1. GPR56/ADGRG1 Activation Promotes Melanoma Cell Migration via NTF Dissociation and CTF-Mediated Gα12/13/RhoA Signaling 
Nien-Yi Chiang, Yen-Ming Peng, Horng-Heng Juang, Tse-Ching Chen, Hsiao-Lin Pan, Gin-Wen Chang, Hsi-Hsien Lin 
Journal of Investigative Dermatology 
Volume 137, Issue 3, Pages (March 2017)
DOI: /j.jid
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2. Figure 1
CG4-induced GPR56 activation depends on GPR56/CD9/CD81 interaction and requires proper GPS proteolysis and the 7TM domain. (a) GPR56 activation in MeWo cells by immobilized CG4 specifically enhances IL-6 secretion. (b-d) The IL-6 level of MeWo cells stably expressing the GPR56-shRNA (sh615 or sh833) (b), tetraspanin-knockdown MeWo cells (c), and indicated stable A375 cells (d) was determined by ELISA. (e) Analysis of the IL-6 promoter activity. The pGL3/IL-6 promoter-luciferase reporter construct (0.4 μg) was cotransfected with 0, 0.1, 0.2, and 0.3 μg of pcDNA3.1-GPR56 into A375 cells. The luciferase activity was measured 24 hours after transfection. (f, g) The IL-6 level of indicated stable A375 cells (f) and stable A375/GPR56-WT cells transfected with the indicated siRNAs (g). GPR56, G protein-coupled receptor 56; GPS, GPCR proteolysis site; 7TM, seven transmembrane; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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3. Figure 2
IL-6 promotes cell migration and invasion of A375-GPR56 cells. (a, c, d) The migration abilities of indicated stable A375 melanoma cell lines were evaluated using the Boyden chamber assay (a) and the gap-closure assay (c, d) (n = 3, mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; A375-Neo vs. A375-GPR56/WT cells, or control IgG1 vs. CG4). T indicated the time points (hour) when cell migration was evaluated. (b) The promoting effect of IL-6 on cell migration (left panel) and invasion (right panel) of stable A375 cells was confirmed by treating cells with a functional blocking anti-IL-6 mAb or a control mIgG2b (10 μg/ml) (n = 3, mean ± SD; ∗P < 0.05, ∗∗P < 0.01). GPR56, G protein-coupled receptor 56; mAb, monoclonal antibody; SD, standard deviation; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
CG4-induced GPR56 cross-linking facilitates NTF dissociation and leads to CTF self-activation. (a–c) FACS analysis of the surface GPR56 levels of A375/GPR56-WT and A375/GPR56-T383A cells incubated with the indicated mAbs (n = 3, mean ± SD; ∗P < 0.05; ∗∗P < 0.01). (d, f) WB analysis of transiently transfected A375 cells as indicated. (e, g) The IL-6 levels secreted by the transiently transfected A375 cells cultured on immobilized mAbs as indicated (n = 3, mean ± SD; ∗∗P < 0.01, ∗∗∗P < vs. respective controls). CTF, C-terminal fragment; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPR56, G protein-coupled receptor 56; HA, hemagglutinin; mAbs, monoclonal antibodies; NTF, N-terminal fragment; SD, standard deviation; WB, western blotting; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
The interaction between the Stachel peptide sequence and CTF is critical for GPR56 activation and signaling. (a) Depiction of specific point mutations within the conserved GPR56 Stachel sequence and CTF. Both the GPR56 full-length and Met-CTF receptor forms are shown. (b, d) The IL-6 levels secreted by the transiently transfected A375 cells expressing the full-length GPR56 receptor with distinct Stachel peptide point mutations and incubated with immobilized CG3 (white bar) or CG4 (black bar) (n = 3, mean ± SD; ∗∗P < 0.01, ∗∗∗P < 0.001). (c, e) The IL-6 levels secreted by the transiently transfected A375 cells expressing Met-CTF only receptors with distinct Stachel peptide point mutations (n = 3, mean ± SD; ∗∗P < 0.01, ∗∗∗P < 0.001). The numbers represent the point mutants as shown in (a). CTF, C-terminal fragment; GPR56, G protein-coupled receptor 56; GPS, GPCR proteolysis site; SD, standard deviation; WT, wild-type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
GPR56-induced IL-6 secretion is mediated by the Gα12/13/RhoA pathway. (a) Western blotting analysis of siRNA-mediated knockdown of specific Gα proteins in A375/GPR56 cells. (b, c) Analysis of the IL-6 levels of A375/GPR56 (b) and MeWo (c) cells transfected with the Gα protein-specific siRNAs as indicated (n = 3, mean ± SD; ∗∗P < 0.01, ∗∗∗P < 0.001; scrambled siRNA vs. specific Gα-siRNA). (d) The IL-6 level in the CM of A375/GPR56 cells treated with or without C3 exoenzyme (n = 3, mean ± SD; ∗∗∗P < 0.001). (e) The IL-6 level in the CM of A375/GPR56 (left panel) and MeWo (right panel) cells transfected with or without the RhoA-N19 construct (n = 3, mean ± SD; ∗∗∗P < 0.001; CG3 vs. CG4; mock control vs. RhoA-N19). Cells were cultured on mIgG1-, CG3-, or CG4-coated plates as indicated. CM, conditioned medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPR56, G protein-coupled receptor 56; SD, standard deviation.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions