Extended Data Figure 2: Binding of [3H]-NNC0640 and 125I-labelled glucagon to wild-type and mutant GCGRs and glucagon-induced cAMP assays.
From: Structure of the full-length glucagon class B G-protein-coupled receptor
a, Binding of [3H]-NNC0640 to membrane preparations from Sf9 cells expressing wild-type (WT) and the engineered GCGR used for crystallization. Data are shown as means ± s.e.m. from three independent experiments performed in duplicate. ‘Construct’ indicates the construct used for crystallization, containing the T4L fusion at ICL2 and 45 residues truncated at the C terminus of the receptor. b, Binding of [3H]-NNC0640 to membrane preparations from HEK293T cells expressing wild-type or mutant GCGRs. Data are shown as means ± s.e.m. from three independent experiments performed in duplicate. The IC50 values for the wild-type and mutant GCGRs from at least three independent experiments are listed in Extended Data Table 2. c, Glucagon-induced cAMP accumulation measurement of the mutants V130C/L210C, V130C and L210C and the wild-type GCGR. d, Glucagon-induced cAMP accumulation measurement of the mutants V130C/L210C, V130C and L210C and the wild-type GCGR in the presence of 1 mM DTT. Dose–response curves of cAMP accumulation assays generated from three independent experiments performed in duplicate. Data are shown as means ± s.e.m. e–g, Disulfide cross linking assays of the GCGR mutant Q113C/D209C (e) and the controls, single-site mutants Q113C (f) and D209C (g). Dose–response curves of 125I-labelled-glucagon-binding assay generated from three independent experiments performed in duplicate. Data are shown as means ± s.e.m.
