Article Figures & Data
Figures
- Fig. 1.
Gene structure, expression, processing, degradation, and elimination of proglucagon. The proglucagon gene is located in human chromosome 2 and transcribed as one single mRNA in three major tissues, namely, the pancreas, the intestine, and the CNS. The mRNA is first translated into one single protein and then processed by prohormone convertase (PC) in different tissues. In the pancreatic α cells, proglucagon protein is processed by PC2 into glicentin-related polypeptide (GRPP), glucagon (Gluc), intervening peptide-1 (IP-1), and major proglucagon fragment, whereas in L cells of the small intestine and the brain, proglucagon is processed by PC1/3 into oxyntomodulin, intervening peptide-2 (IP-2), GLP-1, and GLP-2. GLP-1 is degraded by DPP-4 via cleavage of two amino acids from the N terminus, or by NEP-24.11 through cleavage of the C terminus in vivo. The cleaved products are eventually eliminated in the kidney. UTR, untranslated region.
- Fig. 2.
Structural characteristics of GLP-1 and its cognate receptor. (A) GLP-1–bound full-length GLP-1R homology model based on a previously published full-length glucagon-bound GCGR model combining structural and experimental information from the GCGR 7TMD crystal structure (PDB: 4L6R), the GCGR ECD structure (PDB: 4ERS), and the ECD structure of GLP-1–bound GLP-1R (PDB: 3IOL), complemented by site-directed mutagenesis, electron microscopy, hydrogen-deuterium exchange, and cross-linking studies (Siu et al., 2013; Yang et al., 2015b, 2016). The C-terminal helix of GLP-1 bound to the ECD region of GLP-1R is depicted as cartoon, whereas the atoms of the flexible N-terminal region of GLP-1 predicted to be bound to the 7TMD of GLP-1R are depicted as spheres. GLP-1 is color coded according to mutation effects (blue: <fourfold effect, orange: 4- to 10-fold effect, red: >10-fold effect IC50; see II. Glucagon-Like Peptide-1); mutation effects of GLP-1R are reported in Fig. 3 and Table 1. The Cα/Cβ atoms of GLP-1/GLP-1R residue pairs identified in photo cross-linking studies (Chen et al., 2009, 2010b; Miller et al., 2011) are depicted as green-colored spheres. (B) Structural alignment of the ECD structures of GLP-1 and exendin9–39–bound GLP-1R (PDB: 3IOL, 3C59), GIP-bound GIPR (PDB: 2QKH), and the mAb23-bound GCGR ECD structure (PDB: 4ERS). Comparison of the crystal structure binding modes of (C) GLP-1 and (D) exendin9–39. The surfaces of GLP-1R residues involved in important apolar interactions with GLP-1/apolar are colored pink, whereas residues involved in polar interactions described in II. Glucagon-Like Peptide-1 are also depicted as sticks (and their H-bond interaction networks are depicted as dashed lines). (E) Structure-based sequence alignment of GLP-1, exendin9–39, glucagon, GIP, and GLP-2. The regions of the peptide ligands solved in ECD–ligand complex crystal structures are marked above the amino acid sequences using the same color coding as in (B). Amino acids of GLP-1 are marked according to mutation study effects, as indicated in (A). The residues that are boxed are found in an α-helical conformation in the crystal structure complex (solid lines: GLP-1, exendin9–39, GIP) or in NMR studies in micelle DPC (dashed lines: glucagon, GLP-2), as described in II. Glucagon-Like Peptide-1.
- Fig. 3.
Summary of GLP-1R mutagenesis. A snake plot of GLP-1R from http://www.gpcrdb.org that has been colored (see Key) to highlight the location of signal peptide, glycosylation, and phosphorylation sites, as well as the mutated residues in Table 1. It should be noted that the color coding of 74 of the 195 mutated residues (W39, W72, W87, W91, W110, F169-C174, R176, N177, H180, N182, A200-Q213, W214-G225, R227-F230, L232, M233, E262-L268, F321-I332, K334-K336, and R348-K351) reflects the effects of rat GLP-1R mutations projected on the hGLP-1R amino acid sequence. The color coding of 27 of the 185 residues (K202, W203, S206-Q211, Q213, W214, G216-Q221, S223-G225, R227-F230, L332, M233, I325, and F326) reflects the effect of double mutations, not single-point mutations. Information on the fold change in ligand affinity and potency as well as expression levels of the GLP-1R mutants is reported in Table 1.
- Fig. 4.
Summary of the main characterized pathways of glucose and GLP-1 signaling in the pancreatic β cell. Glucose enters the cell through glucose transporter 2 and undergoes glycolysis to produce pyruvate that enters the mitochondria for oxidative metabolism and ATP production. This increase in cytosolic ATP closes the KATP channels, depolarizing the membrane and opening the voltage-dependent calcium channels, increasing calcium influx into the cell, causing insulin exocytosis. GLP-1 increases insulin exocytosis through a number of mechanisms. GLP-1R couples to Gαs, activates adenylate cyclase that converts ATP to cAMP, and mobilizes two downstream effectors, PKA and Epac. These have a range of effects, including closing KATP channels, enhancing fusion of insulin secretory granules with the membrane, whereas PKA also closes Kv channels, inhibiting membrane repolarization. PKA and Epac also increase intracellular calcium by facilitating CICR through the opening of inositol 1,4,5-trisphosphate receptor and ryanodine receptor calcium channels, respectively. This increase in calcium has also been proposed to upregulate mitochondrial ATP production and activate calcineurin, so nuclear factor of activated T cells promotes insulin gene transcription to increase insulin stores. Alongside increasing insulin synthesis and exocytosis, GLP-1 signals through a number of pathways to increase β cell mass. PKA reduces ER stress through ATF-Gadd34 signaling, increases β cell neogenesis by activating cyclin D, and elevates the expression of insulin receptor substrate 2 (IRS2), a β cell survival factor, as well as anti-apoptotic proteins Bcl-2 and Bcl-xL through CREB. PI3K is activated by IRS2 and transactivation of epidermal growth factor receptor, and this further promotes increased β cell mass through upregulation of PDX-1 and nuclear factor κB, which upregulates anti-apoptotic Bcl-2 /Bcl-xL and inhibitor of apoptosis protein-2.
- Fig. 5.
Web of bias illustrating distinctions in the pattern of signaling of different peptide agonists (left panel) or nonpeptidic modulators (right panel) at GLP-1R. The web of bias plots ΔΔτ/KA values on a logarithmic scale for each ligand and for every signaling pathway tested. Formation of these values included normalization to the reference ligand GLP-17–36 amide and the reference pathway, cAMP accumulation. The plots do not provide information on absolute potency, but on relative efficacy for signaling of individual pathways in comparison with that for cAMP. Data are from Koole et al., 2010; Willard et al., 2012a; and Wootten et al., 2013b.
- Fig. 6.
Homology model of GLP-1R illustrating the relative position of key residues involved in the receptor-signaling bias. The modeling indicates that these residues reside at a fulcrum position of the receptor transmembrane bundle, where the splayed helices of the open extracellular face of the receptor converge, with the residues that contribute to ligand-dependent signaling forming a central interaction network (space fill, red) and the smaller polar residues that are globally important for signaling external to this core (space fill, blue). The receptor is displayed in three views at different horizontal rotation. Transmembrane helices are numbered with Roman numerals.
- Fig. 7.
Nonpeptidic GLP-1R modulators and peptide mimetics. Liraglutide (Novo Nordisk) was the first approved human GLP-1 analog to teat diabetes (European Union, 2009; United States, 2010) and obesity (United States, 2014; European Union, 2015). Liraglutide has 97% sequence homology to human GLP-1 and was designed to reversibly bind to albumin by attachment of palmatic acid via a L-γ-glutamic linker to lys26 in Arg34 GLP-17–37. The modification of Lys34 to Arg34 made it possible to produce Arg34 GLP-17–37 in yeast, followed by acylation of Lys26. The native GLP-1 peptide has a half-life of approximately 2 minutes due to rapid cleavage of GLP-17–37 to GLP-19–37 by DPP-4 (Deacon et al., 1995a,b; Vilsboll et al., 2003). Liraglutide comprises the natural GLP-1 N terminus, but has a half-life of about 11 hours after s.c. dosing to humans combined with a delayed absorption from sub cutis that gives a pharmacokinetic profile applicable for once-daily administration. The reason for extended circulation is due to reversible albumin binding that protects from DPP-4 degradation and glomerular filtration, whereas the delayed absorption is explained by the ability of liraglutide to form heptamers by self-assemble controlled by the fatty acid side-chain at position 26. Liraglutide is well tolerated and capable of substantially improving glycemic control with low risk of hypoglycemia and weight loss benefit (Knudsen et al., 2000; Knudsen, 2004; Madsen et al., 2007; Steensgaard et al., 2008; Dharmalingam et al., 2011; Wang et al., 2015).
Tables
- TABLE 1
Summary of effects on GLP-1 pharmacology (affinity and ability to activate cAMP pathway) in published site-directed mutagenesis studies of GLP-1R
WT (wild-type) refers to mutations that resulted in either <fivefold or no statistically significant change from wild-type GLP-1R. ND (not determinable) refers to a property that was measured, but for which a value was not determinable. Blank cells mean that the assays used to estimate that particular pharmacological property were not carried out in the cited work. Residues with symbol † refer to data from rat GLP-1R (if different, the equivalent human residue number is displayed in the table to aid comparison). GLP-1 affinity or potency fold-change values with suffix M are from membrane preparations, whereas suffix C is from whole-cell assays. Cell surface expression values below 75% of WT are shown (>75% are shown as WT): a suffix E represents estimation from ELISAs; suffix mic was evaluated from immunofluorescent microscopy; suffix cyt was evaluated by flow cytometry with an anti-Flag antibody; suffix Ag refers to affinity or cell surface expression levels determined from agonist radioligand-binding assays, whereas suffix Ant was from antagonist radioligand-binding assays. ΔLog τc values relative to WT are shown where >0.5 and were calculated from data where the expression-corrected efficacy term τc had been calculated using the operational model of agonism, as defined in Wootten et al. (2013c). Residues with transmembrane helices are numbered according to Wootten et al. (2013c).
Residue Mutated to -Fold Reduction Affinity -Fold Reduction Potency‡ Cell Surface Expression (% Wild-Type) Comments and/or Other Observed Effects Reference P7ECD L WTC,Ant WT WTE Koole et al., 2011 R20ECD K WTC,Ant WT WTE Koole et al., 2011 L32ECD A WTM,Ag WT WTAg Underwood et al., 2010 L32ECD A WT Patterson et al., 2013 W33ECD S WTC,Ant Species change (human to rat)— the expected lack of effect on GLP-1 pharmacology was implied in text, but no data are shown Tibaduiza et al., 2001 T35ECD A WT 7%Ag Underwood et al., 2010 Val-36ECD A WTM,Ag WT WTAg Underwood et al., 2010 W39ECD† NDC,Ag Membrane expression confirmed via WB Wilmen et al., 1997 R44ECD H WTC,Ant WT WTE Koole et al., 2011 N63ECD L WTC,Ag WT WTAg Chen et al., 2010a E68ECD A WTM,Ag WT WTAg Underwood et al., 2010 E68ECD K 8 Day et al., 2011 Y69ECD A NDM,Ag ND NDAg Underwood et al., 2010 W72ECD† NDC,Ag Membrane expression confirmed via WB Wilmen et al., 1997 N82ECD L WTC,Ag WT WTAg Chen et al., 2010a W87ECD WTC,Ag WT 24–62% Transient and stable cell lines analyzed Wilmen et al., 1997 Y88ECD A NDM,Ag ND NDAg Underwood et al., 2010 L89ECD A NDM,Ag ND NDAg Underwood et al., 2010 Pro-90ECD A WTM,Ag WT WTAg Underwood et al., 2010 W91ECD† NDC,Ag Membrane expression confirmed via WB Wilmen et al., 1997 W110ECD† NDC,Ag Membrane expression confirmed via WB Wilmen et al., 1997 N115ECD L WTC,Ag WT WTAg Chen et al., 2010a W120ECD† NDC,Ag Membrane expression confirmed via WB Wilmen et al., 1997 R121ECD A WTM,Ag WT WTAg Underwood et al., 2010 L123ECD A WTM,Ag WT 47%Ag Underwood et al., 2010 E127ECD A WTM,Ag WT WTAg Underwood et al., 2010 E127ECD A WT Patterson et al., 2013 E127ECD E WTM,Ag WT WTAg Underwood et al., 2010 E128ECD A WTM,Ag WT WTAg Underwood et al., 2010 E128ECD A WT Patterson et al., 2013 E128ECD Q WTM,Ag WT WTAg Underwood et al., 2010 E128ECD M WT Day et al., 2011 R1311.26b N WTC,Ant WT WTE Koole et al., 2011 L1411.36b A WTC,Ag WT 67%cyt Yang et al., 2016 Y1451.40b A WTC,Ag WT 47%cyt Yang et al., 2016 Y1481.43b A NDC,Ag 26 WTcyt Yang et al., 2016 Y1481.43b N 15C,Ag 8 WTcyt Yang et al., 2016 Y1481.43b F NDC,Ag 14 WTcyt Yang et al., 2016 Y1481.43b F 10C,Ant 14 WTcyt Yang et al., 2016 T1491.44b M 60C,Ant 14–33 WTE & Ant Beinborn et al., 2005 T1491.44b M* 250C,Ant 160 <50%E Emax = ND Koole et al., 2011 For additional residue substiutions, see Koole et al., 2015 T1491.43b M NDC,Ag 59 WTcyt Yang et al., 2016 T1491.43b A NDC,Ag 82 WTcyt Yang et al., 2016 T1491.43b S WTC,Ag WT WTcyt Yang et al., 2016 Y1521.47b A 30M,Ant ND 7%Ant Coopman et al., 2011 Y1521.47b H WTC,Ag WT WTcyt Yang et al., 2016 S1551.50b A WTC,Ant 10 55%E, 48%Ant ΔLog τc = 0.75 Wootten et al., 2013c G168ICL1 S WTC,Ant WT <50%E Koole et al., 2011 F169ICL1† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 R170ICL1† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 H171ICL1† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 L172ICL1† A WTC,Ag 35%Ag WT cAMP 10−7 M GLP-1 Mathi et al., 1997 H173ICL1† A WTC,Ag 48%Ag WT cAMP 10−7 M GLP-1 Mathi et al., 1997 C174ICL1† A WTC,Ag 45%Ag 37% cAMP 10−7 M GLP-1 Mathi et al., 1997 C174ICL1 A 6 Underwood et al., 2013 C174ICL1 S 7 Underwood et al., 2013 T1752.45b† A WTC,Ag 57%Ag WT cAMP 10−7 M GLP-1 Mathi et al., 1997 R1762.46b† A WTC,Ag 13 WTAg 26% cAMP 10−7 M GLP-1 Mathi et al., 1997 N1772.47b† A WTC,Ag 22%Ag 43% cAMP 10−7 M GLP-1 Mathi et al., 1997 H1802.50b† R 21C,Ag WTmic Heller et al., 1996 H1802.50b A NDC,Ant 12 18%E, NDAnt ΔLog τc = 0.86 Wootten et al., 2013c N1822.52b† A WTC,Ag 24%Ag “36% of WT” cAMP Xiao et al., 2000 S1862.56b A WTC,Ant WT WTE,Ant Wootten et al., 2013c F1872.57b A NDC,Ag ND 5%cyt Yang et al., 2016 R1902.60b† A >20C,Ag 21%Ag “27% of WT” cAMP Xiao et al., 2000 R1902.60b A 32M,Ant 270 7%Ant Coopman et al., 2011 R1902.60b A 20C,Ant 34 53%E, 44Ant ΔLog τc = 0.53 Wootten et al., 2013c R1902.60b A NDC,Ag ND WTcyt Yang et al., 2016 R1902.60b K NDC,Ag 17 WTcyt Yang et al., 2016 R1902.60b K 32C,Ant 17 WTcyt Yang et al., 2016 L1922.62b S WT Underwood et al., 2011 L1922.62b S WTC,Ag WT WTcyt Yang et al., 2016 V1942.64b A WTC,Ag WT WTcyt Yang et al., 2016 F1952.65b L WT 59%Ag Underwood et al., 2011 I1962.66b† S WTC,Ag ND Moon et al., 2012 K1972.67b A 28M,Ant 630 57%Ant Coopman et al., 2011 K1972.67b† A 5C,Ag 30%Ag “25% of WT” cAMP Xiao et al., 2000 K1972.67b A NDC,Ag ND 51%cyt Yang et al., 2016 K1972.67b I NDC,Ag ND 51%cyt Yang et al., 2016 K1972.67b I 28C,Ant ND 51%cyt Yang et al., 2016 K1972.67b Q NDC,Ag ND WTcyt Yang et al., 2016 K1972.67b R NDC,Ag 23 WTcyt Yang et al., 2016 D1982.68b† A 10C,Ag 16%Ag “45% of WT” cAMP Xiao et al., 2000 D1982.68b† A 63M,Ant 44 WTAnt López de Maturana and Donnelly, 2002 D1982.68b A 43M,Ant 977 66%Ant Coopman et al., 2011 D1982.68b A NDC,Ag ND WTcyt Yang et al., 2016 D1982.68b† N WTM,Ag López de Maturana and Donnelly, 2002 D1982.68b N NDC,Ag 89 WTcyt Yang et al., 2016 D1982.68b E NDC,Ag 434 WTcyt Yang et al., 2016 D1982.68b E 58C,Ant 434 WTcyt Yang et al., 2016 A200-L201† V/A WTM,Ag WT López de Maturana et al., 2004 L2012.68b A NDC,Ag ND WTcyt Yang et al., 2016 K202ECL1† A WTC,Ag 45%Ag “71% of WT” cAMP Xiao et al., 2000 K202/W203† A/A WTM,Ag WT López de Maturana et al., 2004 W203ECL1 T WTC,Ag WT WTcyt Yang et al., 2016 M204/Y205† A/A 37M,Ant 51 28%Ant López de Maturana et al., 2004 M204/Y205† V/A 23M,Ag 32 WTAnt López de Maturana et al., 2004 M204/Y205† A/V 29M,Ag 87 WTAnt López de Maturana et al., 2004 M204ECL1† A WTM,Ag WT 44%Ant López de Maturana et al., 2004 M204ECL1 A NDC,Ag 334 WTcyt Yang et al., 2016 M204ECL1 R NDC,Ag 93 WTcyt Yang et al., 2016 Y205ECL1† A WTM,Ag WT WTAnt López de Maturana et al., 2004 Y205ECL1 A NDC,Ag 62 WTcyt Yang et al., 2016 S206/T207† A/A WTM,Ag WT López de Maturana et al., 2004 A208/A209† V/V WTM,Ag WT López de Maturana et al., 2004 Q210Q211† A/A WTM,Ag WT López de Maturana et al., 2004 Q211ECL1 D WTC,Ag WT WTcyt Yang et al., 2016 Q211ECL1 R WTC,Ag WT WTcyt Yang et al., 2016 H212ECL1† A WTC,Ag WTAg “WT cAMP” Xiao et al., 2000 H212ECL1 A WTC,Ag WT WTcyt Yang et al., 2016 H212/Q213† A/A WTM,Ag WT López de Maturana et al., 2004 D215ECL1† A WTC,Ag 51%Ag “57% of WT” cAMP Xiao et al., 2000 W214ECL1 V WTC,Ag WT WTcyt Yang et al., 2016 W214/D215† A/A WTM,Ag WT López de Maturana et al., 2004 G216/L217† A/A WTM,Ag WT López de Maturana et al., 2004 L217ECL1 A WTC,Ag WT WTcyt Yang et al., 2016 L218/S219† A/A WTM,Ag WT López de Maturana et al., 2004 Y220/Q221† A/A WTM,Ag WT López de Maturana et al., 2004 Y220ECL1 D NDC,Ag 105 WTcyt Yang et al., 2016 D2223.25b† A WTC,Ag WTAg “82% of WT” cAMP Xiao et al., 2000 D222/S223† A/A WTM,Ag WT López de Maturana et al., 2004 L224/G225† A/A WTM,Ag WT López de Maturana et al., 2004 C2263.29b*† A 25M,Ant 38 65%Ant Mann et al., 2010a C2263.29b A >90 Underwood et al., 2013 R2273.30b† A >20C,Ag 31%Ag “90% WT” cAMP Xiao et al., 2000 R227/L228† A/A WTM,Ag WT López de Maturana et al., 2004 V229/F230† A/A WTM,Ag WT López de Maturana et al., 2004 L232/M233† V/T 10C,Ag 100 Moon et al., 2012 M2333.36b A NDC,Ag 70 WTcyt Yang et al., 2016 M2333.36b A 16C,Ant 70 WTcyt Yang et al., 2016 M2333.36b T NDC,Ag 62 WTcyt Yang et al., 2016 M2333.36b F WTC,Ag WT WTcyt Yang et al., 2016 Q2343.37b A 13M,Ant 45 27%Ant Coopman et al., 2011 Q2343.37b A NDC,Ag 151 35%cyt Yang et al., 2016 Q2343.37b N WTC,Ag WT WTcyt Yang et al., 2016 Q2343.37b E NDC,Ag 29 69%cyt Yang et al., 2016 Q2343.37b E 17C,Ant 29 69%cyt Yang et al., 2016 Y2353.38b A 24M,Ant 23 12%Ant Coopman et al., 2011 C2363.39b A WT Underwood et al., 2013 N2403.43b† A >20C,Ag 14%Ag “8% WT” cAMP Xiao et al., 2000 N2403.43b A WTC,Ant 7 WTE,Ant ΔLog τc = 0.67 Wootten et al., 2013c N2403.43b Q WTC,Ant WT WTE,Ant Wootten et al., 2016 N240/Q394 A/A WTC,Ant WT 71%E,Ant ΔLog τc = 0.70 Wootten et al., 2016 N240/Q394 Q/N 5C,Ant WT 71%E,Ant Wootten et al., 2016 Y2413.44b A WT 42%Ag Underwood et al., 2011 Y2413.44b A WTC,Ag WT WTcyt Yang et al., 2016 E2473.50b A WTC,Ag 64%Ag “39% WT” cAMP Xiao et al., 2000 E2473.50b A NDC,Ant 14 18%E, NDAnt ΔLog τc = 0.99 Wootten et al., 2013c F260ICL2 L WTC,Ant WT <50%E Koole et al., 2011 E262ICL2† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 Q263ICL2† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 R264ICL2† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 I265ICL2† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 F2664.42b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 K2674.43b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 L2684.44b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 L2784.54b M WT Underwood et al., 2011 W2844.60b A 32M,Ant 1349 34%Ant Coopman et al., 2011 G2854.61b A WTC,Ant WT WTE Koole et al., 2012a I2864.62b A WTC,Ant WT WTE Koole et al., 2012a V2874.63b A WTC,Ant WT WTE Koole et al., 2012a I286/V287 A/A WT 85% SBM,Ag Dods and Donnelly, 2015 K2884.64b A 126C,Ant ND WTE Koole et al., 2012a K2884.64b A 23M,Ant 5732 WTAnt ΔLog τc = 1.39 Dods and Donnelly, 2015 K2884.64b† A 79M,Ant 251 Al-Sabah and Donnelly, 2003b K2884.64b† L 63M,Ant 79 Al-Sabah and Donnelly, 2003b K2884.64b L NDC,Ag ND 70%cyt Yang et al., 2016 K2884.64b† R WTM,Ag WT Al-Sabah and Donnelly, 2003b K288/Y289 A, A 2188 28% SBM,Ag Dods and Donnelly, 2015 Y2894.65b A WTC,Ant WT WTE Koole et al., 2012a Y2894.65b A WTM,Ant WT 20%Ant Dods and Donnelly, 2015 L2904.66b A WTC,Ant WT WTE Koole et al., 2012a Y2914.67b A WTC,Ant WT WTE Koole et al., 2012a L290/Y291† A/A WTM,Ag WT Mann et al., 2010a L290-Y291 A/A WT 99% SBM,Ag Dods and Donnelly, 2015 E292ECL2 A 100C,Ant 126 WTE ΔLog τc = 0.57 Koole et al., 2012a E292ECL2 A NDC,Ag 33 39%cyt Yang et al., 2016 D293ECL2 A 25C,Ant 16 62%E Koole et al., 2012a E292/D293† A/A 8M,Ag 79 Mann et al., 2010a E292/D293 A/A 25 75% SBM,Ag Dods and Donnelly, 2015 E294ECL2 A WTC,Ant WT WTE ΔLog τc = 0.65 Koole et al., 2012a G295ECL2 A WTC,Ant WT WTE Koole et al., 2012a E294-G295† A/A WTM,Ag WT Mann et al., 2010a E294-G295 A/A WT 97% SB M,Ag Dods and Donnelly, 2015 C296ECL2*† A 18M,Ant WT 23%Ant Mann et al., 2010a C296ECL2 A 13C,Ant 126 60E Koole et al., 2012a C296ECL2 S NDC,Ag 96 WTcyt Yang et al., 2016 W297ECL2 A 63C,Ant 316 WTE ΔLog τc = 1.00 Koole et al., 2012a W297ECL2 A NDC,Ag ND WTcyt Yang et al., 2016 W297ECL2 H NDC,Ag 50 40%cyt Yang et al., 2016 T298ECL2 A WTC,Ant WT WTE Koole et al., 2012a W297/T298† A/A 100M,Ant 50 Mann et al., 2010a; Donnelly, 2012 W297/T298 A/A 22 57% SBM,Ag Dods and Donnelly, 2015 R299ECL2 A 32C,Ant 85 WTE Koole et al., 2012a R299ECL2 A WTM,Ant WT 40%Ant ΔLog τc = 0.60 Dods and Donnelly, 2015 R299ECL2 S NDC,Ag 43 WTcyt Yang et al., 2016 N300ECL2 A 126C,Ant 501 WTE ΔLog τc = 0.80 Koole et al., 2012a N300ECL2 A 36M,Ant 104 18%Ant Dods and Donnelly, 2015 N300ECL2 A NDC,Ag ND 16%cyt Yang et al., 2016 R299/N300† A/A 25M,Ant >3000 Mann et al., 2010a; Donnelly, 2012 R299/N300 A/A 331 41% SBM,Ag Dods and Donnelly, 2015 S301ECL2 A WTC,Ant WT 63%E Koole et al., 2012a N302ECL2 A 25C,Ant 16 WTE ΔLog τc = 0.53 Koole et al., 2012a S301/N302† A/A WTM,Ag WT Mann et al., 2010a S301/N302 A/A WT 86% SBM,Ag Dods and Donnelly, 2015 N302/M303† V/K WTC,Ag 10 Moon et al., 2012 M303ECL2 A WTC,Ant WT WTE Koole et al., 2012a N304ECL2 A WTC,Ant WT 70%E ΔLog τc = 0.74 Koole et al., 2012a N304ECL2 A WTC,Ag WT WTcyt Yang et al., 2016 M303/N304† A/A WTM,Ag WT Mann et al., 2010a M303/N304 A/A WT 91% SBM,Ag Dods and Donnelly, 2015 Y3055.35b A 79C,Ant 40 52%E Koole et al., 2012a Y3055.35b A WTM,Ant WT 22%Ant Dods and Donnelly, 2015 W3065.36b A NDC,Ant ND ND No receptor expression Koole et al., 2012a W3065.36 A 109M,Ant 206 41%Ant Dods and Donnelly, 2015 W3065.36 A NDC,Ag ND WTcyt Yang et al., 2016 †Y305/W306† A/A 316M,Ant 50 Mann et al., 2010a; Donnelly, 2012 Y305/W306 A/A 263 60% SBM,Ag Dods and Donnelly, 2015 L3075.37b A 13C,Ant 25 47%E Koole et al., 2012a L3075.37b A WT 87% SBM,Ag Dods and Donnelly, 2015 I3085.38b A WT 78% SBM,Ag Dods and Donnelly, 2015 L307/I308† A/A 251M,Ant 6 Mann et al., 2010a; Donnelly 2012 I3095.39b A WTM,Ant WT WTAnt Dods and Donnelly, 2015 R3105.40b A 10M,Ant 1259 17%Ant Coopman et al., 2011 R3105.40b A WTM,Ant 1290 19%Ant ΔLog τc = 0.75 Dods and Donnelly, 2015 I309/R310† A/A 50M,Ant >3000 Mann et al., 2010a; Donnelly, 2012 I309/R310 A/A 13,490 13% SBM,Ag Dods and Donnelly, 2015 L311/P312 A/A WT 91% SBM,Ag Dods and Donnelly, 2015 A3165.46b T WTC,Ant WT <25%E Koole et al., 2011 N3205.50b A 18C,Ant 10 WTE,Ant ΔLog τc = 0.50 Wootten et al., 2013c F3215.51b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 L3225.52b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 I3235.53b† A WTC,Ag 35%Ant WT cAMP 10−7 M GLP-1 Mathi et al., 1997 F3245.54b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 I325/F326† A/A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 V3275.57b† A WTC,Ag 15 WTAg 42% cAMP 10−7 M GLP-1 Mathi et al., 1997 I3285.58b† A WTC,Ag 9 WTAnt 39% cAMP 10−7 M GLP-1 Mathi et al., 1997 C3295.59b† A WTC,Ag WTAnt WT cAMP 10−7 M GLP-1 Mathi et al., 1997 I3305.60b A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 V3315.61b† A WTC,Ag 14 WTAg 45% cAMP 10−7 M GLP-1 Mathi et al., 1997 I3325.62b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 A3335.63b† L WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Mathi et al., 1997 S3335.63b C WTC,Ant WT <50%E For additional residue substiutions, see Koole et al., 2015 Koole et al., 2011 K3345.64b† A WTC,Ag WTAg 28% cAMP 10−7 M GLP-1 Takhar et al., 1996; Mathi et al., 1997 L3355.65b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Takhar et al., 1996; Mathi et al., 1997 K3365.66b† L WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Takhar et al., 1996; Mathi et al., 1997 K334/K351 Deletions WTC,Ag Ag See paper for details Takhar et al., 1996 C3476.36b A WT Underwood et al., 2013 R3486.37b† G 12C,Ag ND WTmic Heller et al., 1996 R3486.37b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Takhar et al., 1996 L3496.38b† A WTC,Ag 76%Ag WT cAMP 10−7 M GLP-1 Takhar et al., 1996 A-3506.39b† E NDC,Ag <10%Ag 5% cAMP 10−7 M GLP-1 Takhar et al., 1996 A-3506.39b† K WTC,Ag 21%Ag 2% cAMP 10−7 M GLP-1 Takhar et al., 1996 K3516.40b† A WTC,Ag WTAg WT cAMP 10−7 M GLP-1 Takhar et al., 1996 T3536.42b A NDC,Ant 22 30%E, NDAnt ΔLog τc = 0.84 Wootten et al., 2013c H3636.52b A 98M,Ant ND 24%Ant Coopman et al., 2011 H3636.52b A 23C,Ant 4 59%E, 53%Ant ΔLog τc = 1.71 Wootten et al., 2013c E3646.53b A 58M,Ant 15 42%Ant Coopman et al., 2011 E3646.53b A 25C,Ant 51%E,Ant ΔLog τc = 0.66 Wootten et al., 2016 E3646.53b A NDC,Ag ND 6%cyt Yang et al., 2016 E3646.53b Q WTC,Ag WT 42%cyt Yang et al., 2016 E3646.53b Q 0.2C,Ant WT 42%cyt Yang et al., 2016 E364/E387 N/Q NDC,Ag ND WTcyt Yang et al., 2016 E3646.53b Y WTC,Ag WT WTcyt Yang et al., 2016 E3646.53b D WTC,Ag WT 53%cyt Yang et al., 2016 F3676.56b A NDC,Ag 72 WTcyt Yang et al., 2016 F3676.56b A 32C,Ant 72 WTcyt Yang et al., 2016 F3676.56b I NDC,Ag 20 WTcyt Yang et al., 2016 F3676.56b H 7C,Ag 131 WTcyt Yang et al., 2016 M371ECL3 A WT M,Ant WT 54%Ant Dods and Donnelly, 2015 D372ECL3 A WT M,Ant 59 13%Ant Dods and Donnelly, 2015 E373ECL3 A WTM,Ant WT 28%Ant Dods and Donnelly, 2015 H374ECL3 A WTM,Ant WT 22%Ant Dods and Donnelly, 2015 H374ECL3 A WTC,Ag WT WTcyt Yang et al., 2016 A-375ECL3 A 10M,Ant WT WTAnt Dods and Donnelly, 2015 R376ECL3 G WTM,Ant WT WTAnt Dods and Donnelly, 2015 R376ECL3 Q WTC,Ag WT WTcyt Yang et al., 2016 G377ECL3 A WTM,Ant WT 19%Ant Dods and Donnelly, 2015 T3787.32b A WTM,Ant WT 12%Ant Dods and Donnelly, 2015 L3797.33b R 12C,Ag 141 WTAg Moon et al., 2015 L3797.33b E 11C,Ag 165 WTAg Moon et al., 2015 L3797.33b A WTM,Ant WT 31%Ant Dods and Donnelly, 2015 R3807.34b D 21C,Ag 1853 WTAg Moon et al., 2015 R3807.34b G 4C,Ag 40 WTAg Moon et al., 2015 R3807.34b A 128M,Ant 263 68%Ant Dods and Donnelly, 2015 R3807.34b Q NDC,Ag ND WTcyt Yang et al., 2016 F3817.35b R WT C,Ag WT WTAg Moon et al., 2015 F3817.35b E >200C,Ag 234 WTAg Moon et al., 2015 F3817.35b A WTM,Ant WT 16%Ant Dods and Donnelly, 2015 F3817.35b S WTC,Ag WT WTcyt Yang et al., 2016 I3827.36b A WTM,Ant WT 23%Ant Dods and Donnelly, 2015 K3837.37b A WTM,Ant 56 WTAnt ΔLog τc = 1.18 Dods and Donnelly, 2015 L3847.38b A WTM,Ant WT WTAnt Dods and Donnelly, 2015 L3847.38b A NDC,Ag 41 WTcyt Yang et al., 2016 L3847.38b A 48C,Ant 41 WTcyt Yang et al., 2016 L3847.38b V NDC,Ag 16 WTcyt Yang et al., 2016 F3857.40b A WTM,Ant WT WTAnt Dods and Donnelly, 2015 T3867.41b A WTM,Ant WT 18%Ant Dods and Donnelly, 2015 E3877.42b A WTM,Ant WT 43%Ant ΔLog τc = 0.52 Dods and Donnelly, 2015 E3877.42b A WTM,Ant WT WTAnt Coopman et al., 2011 E3877.42b D 13C,Ag 10 WTcyt Yang et al., 2016 E3877.42b D 12C,Ant 10 WTcyt Yang et al., 2016 E3877.42b N WTC,Ag WT WTcyt Yang et al., 2016 L3887.43b A NDC,Ag 208 WTcyt Yang et al., 2016 L3887.43b I 5C,Ag 81 WTcyt Yang et al., 2016 L3887.43b F WTC,Ag WT WTcyt Yang et al., 2016 T3917.44b A WTM,Ant WT 64%M,Ant Coopman et al., 2011 T3917.44b A WT WTAg Underwood et al., 2011 S3927.47b A WTC,Ant WT WTE, Ant Wootten et al., 2013c Q3947.49b A WTC,Ant WT WTE, Ant Wootten et al., 2013c Q3947.49b N WTC,Ant WTE,Ant Wootten et al., 2016 M3977.52b L WTC,Ag WT Dong et al., 2012 Y4027.57b A NDC,Ant 10 21%E, NDAnt ΔLog τc = 1.59 Wootten et al., 2013c C4037.58b A 5.4 Underwood et al., 2013 N4067.61b A WTC,Ant WT WTE,Ant Wootten et al., 2013c R421CTT Q WTC,Ant WT <50%E Koole et al., 2011 C438 CTT A WT Underwood et al., 2013 C458 CTT A WT Underwood et al., 2013 C462 CTT A WT Underwood et al., 2013 CTT, C-terminal tail; SB, specific binding of radiolabeled ligand; WB, Western blotting.
↵† Note that potency changes can be due to changes in affinity, efficacy, and/or cell surface expression, and hence should be interpreted with caution, especially when either the affinity and/or expression levels are not known.
↵* Also included in double mutations.
Molecular/Cellular Measurement Description/Outcome References Receptor-binding assays: Radioligand competition Determines binding affinity (IC50 or Kd) by measuring radioactivity remaining on the receptor after competitive inhibition of radioligand with a GLP-1 analog. Mathi et al., 1997; Tibaduiza et al., 2001; Xiao et al., 2001 Time-resolved fluorescence resonance energy transfer (FRET) Determines binding affinity (IC50) by measuring a decrease in FRET signal between Tb-labeled receptor and fluorescent ligand after competitive inhibition with a GLP-1 analog. Maurel et al., 2008; Zwier et al., 2010; Roed et al., 2014 Circular dichroism and fluorescence spectroscopies Determines binding affinity by measuring conformational changes of a receptor protein upon ligand binding. Runge et al., 2007 Isothermal titration calorimetry Determines thermodynamic parameters of binding, such as the dissociation constant, enthalpy change, entropy change, and reaction stoichiometry by measuring heat changes during receptor–ligand interaction. Wiseman et al., 1989; Bazarsuren et al., 2002; Donnelly, 2012 Total-internal reflection fluorescence imaging Determines equilibrium constants and dissociation rates by monitoring fluorescence-labeled single molecule on lipid bilayer. Fox et al., 2009; Myers et al., 2012 Surface plasmon resonance Determines dissociation constants by real-time measurement of receptor–ligand interaction. Bazarsuren et al., 2002; Schroder-Tittmann et al., 2010; Locatelli-Hoops et al., 2013 Photoaffinity labeling Identifies residues of peptide and receptor at binding interface that are spatially proximal to each other. Chowdhry and Westheimer, 1979; Ji et al., 1997; Vodovozova, 2007; Chen et al., 2009; Miller et al., 2011 Receptor functional assays: Homogeneous time-resolved fluorescence (HTRF) or alpha screen cAMP assays HTRF technology is an immunoassay based on a FRET between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanin used as acceptor. Alpha technology is a bead-based proximity assay. Gesellchen et al., 2006; Einhorn and Krapfenbauer, 2015 Radioimmunoassay Determines receptor-stimulating potency (EC50) by quantitative analysis of cAMP production with immobilized anti-cAMP or anti-cGMP antibodies and radiolabeled cAMP/cGMP. Farmer et al., 1975; Wheeler et al., 1995 FRET-based cAMP assay Determines receptor-stimulating potency (EC50) by measuring a decreased FRET signal between fluorescent proteins (CFP and YFP) and Epac protein. Holz, 2004; Nikolaev et al., 2004; Landa et al., 2005; Harbeck et al., 2006; Sloop et al., 2010 Luciferase reporter assay Determines receptor-stimulating potency (EC50) by measuring luminescence that is increased by transcription of transfected luciferase reporter plasmid linked to cAMP response element. Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999; Miranda et al., 2008; Murage et al., 2008; Smale, 2010 Intracellular calcium ion Determines receptor activation by measuring intracellular Ca2+ level with calcium-sensitive dye, Fura-2. Grynkiewicz et al., 1985; Cullinan et al., 1994; Bode et al., 1999 Determination of incretin effects: Glucose tolerance test (GTT; oral GTT, OGTT; intraperitoneal GTT, IPGTT; intravenous GTT, IVGTT) Determines insulinotropic action of GLP-1 analogs by measuring glucose level after their administration. Kreymann et al., 1987; Toft-Nielson et al., 1996 Insulin secretion Determines potency of GLP-1 analogs by measuring insulin secretagogue action. Albano et al., 1972; Andersen et al., 1993; Goke et al., 1993b; Toft-Nielson et al., 1996; Kjems et al., 2003; Peyot et al., 2009 - TABLE 3
Peptidic GLP-1R agonists that have been launched or are in late-stage clinical trials
Analog Description Company Dosing Regimen Status Exenatide Exendin-4 Amylin/Eli Lilly; AstraZeneca Twice daily Launched Exenatide once weekly Exendin-4 Amylin/Alkermes/Eli Lilly; AstraZeneca Once weekly Launched Lixisenatide Exendin-4 analog Sanofi-Aventis Once daily Launched Liraglutide GLP-1 analog linked with a fatty acid Novo Nordisk Once daily Launched Albiglutide GLP-1 analog fused to albumin GlaxoSmithKline Once weekly Launched Dulaglutide GLP-1 analog fused to Fc Eli Lilly Once weekly Launched Semaglutide GLP-1 analog linked with a fatty acid Novo Nordisk Once weekly Phase 3 Semaglutide (NN9924) GLP-1 analog linked with a fatty di-acid Novo Nordisk Oral Phase 2 Taspoglutide GLP-1 analog formulated for sustained release Roche Once weekly Suspended
