Extended Data Figure 6: The N-terminal ECD of the CTR.
From: Phase-plate cryo-EM structure of a class B GPCR–G-protein complex
a, Rigid body fitting of the structure of CTR ECD bound to sCT (PDB: 5II0)22 into the corresponding regions of the cryo-EM map revealed additional density (close to residue 130) that may be attributed to glycosylation. b–d, Asp mutation of four consensus glycosylation residues (N28D, N73D, N125D and N130D) reveals the relative unimportance of glycosylation on cell-surface expression (b), determined via cell-surface ELISA for the N-terminal epitope tag. c, Competition radioligand binding studies for sCT in competition with the radiolabelled ligand [125I]sCT(8–32) revealed reduced affinity for N130D, and to a lesser extent N125D, compared to the wild-type CTR. d, Concentration response curves for cAMP accumulation for mutant receptors relative to wild type show that N130D, and to a lesser extent N125D, reduce the potency of sCT in functional experiments. All data are mean + s.e.m. of five independent experiments, conducted in duplicate or triplicate.
