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

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ABSTRACIAn open ps exchange system was used to monitor the nonsteady state and steady state changes in nitrogenase activity (H2 evolution in N2:02 and Ar:02) and respiration (CO2 evolution) in attached, excised, and sliced nodules of soybean (Glycine max L. Menf.) exposed to extenal P02 of 5 to 100%. In attached nodules, increases in external P02 in steps of 10 or 20% resulted in sharp declines in the rates of H2 and CO2 evolution. Recovery been proposed (18). Evidence for a distinct barrier to gas diffusion in the nodule cortex has been obtained experimentally by direct measurements of P02 in the outer and central tissues of soybean nodules (2 1).The exact nature of the diffusion barrier and the manner in which it is regulated are unknown, but it has been suggested that the nodules of many symbiotic associations increase their diffusion resistance to O2 entry when they are exposed to an atmosphere containing 10% C2H2, or one in which N2 is replaced by Ar (25). This increase in diffusion resistance is apparent as a decline in nodule respiration rate, with a concomitant decline in C2H2 reduction or H2 production rate, during the first 30 min of exposure to C2H2 or Ar. The presence or absence of an C2H2-or Ar-induced decline has been correlated with the optimum P02 for N2 fixation within a specific legume-Rhizobium association and, by inference, with the speed with which the diffusion barrier is regulated in that association (25). It has also been suggested that the regulation of the diffusion resistance of the nodule requires physical changes in the diffusion barrier (4), and this may limit the speed with which the nodule responds to changes in its gaseous environment.The aims of this study were (a) to determine whether the gas exchange characteristics of soybean nodules following changes in external P02 are consistent with the presence of a variable diffusion barrier in the nodule cortex, and (b) if so, to identify experimental conditions which could be used to vary this diffusion barrier. An open circuit gas exchange system was used to monitor continuously changes in respiration (CO2 evolution) and nitrogenase activity (H2 evolution in N2:02 and Ar:02) as the P02 surrounding the nodules was varied. Nonsteady state measurements of CO2 production and H2 evolution were used to determine the speed with which the nodules adjust to changes in rhizosphere O2 concentration. Steady

The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4-and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.