Background The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function. Methodology/Principal Findings Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G7]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G7 into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G7]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes. Conclusions These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target.
References
[1]
Kim W, Egan JM (2008) The role of incretins in glucose homeostasis and diabetes treatment. Pharmacological reviews 60: 470–512.
[2]
Renner S, Fehlings C, Herbach N, Hofmann A, von Waldthausen DC, et al. (2010) Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function. Diabetes 59: 1228–1238.
[3]
Lovshin JA, Drucker DJ (2009) Incretin-based therapies for type 2 diabetes mellitus. Nat Rev Endocrinol 5: 262–269.
[4]
Tharakan G, Tan T, Bloom S (2011) Emerging therapies in the treatment of ‘diabesity’: beyond GLP-1. Trends in pharmacological sciences 32: 8–15.
[5]
Ahren B (2009) Islet G protein-coupled receptors as potential targets for treatment of type 2 diabetes. Nature reviews Drug discovery 8: 369–385.
[6]
Drucker DJ, Dritselis A, Kirkpatrick P (2010) Liraglutide. Nature reviews Drug discovery 9: 267–268.
[7]
Widenmaier SB, Kim SJ, Yang GK, De Los Reyes T, Nian C, et al. (2010) A GIP receptor agonist exhibits beta-cell anti-apoptotic actions in rat models of diabetes resulting in improved beta-cell function and glycemic control. PloS one 5: e9590.
[8]
Pratley RE (2010) GIP: an inconsequential incretin or not? Diabetes care 33: 1691–1692.
[9]
Irwin N, O'Harte FP, Gault VA, Green BD, Greer B, et al. (2006) GIP(Lys16PAL) and GIP(Lys37PAL): novel long-acting acylated analogues of glucose-dependent insulinotropic polypeptide with improved antidiabetic potential. Journal of medicinal chemistry 49: 1047–1054.
[10]
Kulkarni RN (2010) GIP: no longer the neglected incretin twin? Science translational medicine 2: 49ps47.
[11]
Parthier C, Kleinschmidt M, Neumann P, Rudolph R, Manhart S, et al. (2007) Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor. Proceedings of the National Academy of Sciences of the United States of America 104: 13942–13947.
[12]
Runge S, Thogersen H, Madsen K, Lau J, Rudolph R (2008) Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain. The Journal of biological chemistry 283: 11340–11347.
[13]
Hoare SR (2005) Mechanisms of peptide and nonpeptide ligand binding to Class B G-protein-coupled receptors. Drug discovery today 10: 417–427.
[14]
Choi C, Fortin JP, McCarthy E, Oksman L, Kopin AS, et al. (2009) Cellular dissection of circadian peptide signals with genetically encoded membrane-tethered ligands. Current biology : CB 19: 1167–1175.
[15]
Fortin JP, Zhu Y, Choi C, Beinborn M, Nitabach MN, et al. (2009) Membrane-tethered ligands are effective probes for exploring class B1 G protein-coupled receptor function. Proceedings of the National Academy of Sciences of the United States of America 106: 8049–8054.
[16]
Neumann JM, Couvineau A, Murail S, Lacapere JJ, Jamin N, et al. (2008) Class-B GPCR activation: is ligand helix-capping the key? Trends in biochemical sciences 33: 314–319.
[17]
Parthier C, Reedtz-Runge S, Rudolph R, Stubbs MT (2009) Passing the baton in class B GPCRs: peptide hormone activation via helix induction? Trends in biochemical sciences 34: 303–310.
[18]
Fortin JP, Schroeder JC, Zhu Y, Beinborn M, Kopin AS (2010) Pharmacological characterization of human incretin receptor missense variants. The Journal of pharmacology and experimental therapeutics 332: 274–280.
[19]
Tseng CC, Zhang XY (1998) The cysteine of the cytoplasmic tail of glucose-dependent insulinotropic peptide receptor mediates its chronic desensitization and down-regulation. Molecular and cellular endocrinology 139: 179–186.
[20]
Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nature reviews Molecular cell biology 10: 513–525.
[21]
Schwarzmann G, von Coburg A, Mobius W (2000) Using biotinylated gangliosides to study their distribution and traffic in cells by immunoelectron microscopy. Methods in enzymology 312: 534–562.
[22]
Summerhill S, Stroud T, Nagendra R, Perros-Huguet C, Trevethick M (2008) A cell-based assay to assess the persistence of action of agonists acting at recombinant human beta(2) adrenoceptors. Journal of pharmacological and toxicological methods 58: 189–197.
[23]
Aurora R, Rose GD (1998) Helix capping. Protein science : a publication of the Protein Society 7: 21–38.
[24]
Inooka H, Ohtaki T, Kitahara O, Ikegami T, Endo S, et al. (2001) Conformation of a peptide ligand bound to its G-protein coupled receptor. Nature structural biology 8: 161–165.
[25]
Doan ND, Bourgault S, Dejda A, Letourneau M, Detheux M, et al. (2011) Design and in vitro characterization of PAC1/VPAC1-selective agonists with potent neuroprotective effects. Biochemical pharmacology 81: 552–561.
[26]
Bourgault S, Vaudry D, Segalas-Milazzo I, Guilhaudis L, Couvineau A, et al. (2009) Molecular and conformational determinants of pituitary adenylate cyclase-activating polypeptide (PACAP) for activation of the PAC1 receptor. Journal of medicinal chemistry 52: 3308–3316.
[27]
Tseng CC, Zhang XY (2000) Role of G protein-coupled receptor kinases in glucose-dependent insulinotropic polypeptide receptor signaling. Endocrinology 141: 947–952.
[28]
Molinari P, Vezzi V, Sbraccia M, Gro C, Riitano D, et al. (2010) Morphine-like opiates selectively antagonize receptor-arrestin interactions. The Journal of biological chemistry 285: 12522–12535.
[29]
Richardson RM, Marjoram RJ, Barak LS, Snyderman R (2003) Role of the cytoplasmic tails of CXCR1 and CXCR2 in mediating leukocyte migration, activation, and regulation. Journal of immunology 170: 2904–2911.
[30]
Estall JL, Koehler JA, Yusta B, Drucker DJ (2005) The glucagon-like peptide-2 receptor C terminus modulates beta-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation. The Journal of biological chemistry 280: 22124–22134.
[31]
Widmann C, Dolci W, Thorens B (1997) Internalization and homologous desensitization of the GLP-1 receptor depend on phosphorylation of the receptor carboxyl tail at the same three sites. Molecular endocrinology 11: 1094–1102.
[32]
Leterrier C, Bonnard D, Carrel D, Rossier J, Lenkei Z (2004) Constitutive endocytic cycle of the CB1 cannabinoid receptor. The Journal of biological chemistry 279: 36013–36021.
[33]
Conn PM, Ulloa-Aguirre A, Ito J, Janovick JA (2007) G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacological reviews 59: 225–250.
[34]
Waehler R, Russell SJ, Curiel DT (2007) Engineering targeted viral vectors for gene therapy. Nature reviews Genetics 8: 573–587.
[35]
Vauquelin G, Charlton SJ (2010) Long-lasting target binding and rebinding as mechanisms to prolong in vivo drug action. British journal of pharmacology 161: 488–508.
[36]
Vauquelin G, Packeu A (2009) Ligands, their receptors and … plasma membranes. Molecular and cellular endocrinology 311: 1–10.
[37]
Clark RB, Allal C, Friedman J, Johnson M, Barber R (1996) Stable activation and desensitization of beta 2-adrenergic receptor stimulation of adenylyl cyclase by salmeterol: evidence for quasi-irreversible binding to an exosite. Molecular pharmacology 49: 182–189.
[38]
Russell-Jones D (2009) Molecular, pharmacological and clinical aspects of liraglutide, a once-daily human GLP-1 analogue. Molecular and cellular endocrinology 297: 137–140.
[39]
Knudsen LB, Nielsen PF, Huusfeldt PO, Johansen NL, Madsen K, et al. (2000) Potent derivatives of glucagon-like peptide-1 with pharmacokinetic properties suitable for once daily administration. Journal of medicinal chemistry 43: 1664–1669.
[40]
Romano R, Bayerl TM, Moroder L (1993) Lipophilic derivatization and its effect on the interaction of cholecystokinin (CCK) nonapeptide with phospholipids. Biochimica et biophysica acta 1151: 111–119.
[41]
Day JW, Ottaway N, Patterson JT, Gelfanov V, Smiley D, et al. (2009) A new glucagon and GLP-1 co-agonist eliminates obesity in rodents. Nature chemical biology 5: 749–757.
[42]
Pocai A, Carrington PE, Adams JR, Wright M, Eiermann G, et al. (2009) Glucagon-like peptide 1/glucagon receptor dual agonism reverses obesity in mice. Diabetes 58: 2258–2266.
[43]
Gault VA, Kerr BD, Harriott P, Flatt P (2011) Administration of an acylated GLP-1 and GIP preparation provides added beneficial glucose-lowering and insulinotropic actions over single incretins in mice with type 2 diabetes and obesity. Clinical science.
[44]
Tibaduiza EC, Chen C, Beinborn M (2001) A small molecule ligand of the glucagon-like peptide 1 receptor targets its amino-terminal hormone binding domain. The Journal of biological chemistry 276: 37787–37793.
[45]
Beinborn M, Lee YM, McBride EW, Quinn SM, Kopin AS (1993) A single amino acid of the cholecystokinin-B/gastrin receptor determines specificity for non-peptide antagonists. Nature 362: 348–350.
