Fig. 2.

Trp33^ECD mediates key interaction with LY3502970 and the surrounding region. (A) Amino acid sequence comparison of the ECD regions for GLP-1Rs of different species. (B) LY3502970 increases cAMP accumulation in HEK293 cells expressing a chimeric form of the mouse GLP-1R (domains shown in green) where the N-terminal 142 residues have been replaced by the corresponding region of the human GLP-1R (domains shown in blue). (C) Mutation of Ser33^ECD to Trp33^ECD in the mouse GLP-1R enables LY3502970 to stimulate receptor-induced cAMP accumulation, while the reciprocal mutation (D) in the human GLP-1R abolishes compound function. The chimera and mutant data are presented as the mean ± SD of three independent experiments. (E) Trp33^ECD interacts with LY3502970 and residues on TM2, ECL1, and ECL2. Residues and ligands are shown by sticks except Trp33^ECD, which is also shown by spheres. Hydrogen bonds are indicated by dashed lines. (F) GLP-1R ECD orientation in the LY3502970 bound structure stabilized by aromatic interactions with ECL1. (G) The unique ECD orientation of ECD in the LY3502970 bound structure as a result of the unique Trp33^ECD position. The structures of GLP-1R bound to LY3502970 (7TM in green, ECD in blue green, LY3502970 in magenta), GLP-1 (yellow, PDB ID code 6VCB), and peptide 5 (gray, PDB ID code 5NX2) are aligned. Trp33^ECD is shown by a stick. Other peptide bound GLP-1R structures (GLP-1, PDB ID code 5VAI; ExP5, PDB ID code 6B3J) are not shown here, but their ECD orientations are very similar to GLP-1 bound structure 6VCB in yellow. (H and I) Competitive inhibition of [¹²⁵I]GLP-1(7-36) binding to membranes isolated from cells expressing the human GLP-1R (H) or human W33S GLP-1R (I). Data are represented as the mean ± SD of three independent experiments.