Endocrine Abstracts

Adaptor protein-2 (AP2) is a heterotetramer of α, β, μ, and σ subunits that is pivotal in clathrin-mediated endocytosis and facilitates internalisation of plasma membrane constituents such as the calcium-sensing receptor (CaSR). AP2 σ subunit (AP2σ) missense mutations (Arg15Cys, Arg15His and Arg15Leu) result in familial hypocalciuric hypercalcaemia type 3 (FHH3) and decrease the sensitivity of CaSR-expressing cells to changes in extracellular calcium concentration. FHH3 is an autosomal dominant disorder and we hypothesized that AP2σ mutations may exert dominant-negative loss-of-function effects on the AP2 complex. We therefore studied the effect of increasing levels of WT and mutant AP2σ in CaSR-expressing cells by transient transfection with WT or mutant AP2S1-pBI-CMV2-GFP bidirectional expression construct that expresses GFP and AP2σ in a 1:1 ratio. Populations of cells with GFP expression varying from 1- to 32-fold over baseline, and thus AP2σ expression, were assessed by measurement of intracellular calcium responses to changes in extracellular calcium concentration, enabling calculation of the half-maximal effective concentration (EC₅₀) values for each cellular population. Linear regression of mean EC₅₀ values (n=8) for increasing expression levels of WT AP2σ demonstrated no deviation from zero (P=0.28, R² =0.27), indicating that progressive over-expression of WT AP2σ does not affect the function of the AP2 complex in CaSR signaling. In contrast, linear regression of mean EC₅₀ values (n=8) for increasing expression levels of mutant AP2σ demonstrated a positive incline (Cys15 P=0.014, R²=0.81; His15 P=0.0083, R²=0.86; Leu15 P=0.028, R²=0.74) indicating that increasing levels of mutant AP2σ proteins progressively impair intracellular responses to variations in extracellular calcium in CaSR-expressing cells. Thus, our results, demonstrate a likely dominant-negative effect of the AP2σ mutants (σ^m), whereby the mutant heterotetramer (AP2αβμσ^m) interferes with the binding of WT heterotetramer (AP2αβμσ) to the activated CaSR, and provide a mechanistic explanation for the dominant inheritance pattern of FHH3.