Short lipidated peptide sequences derived from various intracellular loop regions of G protein-coupled receptors (GPCRs) are named pepducins and act as allosteric modulators of a number of GPCRs. Recently, a pepducin selectively targeting the C-X-C chemokine receptor type 4 (CXCR4) was found to be an allosteric agonist, active in both cell-based assays and in vivo. However, the precise mechanism of action of this class of ligands remains poorly understood. In particular, given the diversity of signaling effectors that can be engaged by a given receptor, it is not clear whether pepducins can show biased signaling leading to functional selectivity. To explore the ligand-biased potential of pepducins, we assessed the effect of the CXCR4 selective pepducin, ATI-2341, on the ability of the receptor to engage the inhibitory G proteins (Gi1, Gi2 and Gi3), G13, and β-arrestins. Using bioluminescence resonance energy transfer-based biosensors, we found that, in contrast to the natural CXCR4 ligand, stromal cell-derived factor-1α, which promotes the engagement of the three Gi subtypes, G13 and the two β-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the β-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and β-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward β-arrestins results from the reduced ability of the pepducin to promote GPCR kinase-mediated phosphorylation of the receptor. In addition to revealing ligand-biased signaling of pepducins, these findings shed some light on the mechanism of action of a unique class of allosteric regulators.BRET | lipid-anchored peptide | beta-arrestin | protein-protein interaction | cell signaling

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the ␤-cells is of major importance. In ␤-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either G␣ s /cAMP/cAMP-dependent protein kinase (PKA) or ␤-arrestin 1, a scaffold protein. Using pharmacological inhibitors, ␤-arrestin 1 small interfering RNA, and islets isolated from ␤-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the ␤-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the ␤-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the ␤-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. ␤-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in ␤-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a ␤-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of ␤-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.GLP-1 (glucagon-like peptide-1), produced by post-translational processing of the proglucagon in enteroendocrine L-cells, is a potent gluco-regulatory peptide hormone. GLP-1 is released into the blood stream in response to nutrient ingestion, such as carbohydrates, amino acids, and fats, during the early postprandial period (1, 2). A major target for GLP-1 actions is the pancreatic ␤-cell. One of the main physiological roles of this endocrine hormone is to enhance insulin secretion in a glucose-dependent manner (1-5). Besides its insulinotropic action, GLP-1 also favors the maintenance of a correct ␤-cell glucose sensing, regulates transcriptional synthesis, induces ␤-cell proliferation, and is protective against apoptosis (6 -9). Strategies based on activating GLP-1 receptor 3 are intensively developed for the treatment of type 2 diabetes and studies aiming at a better and exhaustive understanding of GLP-1 actions within the ␤-cells are of great importance (1-5).GLP-1 exerts its intracellular effects through binding to its specific receptor that spans the plasma membrane. The GLP-1R belongs to the class II (or B) secretin/glucagon/vasoactive intestinal peptide superfamily of heptahelical transmembrane G protein-coupled receptors (GPCRs) (10, 11). The GLP-1R is positively coupled to adenylate cyclase, through G␣ s -containing heterotrimeric G-prot...