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Glucagon-Like Peptide-1 and Its Class B G Protein–Coupled Receptors: A Long March to Therapeutic Successes

Chris de Graaf, Dan Donnelly, Denise Wootten, Jesper Lau, Patrick M. Sexton, Laurence J. Miller, Jung-Mo Ahn, Jiayu Liao, Madeleine M. Fletcher, Dehua Yang, Alastair J. H. Brown, Caihong Zhou, Jiejie Deng and Ming-Wei Wang
RICHARD DEQUAN YE, ASSOCIATE EDITOR
Pharmacological Reviews October 2016, 68 (4) 954-1013; DOI: https://doi.org/10.1124/pr.115.011395

Article Figures & Data

Figures

  • Fig. 1.
    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.
    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.
    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.
    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.
    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.
    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.
    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).

    ResidueMutated to-Fold Reduction Affinity-Fold Reduction Potency‡Cell Surface Expression (% Wild-Type)Comments and/or Other Observed EffectsReference
    P7ECDLWTC,AntWTWTEKoole et al., 2011
    R20ECDKWTC,AntWTWTEKoole et al., 2011
    L32ECDAWTM,AgWTWTAgUnderwood et al., 2010
    L32ECDAWTPatterson et al., 2013
    W33ECDSWTC,AntSpecies change (human to rat)— the expected lack of effect on GLP-1 pharmacology was implied in text, but no data are shownTibaduiza et al., 2001
    T35ECDAWT7%AgUnderwood et al., 2010
    Val-36ECDAWTM,AgWTWTAgUnderwood et al., 2010
    W39ECD†NDC,AgMembrane expression confirmed via WBWilmen et al., 1997
    R44ECDHWTC,AntWTWTEKoole et al., 2011
    N63ECDLWTC,AgWTWTAgChen et al., 2010a
    E68ECDAWTM,AgWTWTAgUnderwood et al., 2010
    E68ECDK8Day et al., 2011
    Y69ECDANDM,AgNDNDAgUnderwood et al., 2010
    W72ECD†NDC,AgMembrane expression confirmed via WBWilmen et al., 1997
    N82ECDLWTC,AgWTWTAgChen et al., 2010a
    W87ECDWTC,AgWT24–62%Transient and stable cell lines analyzedWilmen et al., 1997
    Y88ECDANDM,AgNDNDAgUnderwood et al., 2010
    L89ECDANDM,AgNDNDAgUnderwood et al., 2010
    Pro-90ECDAWTM,AgWTWTAgUnderwood et al., 2010
    W91ECD†NDC,AgMembrane expression confirmed via WBWilmen et al., 1997
    W110ECD†NDC,AgMembrane expression confirmed via WBWilmen et al., 1997
    N115ECDLWTC,AgWTWTAgChen et al., 2010a
    W120ECD†NDC,AgMembrane expression confirmed via WBWilmen et al., 1997
    R121ECDAWTM,AgWTWTAgUnderwood et al., 2010
    L123ECDAWTM,AgWT47%AgUnderwood et al., 2010
    E127ECDAWTM,AgWTWTAgUnderwood et al., 2010
    E127ECDAWTPatterson et al., 2013
    E127ECDEWTM,AgWTWTAgUnderwood et al., 2010
    E128ECDAWTM,AgWTWTAgUnderwood et al., 2010
    E128ECDAWTPatterson et al., 2013
    E128ECDQWTM,AgWTWTAgUnderwood et al., 2010
    E128ECDMWTDay et al., 2011
    R1311.26bNWTC,AntWTWTEKoole et al., 2011
    L1411.36bAWTC,AgWT67%cytYang et al., 2016
    Y1451.40bAWTC,AgWT47%cytYang et al., 2016
    Y1481.43bANDC,Ag26WTcytYang et al., 2016
    Y1481.43bN15C,Ag8WTcytYang et al., 2016
    Y1481.43bFNDC,Ag14WTcytYang et al., 2016
    Y1481.43bF10C,Ant14WTcytYang et al., 2016
    T1491.44bM60C,Ant14–33WTE & AntBeinborn et al., 2005
    T1491.44bM*250C,Ant160<50%EEmax = NDKoole et al., 2011
    For additional residue substiutions, see Koole et al., 2015
    T1491.43bMNDC,Ag59WTcytYang et al., 2016
    T1491.43bANDC,Ag82WTcytYang et al., 2016
    T1491.43bSWTC,AgWTWTcytYang et al., 2016
    Y1521.47bA30M,AntND7%AntCoopman et al., 2011
    Y1521.47bHWTC,AgWTWTcytYang et al., 2016
    S1551.50bAWTC,Ant1055%E, 48%AntΔLog τc = 0.75Wootten et al., 2013c
    G168ICL1SWTC,AntWT<50%EKoole et al., 2011
    F169ICL1†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    R170ICL1†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    H171ICL1†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    L172ICL1†AWTC,Ag35%AgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    H173ICL1†AWTC,Ag48%AgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    C174ICL1†AWTC,Ag45%Ag37% cAMP 10−7 M GLP-1Mathi et al., 1997
    C174ICL1A6Underwood et al., 2013
    C174ICL1S7Underwood et al., 2013
    T1752.45b†AWTC,Ag57%AgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    R1762.46b†AWTC,Ag13WTAg26% cAMP 10−7 M GLP-1Mathi et al., 1997
    N1772.47b†AWTC,Ag22%Ag43% cAMP 10−7 M GLP-1Mathi et al., 1997
    H1802.50b†R21C,AgWTmicHeller et al., 1996
    H1802.50bANDC,Ant1218%E, NDAntΔLog τc = 0.86Wootten et al., 2013c
    N1822.52b†AWTC,Ag24%Ag“36% of WT” cAMPXiao et al., 2000
    S1862.56bAWTC,AntWTWTE,AntWootten et al., 2013c
    F1872.57bANDC,AgND5%cytYang et al., 2016
    R1902.60b†A>20C,Ag21%Ag“27% of WT” cAMPXiao et al., 2000
    R1902.60bA32M,Ant2707%AntCoopman et al., 2011
    R1902.60bA20C,Ant3453%E, 44AntΔLog τc = 0.53Wootten et al., 2013c
    R1902.60bANDC,AgNDWTcytYang et al., 2016
    R1902.60bKNDC,Ag17WTcytYang et al., 2016
    R1902.60bK32C,Ant17WTcytYang et al., 2016
    L1922.62bSWTUnderwood et al., 2011
    L1922.62bSWTC,AgWTWTcytYang et al., 2016
    V1942.64bAWTC,AgWTWTcytYang et al., 2016
    F1952.65bLWT59%AgUnderwood et al., 2011
    I1962.66b†SWTC,AgNDMoon et al., 2012
    K1972.67bA28M,Ant63057%AntCoopman et al., 2011
    K1972.67b†A5C,Ag30%Ag“25% of WT” cAMPXiao et al., 2000
    K1972.67bANDC,AgND51%cytYang et al., 2016
    K1972.67bINDC,AgND51%cytYang et al., 2016
    K1972.67bI28C,AntND51%cytYang et al., 2016
    K1972.67bQNDC,AgNDWTcytYang et al., 2016
    K1972.67bRNDC,Ag23WTcytYang et al., 2016
    D1982.68b†A10C,Ag16%Ag“45% of WT” cAMPXiao et al., 2000
    D1982.68b†A63M,Ant44WTAntLópez de Maturana and Donnelly, 2002
    D1982.68bA43M,Ant97766%AntCoopman et al., 2011
    D1982.68bANDC,AgNDWTcytYang et al., 2016
    D1982.68b†NWTM,AgLópez de Maturana and Donnelly, 2002
    D1982.68bNNDC,Ag89WTcytYang et al., 2016
    D1982.68bENDC,Ag434WTcytYang et al., 2016
    D1982.68bE58C,Ant434WTcytYang et al., 2016
    A200-L201†V/AWTM,AgWTLópez de Maturana et al., 2004
    L2012.68bANDC,AgNDWTcytYang et al., 2016
    K202ECL1†AWTC,Ag45%Ag“71% of WT” cAMPXiao et al., 2000
    K202/W203†A/AWTM,AgWTLópez de Maturana et al., 2004
    W203ECL1TWTC,AgWTWTcytYang et al., 2016
    M204/Y205†A/A37M,Ant5128%AntLópez de Maturana et al., 2004
    M204/Y205†V/A23M,Ag32WTAntLópez de Maturana et al., 2004
    M204/Y205†A/V29M,Ag87WTAntLópez de Maturana et al., 2004
    M204ECL1†AWTM,AgWT44%AntLópez de Maturana et al., 2004
    M204ECL1ANDC,Ag334WTcytYang et al., 2016
    M204ECL1RNDC,Ag93WTcytYang et al., 2016
    Y205ECL1†AWTM,AgWTWTAntLópez de Maturana et al., 2004
    Y205ECL1ANDC,Ag62WTcytYang et al., 2016
    S206/T207†A/AWTM,AgWTLópez de Maturana et al., 2004
    A208/A209†V/VWTM,AgWTLópez de Maturana et al., 2004
    Q210Q211†A/AWTM,AgWTLópez de Maturana et al., 2004
    Q211ECL1DWTC,AgWTWTcytYang et al., 2016
    Q211ECL1RWTC,AgWTWTcytYang et al., 2016
    H212ECL1†AWTC,AgWTAg“WT cAMP”Xiao et al., 2000
    H212ECL1AWTC,AgWTWTcytYang et al., 2016
    H212/Q213†A/AWTM,AgWTLópez de Maturana et al., 2004
    D215ECL1†AWTC,Ag51%Ag“57% of WT” cAMPXiao et al., 2000
    W214ECL1VWTC,AgWTWTcytYang et al., 2016
    W214/D215†A/AWTM,AgWTLópez de Maturana et al., 2004
    G216/L217†A/AWTM,AgWTLópez de Maturana et al., 2004
    L217ECL1AWTC,AgWTWTcytYang et al., 2016
    L218/S219†A/AWTM,AgWTLópez de Maturana et al., 2004
    Y220/Q221†A/AWTM,AgWTLópez de Maturana et al., 2004
    Y220ECL1DNDC,Ag105WTcytYang et al., 2016
    D2223.25b†AWTC,AgWTAg“82% of WT” cAMPXiao et al., 2000
    D222/S223†A/AWTM,AgWTLópez de Maturana et al., 2004
    L224/G225†A/AWTM,AgWTLópez de Maturana et al., 2004
    C2263.29b*†A25M,Ant3865%AntMann et al., 2010a
    C2263.29bA>90Underwood et al., 2013
    R2273.30b†A>20C,Ag31%Ag“90% WT” cAMPXiao et al., 2000
    R227/L228†A/AWTM,AgWTLópez de Maturana et al., 2004
    V229/F230†A/AWTM,AgWTLópez de Maturana et al., 2004
    L232/M233†V/T10C,Ag100Moon et al., 2012
    M2333.36bANDC,Ag70WTcytYang et al., 2016
    M2333.36bA16C,Ant70WTcytYang et al., 2016
    M2333.36bTNDC,Ag62WTcytYang et al., 2016
    M2333.36bFWTC,AgWTWTcytYang et al., 2016
    Q2343.37bA13M,Ant4527%AntCoopman et al., 2011
    Q2343.37bANDC,Ag15135%cytYang et al., 2016
    Q2343.37bNWTC,AgWTWTcytYang et al., 2016
    Q2343.37bENDC,Ag2969%cytYang et al., 2016
    Q2343.37bE17C,Ant2969%cytYang et al., 2016
    Y2353.38bA24M,Ant2312%AntCoopman et al., 2011
    C2363.39bAWTUnderwood et al., 2013
    N2403.43b†A>20C,Ag14%Ag“8% WT” cAMPXiao et al., 2000
    N2403.43bAWTC,Ant7WTE,AntΔLog τc = 0.67Wootten et al., 2013c
    N2403.43bQWTC,AntWTWTE,AntWootten et al., 2016
    N240/Q394A/AWTC,AntWT71%E,AntΔLog τc = 0.70Wootten et al., 2016
    N240/Q394Q/N5C,AntWT71%E,AntWootten et al., 2016
    Y2413.44bAWT42%AgUnderwood et al., 2011
    Y2413.44bAWTC,AgWTWTcytYang et al., 2016
    E2473.50bAWTC,Ag64%Ag“39% WT” cAMPXiao et al., 2000
    E2473.50bANDC,Ant1418%E, NDAntΔLog τc = 0.99Wootten et al., 2013c
    F260ICL2LWTC,AntWT<50%EKoole et al., 2011
    E262ICL2†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    Q263ICL2†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    R264ICL2†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    I265ICL2†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    F2664.42b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    K2674.43b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    L2684.44b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    L2784.54bMWTUnderwood et al., 2011
    W2844.60bA32M,Ant134934%AntCoopman et al., 2011
    G2854.61bAWTC,AntWTWTEKoole et al., 2012a
    I2864.62bAWTC,AntWTWTEKoole et al., 2012a
    V2874.63bAWTC,AntWTWTEKoole et al., 2012a
    I286/V287A/AWT85% SBM,AgDods and Donnelly, 2015
    K2884.64bA126C,AntNDWTEKoole et al., 2012a
    K2884.64bA23M,Ant5732WTAntΔLog τc = 1.39Dods and Donnelly, 2015
    K2884.64b†A79M,Ant251Al-Sabah and Donnelly, 2003b
    K2884.64b†L63M,Ant79Al-Sabah and Donnelly, 2003b
    K2884.64bLNDC,AgND70%cytYang et al., 2016
    K2884.64b†RWTM,AgWTAl-Sabah and Donnelly, 2003b
    K288/Y289A, A218828% SBM,AgDods and Donnelly, 2015
    Y2894.65bAWTC,AntWTWTEKoole et al., 2012a
    Y2894.65bAWTM,AntWT20%AntDods and Donnelly, 2015
    L2904.66bAWTC,AntWTWTEKoole et al., 2012a
    Y2914.67bAWTC,AntWTWTEKoole et al., 2012a
    L290/Y291†A/AWTM,AgWTMann et al., 2010a
    L290-Y291A/AWT99% SBM,AgDods and Donnelly, 2015
    E292ECL2A100C,Ant126WTEΔLog τc = 0.57Koole et al., 2012a
    E292ECL2ANDC,Ag3339%cytYang et al., 2016
    D293ECL2A25C,Ant1662%EKoole et al., 2012a
    E292/D293†A/A8M,Ag79Mann et al., 2010a
    E292/D293A/A2575% SBM,AgDods and Donnelly, 2015
    E294ECL2AWTC,AntWTWTEΔLog τc = 0.65Koole et al., 2012a
    G295ECL2AWTC,AntWTWTEKoole et al., 2012a
    E294-G295†A/AWTM,AgWTMann et al., 2010a
    E294-G295A/AWT97% SB M,AgDods and Donnelly, 2015
    C296ECL2*†A18M,AntWT23%AntMann et al., 2010a
    C296ECL2A13C,Ant12660EKoole et al., 2012a
    C296ECL2SNDC,Ag96WTcytYang et al., 2016
    W297ECL2A63C,Ant316WTEΔLog τc = 1.00Koole et al., 2012a
    W297ECL2ANDC,AgNDWTcytYang et al., 2016
    W297ECL2HNDC,Ag5040%cytYang et al., 2016
    T298ECL2AWTC,AntWTWTEKoole et al., 2012a
    W297/T298†A/A100M,Ant50Mann et al., 2010a;
    Donnelly, 2012
    W297/T298A/A2257% SBM,AgDods and Donnelly, 2015
    R299ECL2A32C,Ant85WTEKoole et al., 2012a
    R299ECL2AWTM,AntWT40%AntΔLog τc = 0.60Dods and Donnelly, 2015
    R299ECL2SNDC,Ag43WTcytYang et al., 2016
    N300ECL2A126C,Ant501WTEΔLog τc = 0.80Koole et al., 2012a
    N300ECL2A36M,Ant10418%AntDods and Donnelly, 2015
    N300ECL2ANDC,AgND16%cytYang et al., 2016
    R299/N300†A/A25M,Ant>3000Mann et al., 2010a;
    Donnelly, 2012
    R299/N300A/A33141% SBM,AgDods and Donnelly, 2015
    S301ECL2AWTC,AntWT63%EKoole et al., 2012a
    N302ECL2A25C,Ant16WTEΔLog τc = 0.53Koole et al., 2012a
    S301/N302†A/AWTM,AgWTMann et al., 2010a
    S301/N302A/AWT86% SBM,AgDods and Donnelly, 2015
    N302/M303†V/KWTC,Ag10Moon et al., 2012
    M303ECL2AWTC,AntWTWTEKoole et al., 2012a
    N304ECL2AWTC,AntWT70%EΔLog τc = 0.74Koole et al., 2012a
    N304ECL2AWTC,AgWTWTcytYang et al., 2016
    M303/N304†A/AWTM,AgWTMann et al., 2010a
    M303/N304A/AWT91% SBM,AgDods and Donnelly, 2015
    Y3055.35bA79C,Ant4052%EKoole et al., 2012a
    Y3055.35bAWTM,AntWT22%AntDods and Donnelly, 2015
    W3065.36bANDC,AntNDNDNo receptor expressionKoole et al., 2012a
    W3065.36A109M,Ant20641%AntDods and Donnelly, 2015
    W3065.36ANDC,AgNDWTcytYang et al., 2016
    †Y305/W306†A/A316M,Ant50Mann et al., 2010a;
    Donnelly, 2012
    Y305/W306A/A26360% SBM,AgDods and Donnelly, 2015
    L3075.37bA13C,Ant2547%EKoole et al., 2012a
    L3075.37bAWT87% SBM,AgDods and Donnelly, 2015
    I3085.38bAWT78% SBM,AgDods and Donnelly, 2015
    L307/I308†A/A251M,Ant6Mann et al., 2010a;
    Donnelly 2012
    I3095.39bAWTM,AntWTWTAntDods and Donnelly, 2015
    R3105.40bA10M,Ant125917%AntCoopman et al., 2011
    R3105.40bAWTM,Ant129019%AntΔLog τc = 0.75Dods and Donnelly, 2015
    I309/R310†A/A50M,Ant>3000Mann et al., 2010a;
    Donnelly, 2012
    I309/R310A/A13,49013% SBM,AgDods and Donnelly, 2015
    L311/P312A/AWT91% SBM,AgDods and Donnelly, 2015
    A3165.46bTWTC,AntWT<25%EKoole et al., 2011
    N3205.50bA18C,Ant10WTE,AntΔLog τc = 0.50Wootten et al., 2013c
    F3215.51b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    L3225.52b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    I3235.53b†AWTC,Ag35%AntWT cAMP 10−7 M GLP-1Mathi et al., 1997
    F3245.54b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    I325/F326†A/AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    V3275.57b†AWTC,Ag15WTAg42% cAMP 10−7 M GLP-1Mathi et al., 1997
    I3285.58b†AWTC,Ag9WTAnt39% cAMP 10−7 M GLP-1Mathi et al., 1997
    C3295.59b†AWTC,AgWTAntWT cAMP 10−7 M GLP-1Mathi et al., 1997
    I3305.60bAWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    V3315.61b†AWTC,Ag14WTAg45% cAMP 10−7 M GLP-1Mathi et al., 1997
    I3325.62b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    A3335.63b†LWTC,AgWTAgWT cAMP 10−7 M GLP-1Mathi et al., 1997
    S3335.63bCWTC,AntWT<50%EFor additional residue substiutions, see Koole et al., 2015Koole et al., 2011
    K3345.64b†AWTC,AgWTAg28% cAMP 10−7 M GLP-1Takhar et al., 1996;
Mathi et al., 1997
    L3355.65b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Takhar et al., 1996;
Mathi et al., 1997
    K3365.66b†LWTC,AgWTAgWT cAMP 10−7 M GLP-1Takhar et al., 1996;
Mathi et al., 1997
    K334/K351DeletionsWTC,AgAg See paper for detailsTakhar et al., 1996
    C3476.36bAWTUnderwood et al., 2013
    R3486.37b†G12C,AgNDWTmicHeller et al., 1996
    R3486.37b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Takhar et al., 1996
    L3496.38b†AWTC,Ag76%AgWT cAMP 10−7 M GLP-1Takhar et al., 1996
    A-3506.39b†ENDC,Ag<10%Ag5% cAMP 10−7 M GLP-1Takhar et al., 1996
    A-3506.39b†KWTC,Ag21%Ag2% cAMP 10−7 M GLP-1Takhar et al., 1996
    K3516.40b†AWTC,AgWTAgWT cAMP 10−7 M GLP-1Takhar et al., 1996
    T3536.42bANDC,Ant2230%E, NDAntΔLog τc = 0.84Wootten et al., 2013c
    H3636.52bA98M,AntND24%AntCoopman et al., 2011
    H3636.52bA23C,Ant459%E, 53%AntΔLog τc = 1.71Wootten et al., 2013c
    E3646.53bA58M,Ant1542%AntCoopman et al., 2011
    E3646.53bA25C,Ant51%E,AntΔLog τc = 0.66Wootten et al., 2016
    E3646.53bANDC,AgND6%cytYang et al., 2016
    E3646.53bQWTC,AgWT42%cytYang et al., 2016
    E3646.53bQ0.2C,AntWT42%cytYang et al., 2016
    E364/E387N/QNDC,AgNDWTcytYang et al., 2016
    E3646.53bYWTC,AgWTWTcytYang et al., 2016
    E3646.53bDWTC,AgWT53%cytYang et al., 2016
    F3676.56bANDC,Ag72WTcytYang et al., 2016
    F3676.56bA32C,Ant72WTcytYang et al., 2016
    F3676.56bINDC,Ag20WTcytYang et al., 2016
    F3676.56bH7C,Ag131WTcytYang et al., 2016
    M371ECL3AWT M,AntWT54%AntDods and Donnelly, 2015
    D372ECL3AWT M,Ant5913%AntDods and Donnelly, 2015
    E373ECL3AWTM,AntWT28%AntDods and Donnelly, 2015
    H374ECL3AWTM,AntWT22%AntDods and Donnelly, 2015
    H374ECL3AWTC,AgWTWTcytYang et al., 2016
    A-375ECL3A10M,AntWTWTAntDods and Donnelly, 2015
    R376ECL3GWTM,AntWTWTAntDods and Donnelly, 2015
    R376ECL3QWTC,AgWTWTcytYang et al., 2016
    G377ECL3AWTM,AntWT19%AntDods and Donnelly, 2015
    T3787.32bAWTM,AntWT12%AntDods and Donnelly, 2015
    L3797.33bR12C,Ag141WTAgMoon et al., 2015
    L3797.33bE11C,Ag165WTAgMoon et al., 2015
    L3797.33bAWTM,AntWT31%AntDods and Donnelly, 2015
    R3807.34bD21C,Ag1853WTAgMoon et al., 2015
    R3807.34bG4C,Ag40WTAgMoon et al., 2015
    R3807.34bA128M,Ant26368%AntDods and Donnelly, 2015
    R3807.34bQNDC,AgNDWTcytYang et al., 2016
    F3817.35bRWT C,AgWTWTAgMoon et al., 2015
    F3817.35bE>200C,Ag234WTAgMoon et al., 2015
    F3817.35bAWTM,AntWT16%AntDods and Donnelly, 2015
    F3817.35bSWTC,AgWTWTcytYang et al., 2016
    I3827.36bAWTM,AntWT23%AntDods and Donnelly, 2015
    K3837.37bAWTM,Ant56WTAntΔLog τc = 1.18Dods and Donnelly, 2015
    L3847.38bAWTM,AntWTWTAntDods and Donnelly, 2015
    L3847.38bANDC,Ag41WTcytYang et al., 2016
    L3847.38bA48C,Ant41WTcytYang et al., 2016
    L3847.38bVNDC,Ag16WTcytYang et al., 2016
    F3857.40bAWTM,AntWTWTAntDods and Donnelly, 2015
    T3867.41bAWTM,AntWT18%AntDods and Donnelly, 2015
    E3877.42bAWTM,AntWT43%AntΔLog τc = 0.52Dods and Donnelly, 2015
    E3877.42bAWTM,AntWTWTAntCoopman et al., 2011
    E3877.42bD13C,Ag10WTcytYang et al., 2016
    E3877.42bD12C,Ant10WTcytYang et al., 2016
    E3877.42bNWTC,AgWTWTcytYang et al., 2016
    L3887.43bANDC,Ag208WTcytYang et al., 2016
    L3887.43bI5C,Ag81WTcytYang et al., 2016
    L3887.43bFWTC,AgWTWTcytYang et al., 2016
    T3917.44bAWTM,AntWT64%M,AntCoopman et al., 2011
    T3917.44bAWTWTAgUnderwood et al., 2011
    S3927.47bAWTC,AntWTWTE, AntWootten et al., 2013c
    Q3947.49bAWTC,AntWTWTE, AntWootten et al., 2013c
    Q3947.49bNWTC,AntWTE,AntWootten et al., 2016
    M3977.52bLWTC,AgWTDong et al., 2012
    Y4027.57bANDC,Ant1021%E, NDAntΔLog τc = 1.59Wootten et al., 2013c
    C4037.58bA5.4Underwood et al., 2013
    N4067.61bAWTC,AntWTWTE,AntWootten et al., 2013c
    R421CTTQWTC,AntWT<50%EKoole et al., 2011
    C438 CTTAWTUnderwood et al., 2013
    C458 CTTAWTUnderwood et al., 2013
    C462 CTTAWTUnderwood 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.

  • TABLE 2

    Summary of traditional biologic assays for GLP-1 analogs

    Molecular/Cellular MeasurementDescription/OutcomeReferences
    Receptor-binding assays:
    Radioligand competitionDetermines 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 spectroscopiesDetermines binding affinity by measuring conformational changes of a receptor protein upon ligand binding.Runge et al., 2007
    Isothermal titration calorimetryDetermines 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 imagingDetermines equilibrium constants and dissociation rates by monitoring fluorescence-labeled single molecule on lipid bilayer.Fox et al., 2009; Myers et al., 2012
    Surface plasmon resonanceDetermines 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 labelingIdentifies 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 assaysHTRF 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
    RadioimmunoassayDetermines 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 assayDetermines 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 assayDetermines 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 ionDetermines 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 secretionDetermines 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

    AnalogDescriptionCompanyDosing RegimenStatus
    ExenatideExendin-4Amylin/Eli Lilly; AstraZenecaTwice dailyLaunched
    Exenatide once weeklyExendin-4Amylin/Alkermes/Eli Lilly; AstraZenecaOnce weeklyLaunched
    LixisenatideExendin-4 analogSanofi-AventisOnce dailyLaunched
    LiraglutideGLP-1 analog linked with a fatty acidNovo NordiskOnce dailyLaunched
    AlbiglutideGLP-1 analog fused to albuminGlaxoSmithKlineOnce weeklyLaunched
    DulaglutideGLP-1 analog fused to FcEli LillyOnce weeklyLaunched
    SemaglutideGLP-1 analog linked with a fatty acidNovo NordiskOnce weeklyPhase 3
    Semaglutide (NN9924)GLP-1 analog linked with a fatty di-acidNovo NordiskOralPhase 2
    TaspoglutideGLP-1 analog formulated for sustained releaseRocheOnce weeklySuspended
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Review ArticleReview Article

Glucagon-Like Peptide-1 and Its Receptors

Chris de Graaf, Dan Donnelly, Denise Wootten, Jesper Lau, Patrick M. Sexton, Laurence J. Miller, Jung-Mo Ahn, Jiayu Liao, Madeleine M. Fletcher, Dehua Yang, Alastair J. H. Brown, Caihong Zhou, Jiejie Deng and Ming-Wei Wang
Pharmacological Reviews October 1, 2016, 68 (4) 954-1013; DOI: https://doi.org/10.1124/pr.115.011395
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