Prolonged signaling of backbone-modified glucagon-like peptide-1 analogues with diverse receptor trafficking

Signal duration and subcellular location are emerging as important facets of G protein-coupled receptor (GPCR) function. The glucagon-like peptide-1 receptor (GLP-1R), a clinically relevant class B1 GPCR, stimulates production of the second messenger cAMP upon activation by the native hormone, GLP-1. cAMP production continues after the hormone-receptor complex has been internalized via endocytosis. Here, we report GLP-1 analogues that induce prolonged signaling relative to GLP-1. A single {beta}-amino acid substitution at position 18, with the residue derived from (S,S)-trans-2-aminocyclopentanecarboxylic acid (ACPC), enhances signaling duration with retention of receptor endocytosis. Pairing ACPC at position 18 with a second substitution, -aminoisobutyric acid (Aib) at position 16, abrogates endocytosis, but prolonged signaling is maintained. Prolonged signaling is sensitive to the structure of the {beta} residue at position 18. Cryo-electron microscopy (cryo-EM) structures of two GLP-1 analogues bound to the GLP- 1R:Gs complex suggest substantial alterations to bound peptide structure and dynamics compared to the GLP-1:GLP-1R complex. These structural findings strengthen an emerging view that agonist dynamics in the receptor-bound state influence signaling profile. Our results advance understanding of the structural underpinnings of receptor activation and introduce new tools for exploring the impact of spatiotemporal signaling profiles following GLP-1R activation.