Oxyntomodulin Differentially Affects Glucagon-Like Peptide-1 Receptor -Arrestin Recruitment and Signaling through G

The glucagon-like peptide (GLP)-1 receptor is a promising target for the treatment of type 2 diabetes and obesity, and there is great interest in characterizing the pharmacology of the GLP-1 receptor and its ligands. In the present report, we have applied bioluminescence resonance energy transfer assays to measure agonist-induced recruitment of arrestins and G-protein-coupled receptor kinase (GRK) 2 to the GLP-1 receptor in addition to traditional measurements of second messenger generation. The peptide hormone oxyntomodulin is described in the literature as a full agonist on the glucagon and GLP-1 receptors. Surprisingly, despite being full agonists in GLP-1 receptor-mediated cAMP accumulation, oxyntomodulin and glucagon were observed to be partial agonists in recruiting arrestins and GRK2 to the GLP-1 receptor. We suggest that oxyntomodulin and glucagon are biased ligands on the GLP-1 receptor. A great deal of the signaling molecules implicated in appetite regulation and energy homeostasis exert their effect through seven-transmembrane (7TM) G-protein-coupled receptors. One of the most interesting new targets in the management of diabetes and obesity is the 7TM glucagon-like peptide (GLP)-1 receptor (Holst, 2004; Davidson et al., 2005). GLP-1 is created through posttranslational processing of the proglucagon peptide, a processing that also creates the peptides GLP-2, glucagon, oxyntomodulin, and glicentin (Mayo et al., 2003; Sinclair and Drucker, 2005). The 37-amino acid residue oxyntomodulin peptide is an eight-residue C-terminally extended glucagon peptide that is cosecreted with GLP-1 from intestinal L-cells as a response to food intake (Mayo et al., 2003). In vitro data demonstrates that oxyntomodulin binds to and activates both the glucagon and GLP-1 receptor although with severely reduced affinity compared with the cognate agonists (Bataille et al., 1982; Baldissera et al., 1988; Gros et al., 1993; Schepp et al., 1996; Baggio et al., 2004). Oxyntomodulin is receiving an increasing amount of attention after in vivo data have demonstrated that it reduces food intake in mice, rats, and human subjects (Dakin et al., 2001, 2002, 2004; Cohen et al., 2003; Baggio et al., 2004; Wynne et al., 2005). We describe that oxyntomodulin and glucagon are full agonists in GLP-1 receptor-mediated cAMP accumulation but partial agonists in recruiting G-proteincoupled receptor kinase (GRK) 2, arr1, and arr2 to the receptor, suggesting that oxyntomodulin and glucagon are biased ligands on the GLP-1 receptor. Materials and Methods Molecular Biology and Peptides. Human arr2 N-terminally tagged with green fluorescent protein (GFP) and the Renilla luciferase (RLuc) cDNA were purchased from PerkinElmer Life and Analytical Sciences (Wellesley, MA). Human arr1 cDNA was purchased from Origene (Rockville, MD) and subcloned in a 3 position of GFP lacking the stop codon. Human GRK2 and GRK5 were cloned from a hypothalamic cDNA library. GRK2 lacking the stop codon was made by standard molecular biology techniques and inserted in a 5 position of GFP. Human GLP-1 receptor and rat glucagon receptor constructs lacking the stop codon were made by standard molecular biology techniques and subcloned in a 5 position of Renilla luciferase cDNA. Mutations (R393E and R395E) in arr2 were introduced in human GFParr2 using the QuikChange sitedirected mutagenesis kit (Stratagene, La Jolla, CA). All cDNA clones were verified by sequencing. All peptides were obtained from Bachem (Bubendorf, Switzerland). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.120006. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: 7TM, seven transmembrane; GLP, glucagon-like peptide; GRK, G-protein-coupled receptor kinase; GFP, green fluorescent protein; arr, -arrestin; RLuc, Renilla luciferase; BRET, bioluminescence resonance energy transfer; wt, wild type; PTH, parathyroid hormone; MAP, mitogen-activated protein. 0022-3565/07/3221-148–154$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 1 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 120006/3214707 JPET 322:148–154, 2007 Printed in U.S.A. 148 http://jpet.aspetjournals.org/content/suppl/2007/04/02/jpet.107.120006.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M ay 5, 2017 jpet.asjournals.org D ow nladed from Tissue Culture and cAMP Measurements. HEK293 and COS-7 cells were obtained from the European Collection of Animal Cell Cultures and maintained according to protocol. Agonist-induced cAMP accumulation was measured as described previously (Elling et al., 1999). Bioluminescence Resonance Energy Transfer Assays. We measured bioluminescence resonance energy transfer (BRET) by using the GFP blue-shifted variant of GFP. BRET measurement was done using a Mithras LB 940 plate reader (Berthold Technologies, Bad Wildbad, Germany). To enhance the BRET signal in the arr2 recruitment assay, a GFParr2(R393E;R395E) mutant was used (Vrecl et al., 2004) (except in Fig. 3). Receptor-RLuc cDNA was transiently transfected into HEK293 cells stably expressing GFParr2(R393E;R395E) (hereafter referred to as arr2) for measurements of arr2 recruitment or transiently cotransfected with GFParr1 or GRK2-GFP into HEK293 cells for measurements of arr1 or GRK2 recruitment, respectively. The commercial use of the arrestin2(R393E;R395E) mutant in BRET assays requires a license from 7TM Pharma. To potentiate the BRET signal as described previously (Jorgensen et al., 2005), human GRK5 cDNA was cotransfected in a receptor-RLuc/GRK5 cDNA ratio of 1:1 in arr recruitment assays (except in Fig. 3). The reading time was 10 min after agonist addition for arr recruitment and 5 min for GRK2 recruitment. For measurements of the antagonistic effect of oxyntomodulin or exendin(9–39), the antagonist was added 2 min before the agonist (10 nM GLP-1). For the kinetic study, variable reading times were used, and agonist was added with an injector. The background signal from RLuc was determined by coexpressing the RLuc construct with empty vector, and the BRET ratio generated from this transfection was subtracted from all other BRET ratios. Receptor Internalization Assay. GLP-1 receptor internalization assay was based on a protocol described previously (Vrecl et al., 1998). In brief, HEK293 cells transiently transfected with GLP-1 receptor cDNA were first incubated in serum-free Hepes-modified Dulbecco’s modified Eagle’s medium containing 0.1% bovine serum albumin, pH 7.4, before GLP-1 receptor internalization was induced by 0.1 M GLP-1 or 10 M oxyntomodulin at 37°C for time intervals ranging from 2 min to 1 h. Stimulation was stopped by washing the cells with ice-cold phosphate-buffered saline followed by a 6-min acid wash (50 mM acetic acid and 150 mM NaCl, pH 2.8) to remove surface-bound ligand. Cells were then subjected to I-exendin(9– 39) (PerkinElmer Life and Analytical Sciences) binding at 4°C for 3 h, and the radioactivity was measured. The time-dependent loss of the surface GLP-1 receptor was determined relative to unstimulated cells. Nonspecific binding for each time point was determined in the presence of 1 M GLP-1. Data Analysis. Data were analyzed using Prism (GraphPad Software Inc., San Diego, CA).