Endocrine Abstracts

The calcium-sensing receptor (CaSR), a class C G-protein coupled receptor (GPCR) is critical for calcium homeostasis. The presence of CaSR at the plasma membrane (PM) is regulated by a balance between internalisation via clathrin-mediated endocytosis, and agonist-induced PM insertion from intracellular receptor pools, in a mechanism known as agonist-driven insertional signalling. Recently, mutations of the clathrin-mediated endocytic adaptor protein-2 sigma subunit, AP2σ, have been identified in patients with familial hypocalciuric hypercalcaemia type-3 (FHH3). To date, AP2σ mutations have only been identified at one residue, Arg15, and consist of substitutions to Cys, His or Leu. We investigated the effect of AP2σ mutations on CaSR PM expression using total internal reflection fluorescence microscopy. Imaging was performed in HEK293 cells stably expressing AP2σ-WT or FHH3-associated AP2σ-mutants R15C, R15H or R15L (n=33–48 cells), and transiently transfected with a CaSR construct with an N-terminal tag containing: a fluorescent bungarotoxin (BTx-594) binding site to monitor endocytosis; and pH-sensitive superecliptic pHluorin to simultaneously measure total PM CaSR. We observed an increase in net PM abundance in all cells on exposure to Ca²⁺, confirming agonist-induced insertion of CaSR following receptor stimulation. Levels of BTx-594 at the cell surface declined rapidly in AP2σ-WT cells consistent with constitutive internalisation of CaSR. Cells expressing AP2σ-mutants R15H and R15L had significantly longer BTx-594 cell surface labelling, indicative of delayed internalisation of the receptor (P<0.02). Furthermore, the time to 25% of CaSR internalisation was longer for all cells harbouring AP2σ mutations than AP2σ-WT expressing cells (393.4±63.4s (WT), 1003.3±102.2s (R15H), 688.6±112.1s (R15L), 670.1±99.3s (R15C), P<0.02 in all). CaSR activation also induced a second rapid internalisation step in AP2σ-WT expressing cells, that was absent or severely reduced in cells expressing AP2σ-mutant proteins. Thus, FHH3-associated AP2σ-mutations result in delayed CaSR internalisation, indicating an important role for CaSR endocytosis in regulating CaSR cell surface expression and signalling.