N-ethyl-N-nitrosourea-Induced Adaptor Protein 2 Sigma Subunit 1 (Ap2s1) Mutations Establish Ap2s1 Loss-of-Function Mice

The adaptor protein-2 sigma subunit (AP2s), encoded by AP2S1, forms a heterotetrameric complex, with AP2a, AP2b, andAP2msubunits, that is pivotal for clathrin-mediated endocytosis, and AP2s loss-of-function mutations impair internalization of the calcium-sensing receptor (CaSR), a G-protein-coupled receptor, and cause familial hypocalciuric hypercalcemia type-3 (FHH3). Mice with AP2s mutations that would facilitate investigations of the in vivo role of AP2s, are not available, and we therefore embarked on establishing suchmice. We screened >10,000 mice treated with the mutagen N-ethyl-N-nitrosourea (ENU) for Ap2s1 mutations and identified 5 Ap2s1 variants, comprising 2 missense (Tyr20Asn and Ile123Asn) and 3 intronic base substitutions, one of which altered the invariant donor splice site dinucleotide gt to gc. Three-dimensionalmodeling andcellular expression of themissense Ap2s1 variants did not reveal them to alterAP2s structure or CaSR-mediated signaling, but investigation of the donor splice site variant revealed it to result in an in-frame deletion of 17 evolutionarily conserved amino acids (del17) that formed part of theAP2sa1-helix, alpha 1-beta 3 loop, and beta 3 strand. Heterozygousmutantmice (Ap2s1+/del17) were therefore established, and these had AP2s haplosufficiency but were viable with normal appearance and growth. Ap2s1+/del17 mice, when compared with Ap2s1(+/+) mice, also had normal plasma concentrations of calcium, phosphate, magnesium, creatinine, urea, sodium, potassium, and alkaline phosphatase activity; normal urinary fractional excretion of calcium, phosphate, sodium, and potassium; and normal plasma parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D (1,25(OH)(2)) concentrations. However, homozygous Ap2s1del17/del17mice were non-viable and died between embryonic days 3.5 and 9.5 (E3.5-9.5), thereby indicating that AP2s likely has important roles at the embryonicpatterning stages andorganogenesis of the heart, thyroid, liver, gut, lungs, pancreas, and neural systems. Thus, our studies have established a mutant mouse model that is haplosufficient for AP2s. (C) 2017 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.