Structure/Activity Characterization of Glucagon‐Like Peptide‐1

Abstract

Glucagon-like peptide-1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic p-cell. To characterize the side-chain groups required for interaction of glucagon-like peptide-1 with its receptor, we performed binding studies with alanine-substituted glucagon-like peptide-1 analogues on RINmSF insulinoma cells. The binding affinity and biological activity of glucagon-like peptide-1 have been found to be sensitive to alanine exchanges in the N-terminal positions 1, 4, 6 and the C-terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10-12, 14, 16-21 and 25-30 do not change receptor affinity. These findings could be exploited to design glucagon-like peptide-1 agonists and probably antagonists for further physiological studies.Glucagon-like peptide-1 is a 30-residue gastrointestinal hormone released from the enteroglucagon cells (L-cells) in the small intestine [l -31. The post-translational processing of proglucagon in the small intestine leads to the generation of glucagon-like peptide-1, corresponding to positions 78 -108 of the human proglucagon precursor [3, 41. In v i m , glucagon-like peptide-1 increases insulin secretion from the isolated rat [S] and pig pancreas [2, 61, and from isolated rat islets [7]. In man, glucagon-like peptide-1 is released into the circulation postprandially, and glucagon-like peptide-1 infusions stimulate insulin release [ 81. Therefore, glucagon-like peptide-1 plays an important role in the postprandial regulation of insulin secretion. Recent studies have shown that glucagon-like peptide-1 also has an anti-diabetogenic effect by reducing the isoglycaemic meal-related requirement for insulin in patients with non-insulin-dependent diabetes mellitus [9][10][11].Receptors for glucagon-like peptide-1 have been characterized in RINm5F cells [12-141, a Abbreviations. Fmoc, N-9-fluorenylmethoxycarbonyl; [HlAIglucagon-like peptide-1 , glucagon-like peptide-I , with an alanine exchange in position 1 for the naturally occurring histidine residue. All other glucagon-like peptide-1 analogues are named accordingly with the first letter giving the naturally occurring amino acid in the glucagon-like peptide-1 sequence and the number following the first letter giving the position of the exchanged amino acid.Note. This work is dedicated to Werner Creutzfeldt, Professor emeritus of Medicine, Georg-August University of Gottingen, Germany, on the occasion of his 70th birthday. the gene encoding the receptor has been localized in humans [17]. So far, little is known about the structural requirements for glucagon-like peptide-1 binding to its receptor. Binding studies with N-terminal and C-terminal glucagon-like peptide-1 fragments have shown that the C-terminal domains of the glucagon-like peptide-1 molecule are important for receptor binding. The N-terminal fragment glucagon-like peptide-1 (7 -2.5) did not show receptor binding, indicating that longer N-terminal fragments are needed for receptor recognition [ 1 31. The glucag...

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

“…However, the mechanisms underlying binding and activation of this receptor and other members of this family are poorly understood and the development of small molecule ligands is problematic. This has been approached by previous structure-activity, mutagenesis and chimeric studies (18,(27)(28)(29)(30)(31)(32)(33)(34)(35). In this study, we used photoaffinity labeling to demonstrate that the carboxyl terminus of GLP1 is in approximation with the amino terminus of the receptor when docked.…”

“…Alanine-scanning of GLP1 has implicated positions 7, 10, 12, 13, 15, 26, 28, and 29 as being critical for receptor binding (18,30), whereas substitutions at positions 24 and 35 have minimal effects on GLP1 receptor binding and activation (18). This is consistent with our incorporation of a Bpa in these positions.…”

Ligand Binding Pocket Formed by Evolutionarily Conserved Residues in the Glucagon-like Peptide-1 (GLP-1) Receptor Core Domain

“…, Gly 4 , Thr 7 , and Asp 9 are important for receptor binding and activation (30,31 7 . Thus, similarities in the ligand binding pocket structures of GLP1R, GCGR, GCRPR, and GLP2R may accommodate the conserved residues in the N-terminal moieties of the peptides.…”

“…Alanine scanning analysis revealed that His 1 , Gly 4 , Thr 7 , and Asp 9 in the N-terminal portion of GLP-1 are important for receptor binding and activation (30,31). Recently, by using chimeric GLP1R/GIPR together with chimeric GLP-1/GIP peptides, we identified interactions of His 1 and Thr 7 of GLP-1 with Ile 196 /Lys 197 at TMH2, Met 233 at ECL1, and Asn 302 at ECL2 of GLP1R (32).…”

MOLECULAR EVOLUTION OF GPCRS: Secretin/secretin receptors

“…Asp at position 3 is conserved across all the mature SCT peptides and this residue has a role in adenyl cyclase (AC) stimulation and interacts with the basic residues in the second transmembrane (TM) helix of the secretin GPCRs (Cardoso et al 2010). Other conserved residues such as His1 and Phe6 are key amino acids in secretin's GPCR-binding affinity (Gourlet et al 1991 Gallwitz et al 1994, Irwin 2001, Bourgault et al 2009). …”