Exploring the N-terminal Hydrophobic Faces of Glucagon and Glucagon-like Peptide-1

摘要

Glucagon, Glucagon-like Peptide-1 (GLP-1), as well as human (hCT) and salmon calcitonin (sCT) are all members of the secretin family of hormones that act on distinct G protein-coupled receptors in what are believed to be largely α-helical conformations [1-5]. Approximately 50% sequence homology exists between the two glucagon-related peptides and among the two calcitonins. The amino acid sequences and α-helical projections of these four peptides reveals a strong sequence and structural similarity between three N-terminal hydrophobic residues in GLP-1 (F~5, V~(10) and Y~(13)) and glucagon (F~6, Y~(10) and Y~(13)) and a set of equally-spaced, aromatic residues in hCT (Y~(12), Y~(16) and F~(19)). The sCT sequence differs from this trend with a triad of leucine residues (L~(12), L~(16) and L~(19)). Structural studies have indicated that both of these regions (6, 10, 13 for glucagon & GLP-1 and 12, 16, 19 in hCT & sCT) exhibit a-helical secondary structure. This structure is purported to be the most probable receptor-active conformation for these two regions [2-5]. Fowler et al. (2005) reported the characterization of hCT-sCT chimeric peptides [11. Most crucial to the design of our study was a H~(17)R-hCT peptide substituted with residues K~(11) , L~(12), E~(13), L~(16) and L~(19) of sCT. The resulting peptide (6CH-R) showed a ten-fold increase in receptor binding potency and a two-fold increase in cAMP production when compared to native hCT. This hCT analog was reported to be equipotent with sCT [1]. Given these results and the structural similarities in the aromatic regions of CT and the glucagon-related peptides, we questioned whether a L~6, L~(10) and L~(13) variant of GLP-1 or glucagon would provide an analog with enhanced receptor potency. We characterized this assertion as the leucine hypothesis. The strategy was two-fold; if L~(6,10,13) analogs proved more potent in vitro we would explore the possibility of using aliphatic residues at these positions, and if not, we would determine the extent that these three residues could be modified with aromatic character.