Fig. 9.

Effects on mutation of residues located in the hydrogen bonding network located between TMs 2, 6 and 7 at the cytoplasmic face. (A) TMs 2, 6, 7 and helix 8 (H8) of the apo GLP-1R TM bundle as viewed from the cytoplasmic face, highlighting an extensive hydrogen bond network between R2.46¹⁷⁶, R6.37³⁴⁸, N7.61⁴⁰⁶ and E7.63⁴⁰⁸. Q7.65⁴¹⁰ at the start of H8 is also shown where it forms hydrogen binds with the side chain of N7.61⁴⁰⁷ and the backbone of TM7 at F7.59⁴⁰⁴. (B) TMs 2, 6, 7 and H8 of the GLP-1 docked GLP-1R TM bundle as viewed from the cytoplasmic face with the G_αs peptide fragment indicating the extensive hydrogen bond network between R2.46¹⁷⁶, R6.37³⁴⁸, N7.61⁴⁰⁶ and E7.63⁴⁰⁸ is broken in the activated receptor. Q7.65⁴¹⁰ at the start of H8 is also shown where it still maintains a backbone interaction with F7.59⁴⁰⁴. (C) Cell surface expression of mutations R2.46¹⁷⁶A, R6.37³⁴⁸A, N7.61⁴⁰⁶A, E7.63⁴⁰⁸A and Q7.65⁴¹⁰A relative to the wildtype receptor (as assessed by antibody binding to the N-terminal c-myc epitope tag). (D) Differences in the coupling efficiency (log τ_c) of GLP-1, exendin-4 and oxyntomodulin to three signalling pathways (cAMP production (top), _iCa²⁺ mobilisation (middle), and pERK1/2 (bottom)) for R2.46¹⁷⁶A, R6.37³⁴⁸A, N7.61⁴⁰⁶A, E7.63⁴⁰⁸A and Q7.65⁴¹⁰A compared to the wildtype receptor. These log τ_c were calculated from concentration–response curves presented in Fig. 2, Fig. 3, Fig. 4, and corrected for cell surface expression as measured by antibody labelling recorded in Table 1. Statistical significance of changes in cell surface expression or coupling efficacy in comparison with wildtype were determined by one-way analysis of variance and Dunnett’s post test, and values are indicated with an asterisk (^∗, p < 0.05). All values are ±S.E.M of four to six independent experiments, conducted in duplicate.