Fig. 4. Aβ₄₂ interacts with GABA_B1R/CaSR heterodimers and antagonizes Gi activation.

(A) PTGs from ^PTCApp^−/− (pink squares), ^PTCApp^−/−Casr^−/− (red triangles), ^PTCApp^−/−Gabbr1^−/− (blue diamonds), and ^PTCApp^−/−Casr^−/−Gabbr1^−/− (purple circles) mice were sequentially incubated with increasing doses (from 0. 3 to 1000 nM) of Aβ₄₂ in the presence of 1 mM of [Ca²⁺]_e. mean ± s.e.m., n=8–11 mice per genotype. (B) Averaged time-course of cAMP levels in the PTH-C1 cells co-expressed CaSR, GABA_B1R, and a FRET-based cAMP reporter and were pretreated with cholera toxin. Arrows indicate the times to apply Ca²⁺ (3 mM), Aβ₄₂ (1 μM), and forskolin/IBMX (10 μM), mean ± s.e.m. of n=3 experiments with 10–15 cells/experiment. (C) Detection of endogenous GABA_B1R/CaSR heterodimers by the proximity ligation assay (PLA) and Aβ₄₂ by immunohistochemistry (IHC) and their colocalization in overlayed images of human PTGs from normal donors and parathyroid adenomas from PHPT patients with deficient (<20 ng/ml) or repleted (>30 ng/ml) pre-operative 25OH vitamin D levels (n=4 glands per group). Aβ₄₂ immunoreactivity was visualized with Alex Fluor 488 (green) and GABA_B1R/CaSR with Texas Red (red) signals and counterstained with DAPI (blue) for nuclei. Colocalization of Aβ₄₂ and GABA_B1R/CaSR heterodimers was indicated by yellow signals in overlayed images. Scale bar: 125 μm.