Extended Data Fig. 9: Structural comparisons of receptor and bound G13 protein for GPR56 and LPHN3, and experimental data for G13 N-terminal construct design.
From: The tethered peptide activation mechanism of adhesion GPCRs
a-b, Overall structural comparison of 7TM domains of G protein coupled GPR56 (blue) with active state Family B1 receptors: GLP1R (PDB ID: 5VAI; receptor in brown, GLP1 peptide in tan), GCGR (PDB ID: 6WPW; receptor in dark green, glucagon derivative ZP3780 in light green) and calcitonin receptor (PDB ID: 5UZ7; orange). a, side view showing similarities in 7TM domain topology and b, top view with superposition of B1 agonists with GPR56 TA in the orthosteric site. c, Superposition of glucocorticoid ligand-bound GPR97 (PDB ID: 7D77, light grey) with GPR56 (blue) and LPHN3 (magenta). Arrows are indicating differences in TM1, TM6 and TM7 between the ligand and to TA-bound structures. d, Top view of superimposed GPR56 and LPHN3 complexes showing positioning of mini-G13 N-terminal helix (αN) with respect to the receptor TMs. e, Superposition of GPR56 bound mini-Gα13 (gold) vs. 5HT1A (PDB ID: 6G79) bound mini-Gαo (green). f, GTPγS binding assay for recombinant G13 proteins in which the authentic N-terminus of Gα13 was replaced with 15 or 29 residues of the Gαi2 αN to improve expression, stability and ability to interact with receptor. Stimulation of G13/i29 nucleotide exchange by both receptors GPR56 (blue) and LPHN3 (magenta) was reduced substantially when compared to wild type G13 or G13/i15. Receptor constructs used in this assay are the TA-decrypted GPR56 and LPHN3. Data displayed as mean of reactions (n = 18 for all except GPR56 + G13/i29, n = 17, and LPHN3 + G13/i29, n = 16) with error bars representing +/− S.E.M. Statistical significance between experimental condition and corresponding control group was calculated using Mann-Whitney analysis, n.s. = not significant, **** = p < 0.0001. g, G protein binding through α5 helix of mini-Gα13 (gold) by GPR56 (blue) and mini-Gαo (red) by GPR97 (PDB ID: 7D77, in white) showing substantially greater opening of TM5-6 in the GPR56 TA-bound structure.
