MS4A3 mediates GM-CSF/IL-3-induced receptor endocytosis and downstream signaling in LAMA-84 cells. (A) Western blotting of pERK and pSTAT5^Y694 after GM-CSF/IL-3 stimulation, with or without pretreatment of the endocytosis blocker, dynasore (a dynamin inhibitor). (B) Flow cytometry plots of MS4A3 and surface CD116 in LAMA-84 cells with endogenous dox-shMS4A3 (KD), MS4A3 (basal), or MS4A3 overexpression (OE). Right: representative plots of GM-CSF-induced CD116 endocytosis and the effect of MS4A3 KD. (C) GM-CSF-induced CD116 endocytosis analyzed by flow cytometry. MFI: mean fluorescent intensity (surface). (D-E) Endocytosis and recycling of GM-CSFR α chain and β chain after GM-CSF stimulation and wash off. The changes of MFI compared with before stimulation values were plotted. (F-G) Two-dimensional and 3D illustrations of MS4A3 protein structure and features. Two-dimensional structure and transmembrane domain depiction were generated by TOPO2 (Johns S.J., TOPO2, Transmembrane protein display software, http://www.sacs.ucsf.edu/TOPO2/). The 3D structure is predicated by the world-leading AI-based protein folding engine, AlphaFold.⁸³ Color-coding indicates the prediction confidence level, with higher confidence trending blue and lower trending yellow. Arrows point at the color-coded mutant regions in panel H-J. (H) Baseline CD116 surface level in cells transfected with various MS4A3 mutants. (I-J) GM-CSF-induced GM-CSFR endocytosis as shown by relative changes in CD116 and CD131 surface levels in cells transfected with various MS4A3 mutants. (K) IL-3-induced CD123 endocytosis analyzed by flow cytometry. (L) Western blotting of GM-CSF/IL-3-induced signaling activation in cells with endogenous MS4A3, dox-shMS4A3, or MS4A3 overexpression. Quantifications of signal intensities from 4 independent experiments are provided on the right.