Fig. 1: The adhesion GPCR LPHN2 is a TGF-β–independent receptor of LRG1. | Experimental & Molecular Medicine

Fig. 1: The adhesion GPCR LPHN2 is a TGF-β–independent receptor of LRG1.

From: Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function

Fig. 1

a, b Tube formation assay (a) and transwell cell migration assays (b) using HUVECs treated with the indicated concentrations of the following proteins: PBS (negative control), LRG1 (1 μg/ml), TGF-β1 (5 ng/ml), LRG1 (1 μg/ml) + TGF-β1 (5 ng/ml), LRG1 (1 μg/ml) + anti-TGF-β1 Ab (10 μg/ml), and LRG1 (1 μg/ml) + TGF-β1 (5 ng/ml) + anti-TGF-β1 Ab (10 μg/ml). Left: Representative images. Scale bars, 200 µm. Right: Master junctions and migrated cells were quantified using ImageJ, and the results are presented as the means ± SEM (n = 4). The relative ratio of the control group was defined as 1. c Cell surface binding of LRG1-YFP or YFP to HEK293T cells (top) and HUVECs (bottom) was assessed after treatment with LRG1-YFP (10 μg/ml) or YFP (10 μg/ml). Nuclei were labeled with 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 100 µm. d Schematic diagram of LRC-TriCEPS technique to identify a TGF-β-independent receptor of LRG1. e Volcano plots showing FDR-adjusted P values plotted against fold changes between samples comparing TriCEPS-coupled LRG1 or Transferrin with the glycine-quenched TriCEPS reagent control sample. The three proteins LPHN2, LEG3 and NDUA5 represent receptor candidates and were defined as those with an enrichment factor greater than 4 and an FDR-adjusted P value < 0.05. f LPHN2 expression in HEK293T cells and HUVECs after infection with scramble shRNA control (shCon, 5 × 104 TU/ml) or LPHN2 knockdown shRNA lentivirus (shNPHN2, 5 × 104 TU/ml) was analyzed by Western blotting. g Immunoprecipitation (IP) of LPHN2 in whole HUVEC lysates treated with or without LRG1 (1 μg/ml) for 10 min followed by immunoblot analysis to detect LRG1 and LPHN2. Data are the means ± SEM (n = 3). h Cell surface binding of LRG1-YFP or YFP to control (shCon-expressing) or LPHN2-knockdown (shLPHN2-expressing) HEK293T cells (left) and HUVECs (right) after treatment with LRG1-YFP (10 μg/ml) or YFP (10 μg/ml). Nuclei were stained with DAPI (blue). Scale bar, 100 µm. *P < 0.05; **P < 0.01 (Student’s t test). N.S., not significant.

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