Glucagon-like peptide-1 (GLP-1) is a major player in the regulation of glucose homeostasis. It acts on pancreatic beta cells to stimulate insulin secretion and on the brain to inhibit appetite. Thus, it may be a promising therapeutic agent for the treatment of type 2 diabetes mellitus and obesity. Despite the physiological and clinical importance of GLP-1, molecular interaction with the GLP-1 receptor (GLP1R) is not well understood. Particularly, the specific amino acid residues within the transmembrane helices and extracellular loops of the receptor that may confer ligand-induced receptor activation have been poorly investigated. Amino acid sequence comparisons of GLP-1 and GLP1R with their orthologs and paralogs in vertebrates, combined with biochemical approaches, are useful to determine which amino acid residues in the peptide and the receptor confer selective ligand-receptor interaction. This article reviews how the molecular evolution of GLP-1 and GLP1R contributes to the selective interaction between this ligand-receptor pair, providing critical clues for the development of potent agonists for the treatment of diabetes mellitus and obesity.
Glucagon-like peptide-1 (GLP-1) is an intestinal incretin released in response to nutrient ingestion that stimulates insulin secretion in a glucose-dependent manner. The insulinotropic effect of GLP-1 on the pancreas has been demonstrated to be preserved in animal models of diabetes by stimulating insulin exocytosis (Shen et al., 1998; Drucker, 2001; MacDonald et al., 2002), and promoting insulin biosynthesis (Fehmann and Habener, 1992; Perfetti and Merkel, 2000; Moon et al., 2011). Recently, direct effects of GLP-1 on growth, survival (Xu et al., 1999; Stoffers et al., 2000; List and Habener, 2004), and differentiation of β-cells have been reported (Drucker, 2003). Beside its insulinotropic effects, GLP-1 inhibits glucagon (GCG) secretion in pancreatic α-cells (Nauck et al., 2002), attenuates gastric emptying, and ameliorates glucose excursion in the gastrointestinal tract (Nauck et al., 2002).
GLP-1 exerts its action through the G protein-coupled receptor (GPCR), GLP1R. This receptor belongs to the class B (or secretin-like) GPCR family. This GPCR family has only 15 members in humans and is characterized by a relatively long N-terminal extracellular domain (ECD) containing six conserved Cys residues that form a constraint structure necessary for ligand binding (Couvineau et al., 2004). GLP1R is mainly expressed in pancreatic β-cells, and upon stimulation by GLP-1, it induces the accumulation of cAMP and the influx of intracellular calcium which accelerate insulin release from secretory granules (Drucker et al., 1987; Fehmann et al., 1995).
GLP-1 and GLP1R are also expressed in the central nervous system. GLP-1 is synthesized largely in the brainstem and transported along axonal networks to diverse brain regions, including the hypothalamus (Vrang et al., 2007; Hisadome et al., 2010). GLP1R is expressed in cerebral cortex, hypothalamus, hippocampus, thalamus, caudate-putamen, and globus pallidum (Alvarez et al., 2005). In the brain, GLP-1 is known to reduce appetite, leading to significant reductions in body weight (Zander et al., 2002). In addition, GLP-1 is likely neuroprotective and involved in neurite growth and spatial learning ability in the brain (During et al., 2003; Perry et al., 2007).
Due to its combined beneficial effects, GLP-1 has been identified as a potential therapeutic agent for the treatment of diabetes mellitus and obesity. However, the molecular mechanisms leading to high affinity ligand-receptor binding and receptor activation have not been fully understood. Studies using alanine scanning mutagenesis, substitution/modification, and chimeric peptide construction of GLP-1 have explored the bioactive motifs of GLP-1(Adelhorst et al., 1994; Gallwitz et al., 1994; Hinke et al., 2004), yet none have been able to identify the mechanism through which individual residues in the peptide interact with residues in the receptor. Recent studies using X-ray crystallography have demonstrated that residues in the central α-helical region of GLP-1 interact with residues in the N-terminal ECD of GLP1R (Runge et al., 2008; Underwood et al., 2010). However, as ligand-induced receptor activation is mainly governed by interactions between residues in the N-terminus of the peptide and residues within the transmembrane helices (TMH) and extracellular loops (ECL) of the receptor (Thorens et al., 1993; During et al., 2003), this crystal structure only accounts for the peptide binding to the ECD of the receptor. Indeed, virtually no progress has been made in exploring the receptor binding sites for the N-terminal moiety of the peptide which is responsible for ligand-induced receptor activation.
Peptide ligands and their receptors have become diversified through evolutionary processes that ultimately yield families of related, yet distinct, peptides and receptors (Hoyle, 1999; Cho et al., 2007; Lee et al., 2009; Kim et al., 2011, 2012). Specific diversification of peptides, conservation within orthologs but variation among paralogs, would often confer the selective interaction with their cognate receptors, allowing discrimination of paralogous receptors (Acharjee et al., 2004; Wang et al., 2004; Li et al., 2005). Thus, amino acid sequence comparison of the peptide and receptor with their orthologs and paralogs, along with mutational mapping approaches, are useful tools helping to determine specific residues in the peptide ligands and receptors that are essential for maintaining selective ligand-receptor interaction. Indeed, ligand binding domains identified in mammalian receptors are highly conserved in orthologous non-mammalian receptors, indicating that there is high evolutionary selection pressure to maintain selectivity for their ligands (Acharjee et al., 2004; Wang et al., 2004; Li et al., 2005). Recently, we reported that evolutionarily conserved amino acid residues in GLP-1 and core domains of the GLP1R confer selective ligand-receptor interaction and receptor activation (Moon et al., 2010, 2012). This article reviews how the molecular evolution of GLP-1 and GLP1R contributes to acquiring high affinity interaction between this peptide ligand and receptor.
GLP-1 is a product of the GCG gene which encodes a common GCG-GLP-1-GLP-2 precursor. All three peptides are encoded by different exons of the GCG gene, raising the possibility of exon duplications during early vertebrate evolution (Sherwood et al., 2000). The GCG gene produces one or two mature peptides by a tissue-specific alternative post-translational process (Kieffer and Habener, 1999; Irwin, 2001, 2009). For instance, in pancreatic α-cells mature GCG, but not GLP-1 and GLP-2, is produced. In intestinal L cells, however, mature GLP-1 and GLP-2, but not GCG, are generated. One GLP-1 paralog is the glucose-dependent insulinotropic polypeptide (GIP) which is independently encoded by the GIP gene (McIntosh et al., 2009). GLP-1 and GIP share a high degree of amino acid sequence identity, particularly in their N-terminal moiety, and function similarly by inducing insulin secretion from β-cells. However, these peptides act through distinct yet related receptors, GLP1R and GIP receptor (GIPR), respectively. In addition, albeit absent in mammals, another GLP-1 paralog is exendin, which was first discovered in Gila monsters (Heloderma suspectum; Göke et al., 1993). Recently, the full-length and/or partial cDNAs for exendin were characterized in a few species, such as Xenopus, chicken, and Gila monster (Irwin and Prentice, 2011). Although the receptor for exendin has not yet been identified in non-mammals, exendin exhibits high affinity binding for mammalian GLP1R (Göke et al., 1993). Recent evidence suggests that the GCG, GIP, and exendin genes were generated by genome duplication events during early vertebrate evolution, as these genes are flanked by similar neighboring genes in the genomes of vertebrates (Irwin, 2002; Irwin and Prentice, 2011).
GLP-1 and its family peptides are 30 ∼ 40 amino acids in length and share similarities in amino acid sequence and secondary structure. All these peptides tend to be disordered in aqueous solutions but exhibit a marked propensity to form α-helices under mild ambient conditions, such as in the presence of organic solvents or lipids (Braun et al., 1983; Gronenborn et al., 1987; Thornton and Gorenstein, 1994; Inooka et al., 2001; Neidigh et al., 2001; Chang et al., 2002; Tan et al., 2006; Alana et al., 2007), or upon crystallization (Sasaki et al., 1975). It is now well known that the N-terminal domains of GLP-1-related peptides form
