Endocrine Abstracts

Familial hypocalciuric hypercalcaemia type-3 (FHH3) is caused by loss-of-function mutations of the sigma subunit of adaptor protein-2 (AP2), a ubiquitously expressed heterotetrameric protein with a fundamental role in endocytosis of transmembrane proteins. FHH3-associated AP2σ mutations impair internalisation of calcium-sensing receptor (CaSR) giving rise to FHH. CaSR predominantly signals via Gα_q/11 leading to intracellular calcium release, and activation of mitogen-activated protein kinase (MAPK) pathways. We have previously shown AP2σ mutations impair intracellular calcium release and therefore hypothesised that MAPK signalling may also be altered. To determine the effect of AP2σ mutations on MAPK signalling we undertook two approaches: examination of immediate signalling events by measuring phosphorylated ERK1/2 (pERK1/2) using AlphaScreen assays; and examination of late signalling events using a serum-response element (SRE) luciferase reporter in HEK293 cells stably expressing AP2σ-WT or AP2σ-mutant (R15C, R15H or R15L) proteins and transiently expressing CaSR. Exposure to increasing (0–10 mM) extracellular calcium concentrations ([Ca²⁺]_e) led to increased AlphaScreen pERK1/2 responses in a dose-dependent manner; however, AP2σ-mutant cells had significantly reduced responses at higher [Ca²⁺]_e compared to AP2σ-WT (P<0.05 n=4). Furthermore, activation of the CaSR with 5mM extracellular calcium induced increased SRE reporter expression in all cell-lines; however, reporter activity in AP2σ-mutant cells was significantly reduced compared to AP2σ-WT (P<0.001, n=4). Finally, we investigated the effect on ERK1/2 signalling in lymphoblastoid cell-lines derived from leukocytes of FHH3 patients with AP2σ-R15C mutations, alongside control cells from unaffected family members. Western blot analyses of lymphoblastoid cells exposed to 5mM extracellular calcium demonstrated an increase in pERK1/2, but responses were significantly reduced in AP2σ-R15C expressing cells (P<0.05). Furthermore, AlphaScreen analyses confirmed that AP2σ-R15C lymphoblastoid cells had reduced pERK1/2 responses compared to AP2σ-WT cells (P<0.001, n=4). In conclusion, our studies demonstrate FHH3-associated AP2σ mutations impair MAPK signalling which may have implications for cell proliferation and survival.