Extended Data Fig. 6: Effects of mutations in the FXφφφXφ motif in ADGRG2-β, ADGRG4-β, ADGRG5-β and ADGRF5-β on their basal activity.

a, The calculated energy contribution of the individual residues within the tethered Stachel sequence region bound in the orthosteric binding pocket of ADGRG4-β. Values are the mean ± s.e.m. of three independent experiments (n = 3). The top five contributing residues are highlighted in red. b, d, e, Effects of mutations in the FXφφφXφ motifs of Stachel sequence in ADGRG4 (GPR112)-β (b), ADGRG5 (GPR114)-β (d) or ADGRF5 (GPR116)-β (e) on their constitutive activities determined by measuring their cAMP levels. Represented differences between wild-type ADGRG4 (GPR112)-β, ADGRG5 (GPR114)-β or ADGRF5 (GPR116)-β and their mutants expressed at a relative expression level of 0.8 (refer to ELISA data for receptor cell surface expression level in Supplementary Fig. 8c–h) are shown as the mean ± s.e.m. of three experiments (n=3). Data are normalized and presented as the response percentage for wild-type ADGRG2-β, ADGRG5-β or ADGRF5-β, respectively. Statistical differences between wild-type and mutant proteins were determined by two-sided one-way ANOVA with Tukey’s test. ***P < 0.001, **P < 0.01; (P < 0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001 from top to bottom in panel b; P<0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0003, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, <0.0001, 0.0002, <0.0001, <0.0001, <0.0001 from top to bottom in panel d; P = 0.0021, 0.0031, 0.0035, 0.0006, <0.0001, 0.0001, <0.0001, <0.0001, 0.0004, <0.0001, <0.0001, <0.0001 from top to bottom in panel e). FXφφφXφ motif key residues are highlighted in red with blue shades. c, Sequence alignment of Stachel sequence between 33 human adhesion GPCRs. Key residues in the FXφφφXφ motif were labelled by red pentagram. Residues at the positions of FXφφφXφ motif which have large hydrophobic side chains are highlighted in red. Residue numbers following the Stachel sequence (ss) refer to residues related to the position of the GPS. Residues C-terminal to the GPS are numbered starting from 1. f, Cartoon representations of the AlphaFold2 simulated full-length ADGRG4 structure encompassing its GAIN domain. Detailed depictions of the interactions between the buried Stachel sequence of ADGRG4 (pink) in β sheet configuration and its surrounding GAIN domain residues are shown in an enlarged panel on the right. Note that ten hydrophobic residues of the ADGRG4 GAIN domain engaged with the hydrophobic motif of the Stachel sequence of ADGRG4. Through combined MD simulation and chemical labelling, a very recent study⁶ revealed that the Stachel sequence might be released from the GAIN domain owing to its intrinsic structural flexibility and then be accessible to the 7TM domain. According to this model, the Stachel sequences of ADGRG2 and ADGRG4 might be able to slide away from the GAIN domain and transit to the U-shaped configuration for 7TM domain association in response to changes in different physiological contexts and participate in aGPCR activation. (according to previous publications, the GAIN domain structure of a cleavage-deficient aGPCR should be similar to that of wild-type receptor). g, Effects of mutations in the Stachel sequence-interacting GAIN domain of auto-proteolysis deficient ADGRG4 (GAIN-ADGRG4-β-AA) on their constitutive activities were determined by measuring the basal cAMP levels using a GloSensor cAMP assay. Representative curves for constitutive activities of GAIN-ADGRG4-β-AA and its mutants at indicated expression levels (refer to the ELISA data for the receptor cell surface expression level in Supplementary Fig. 11b) are shown. Values are the mean ± s.e.m. of three independent experiments performed in triplicate (n = 3). Statistical differences between GAIN-ADGRG4-β-AA and its mutants were determined by two-sided one-way ANOVA with Tukey’s test. ***P < 0.001; **P < 0.01; *P < 0.05; n.s., no significant difference (P = 0.2855, 0.0450, 0.0001, 0.0006, 0.0002, <0.0001 from left to right for V2683A curve; P = 0.0535, 0.0112, 0.0092, 0.0345, 0.0027, 0.0003 from left to right; I2660A Curve: P = 0.4548, 0.0135, 0.0004, 0.0257, 0.0026, 0.0002 from left to right for L2671A curve).