Impact of Substitution Registry on the Receptor‐Activation Profiles of Backbone‐Modified Glucagon‐like Peptide‐1 Analogues

Abstract

Family BGprotein-coupled receptors play importantp hysiological roles and possess large extracellular domains (ECDs) that aid in binding the long polypeptide hormones that are their natural agonists. We have previously shown that agonist analogues in which subsets of native a-amino acid residues are replaced with b-aminoa cid residues can retain activity while avoiding proteolytic degradation. This study focuses on eight new a/b analogues of glucagon-like peptide 1( GLP-1) that each contain five a-to-b replacements in the C-terminal half of the peptide. This portion of GLP-1 is known to adopt an a-helicalc onformation and contact the ECD. All four registries of the aaab backbone pattern weree valuated;p revious work has shown that the aaab pattern supports adoption of an ahelix-like conformation. Two a-to-b replacement formats were employed, one involving b 3 homologues of the native residues replaced and the other involving ac yclic b residue. GLP-1R response was characterizedi nt erms of stimulation of cAMP production and b-arrestin recruitment.S ome of the backbone-modifiedG LP-1 analogues display biased agonism of the GLP-1R. This study helps to establish the scope of the a!b backbone modification strategy.

Structural and functional diversity among agonist-bound states of the GLP-1 receptor

“…29 We previously showed that GLP-1 analogues with multiple (S,S)-ACPC substitutions in the C-terminal region, which is α-helical when bound to the ECD, 30 display substantial agonist activity. 31,32 In contrast, (R,R)-ACPC (Fig. 1a) destabilizes a right-handed α-helix-like conformation by >1 kcal/mol relative to (S,S)-ACPC or L-Ala. 29 As observed for D-Ala, 24 (R,R)-ACPC can replace Gly to stabilize turn segments.…”

Structural and Functional Diversity among Agonist-Bound States of the GLP-1 Receptor

“…52 We previously showed that GLP-1 analogues with multiple ( S , S )-ACPC substitutions in the C-terminal region, which is α-helical when bound to the ECD, display substantial agonist activity. 53,54 In contrast, ( R,R )-ACPC (Fig. 1a) destabilizes a right-handed α-helix-like conformation by >1 kcal/mol relative to ( S,S )-ACPC or L-Ala. 52 As observed for D-Ala, 55 ( R , R )-ACPC can replace Gly to stabilize turn segments.…”

Phosphorylation Sites of the Gastric Inhibitory Polypeptide Receptor (GIPR) Revealed by Trapped-Ion-Mobility Spectrometry Coupled to Time-of-Flight Mass Spectrometry (TIMS-TOF MS)

“…The cells we used overexpress the Glosensor, , which allowed us to determine whether the Flag tag influences GIPR signal transduction as mediated by intracellular cAMP production. We chose HEK293 cells because this cell line is commonly used in pharmacological studies involving family B GPCRs. − Furthermore, HEK293 cells endogenously express GRK isoforms 2, 3, 4, 5, and 6; GRK2 and GRK5 have previously been shown to regulate GIPR internalization. , …”