak mean fluorescence ratio of the Ca 2+ i transient response after each individual stimulus was measured using Cytomation Summit software (Beckman Coulter) and expressed as a normalized response, as described 11, 12. Nonlinear regression of concentration‐response curves was performed with GraphPad Prism (GraphPad, La Jolla, CA, USA) using the normalized response at each [Ca 2+ ] o for each separate experiment for the determination of EC 50 (ie, [Ca 2+ ] o required for 50% of the maximal response) and Hill coefficient values.…”