Extended Data Fig. 6: The activation of GPR50 by L-LEN and its structure-function relationship.
From: Photo-cross-linking-assisted deorphanization deciphers GPR50–L-LEN pairing in metabolism
Related to Fig. 4. (a) Left, representative images showing the raw fluorescence signal of Ca2+ sensor in buffer and L-LEN and the fluorescence changes (in pseudocolor of ΔF/F0), in GPR50 (50) or vector (VC) expressing cells, co-expressing the indicated chimeric G protein (left). Middle, traces showing the buffer or L-LEN-evoked Ca2+ response in GPR50 (50) or vector (VC) expressing cells co-expressing Gα15. Right, quantification of the peak fluorescence changes to L-LEN in vector-expressing cells with or without the co-expression of Gα15 or Gαqs (n = 15 cells from 3 wells; p = 0.37 for VC and VC + Gα15; p = 0.15 for VC and VC+Gαqs). (b) Left, representative images showing the raw fluorescence of G-Flamp2 at baseline and to the application of forskolin and IBMX and the fluorescence changes (in pseudocolor of ΔF/F0) in GPR50-expressing cells pre-incubated with buffer, L-LEN (10−6 M), L-LEN and pertussis toxin (PTX, 0.01 mg/ml for additional 12 h) or SCG5 (10−6 M). Middle and right, the traces showing the forskolin-evoked fluorescence changes of G-Flamp2 in GPR50-expressing cells treated with indicated drugs. Right, quantification of the inhibition of forskolin-evoked cAMP by indicated drugs in GPR50-expressing cells (n = 3 wells; p = 0.0232 for LL and LL + PTX; p = 0.0093 for LL and SCG5). (c) The fluorescence response of Ca2+ sensor (up) and the inhibition of forskolin-evoked changes in G-Flamp2 (bottom) in GPR50 or GPR171-expressing cells stimulated by L-LEN (LL) or B-LEN (BL) independently (Ca2+: n = 13/15/14/15 cells; p = 5.64 ×10−6 for GPR50, p = 4.47×10−4 for GPR171; cAMP: n = 11/11/8/10 cells; p = 1.81 ×10−9 for GPR50, p = 3.13×10−7 for GPR171). (d) The dose-dependent inhibition of forskolin-evoked cAMP in GPR50-expressing cells by L-LEN variants and B-LEN, n = 370/233/237/294/156/267/164 cells for log[LATPAPQVP] = -5 to -12M); n = 125/123/170/154/143/186/154 cells for log[LETPAPQVP] = -6 to -11M；n = 261/370/367/300/350/201/303 cells for LETPAPQV; n = 350/367/369/473/544/389 cells for log[B-LEN] = -5 to -11M). (e) The dose-dependent inhibition of forskolin-evoked cAMP in GPR50 mutant-expressing cells by L-LEN (n = 341/359/361/435/223/362 cells for log[L-LEN] = -5 to -10M in 172 A; n = 371/217/293/315/348/384/132 cells for log[L-LEN] = -6 to -12M in 185 A; n = 202/249/131/229/293/502/176 cells log[L-LEN] = -6 to -12 M in 255 A; n = 203/157/280/246/183/278/253/284 cells for log[L-LEN] = -5 to -12 in 277 A). Scale bar, 25 μm. Data are shown as mean ± SEM, n.s. not significant; *, p < 0.05; **, p < 0.01. See Supplementary Table 1 for statistics.
