Dose-dependent learning impairment induced by allatostatins.

<p>Percentages of forager bees displaying conditioned proboscis extension responses (PER) in the third and last trial of conditioning, in honeybees injected with either vehicle alone (control bees, CT) or a given concentration of allatostatin A (A), C (B), or CC (C). For easier comparisons, <b>l</b>earning performances of control bees are set at 100% and those of all other groups are relative to CT. N = 120–182 (A), N = 70–80 (B) N = 56–87 (C). Significant decreases of learning performance, as compared to those of saline-injected bees, are indicated (*: p<0.05, **:p<0.01, ***:p<0.001).</p

Aminoacid sequence alignment of Apime-ASTC-R and its <i>Drosophila</i> and human homologs.

<p>Sequence alignments of the predicted allatostatin C receptor in the honey bee <i>Apis mellifera</i> (<i>Apime-ASTC-R</i>, GenBank: XP_006560939.1) and of its homologs in the fruitfly <i>Drosophila melanogaster</i> (<i>Drostar1</i>, GenBank: NP_649040.2 and <i>Drostar2</i>, GenBank: NP_649039.4), and human (SSTR2, NP_001041.1). The amino acid position is indicated on the left. Conserved residues are in red, and conservative changes in blue. Grey bars indicate putative trans-membrane regions (TM1–TM7). Amino acids that are characteristic of class A GPCRs are indicated by *, open diamonds (◊) indicate putative N-linked glycolysation sites, # indicate cysteine residues for disulfide bridge (between TM2—TM3 and TM4 –TM5) or palmitoylation (intracellular domain), and dots (●) indicate conserved putative phosphorylation sites for PKA/C. Sequences are based on transcripts (cDNA).</p

Apime-ASTA-R and Apime-ASTC-R display constitutive inhibitory activity, down-regulating cAMP.

<p>HEK cells transfected with Apime-ASTA-R or Apime-ASTC-R show lower levels of bioluminescence than mock-transfected cells (A). Bars represents mean+/-SD values of 8 transfections in duplicates (mock) or triplicates (Apime-AST-R). Forskolin analog NKH-477 has a reduced ability to increase bioluminescence levels in Apime-ASTA-R and Apime-ASTC-R transfected cells than in mock-transfected cells (B). Bars represent mean +/- SD values of 2 transfections in duplicates or triplicates. Application of increasing doses of Apime-ASTA on Apime-ASTA-R transfected cells induced a dose-dependent increase in bioluminescence (C). Application of increasing doses of Apime-ASTC on Apime-ASTC-R transfected cells did not change bioluminescence levels (D). Application of increasing doses of Apime-ASTCC on Apime-ASTC-R transfected cells increased bioluminescence levels at high concentrations (E). HEK cells transfected with luciferase reporter gene and an empty vector (mock-transfected) show a dose-dependent response to Apime-ASTCC (F). Bars represent mean +/- SD values of 3 transfections in triplicates.</p

Binding of radiolabelled Apime-ASTA <i>in vivo</i> and <i>in vitro</i>.

<p>¹²⁵I-Apime-ASTA binding to bee brain crude membranes was displaced by increasing concentrations of non-labeled Apime-ASTA (A). Competitive binding with Apime-ASTA and increasing concentrations of a somatostatin homolog (SOM230) (B). Bars represent mean+/-SD (n = 3). Curves were obtained by fitting the data using nonlinear regression analysis in the GraphPad Prism software.</p

Intact response to sucrose and odors after allatostatin treatment.

<p>Responses elicited by stimulation of the antennae with increasing sucrose concentrations were tested following injection of saline (controls, <i>CT</i>) or either allatostatin (A). Overall, bees responded more to sucrose (solid line) than water (dashed line, GLM: 4.11±0.58, p<0.001), and the proportion of bees responding to sucrose increased with the concentration (GLM: 0.82±0.14, p<0.001) similarly in all groups, while the proportion of bees responding to water remained constant (GLM: -0.11±0.12, p = 0.34) (CT: N = 31, ASTA: N = 35 ASTC: N = 49, ASTCC: N = 29). Following olfactory appetitive conditioning, bees were tested for their capacity to generalize their learned responses to other odorants, as a way to assess their olfactory capacities (B). Allatostatin treatment had no impact on the response to any of the odors tested (CT: N = 73, ASTA: N = 66, ASTC: N = 75, ASTCC: N = 72). All bees were collected as they were leaving the hive, as for learning experiments.</p

Phylogenetic tree of <i>A</i>. <i>mellifera</i> and <i>D</i>. <i>melanogaster</i> allatostatin receptors with respect to their closest mouse homologs.

<p><i>Drosophila</i> gene names are indicated. For <i>M</i>. <i>musculus</i> receptors, abbreviations are: GALR (Galanin receptor), KISS1R (kisspeptin receptor), OPR (opioid receptor), MCHR (melanin concentrating hormone receptor), NPBWR (Neuropeptide B and W receptor), SSTR (somatostatin receptor). The bottom bar indicates the phylogenetic distance scale. The bootstrap values are displayed in red.</p

Aminoacid sequence alignment of Apime-ASTA-R and its <i>Drosophila</i> and human homologs.

<p>Sequence alignments of the predicted allatostatin A receptor in the honey bee <i>Apis mellifera</i> (Apime-ASTA-R, GenBank: XP_006560262.1) and of its homologs in the fruit fly <i>Drosophila melanogaster</i> (DAR1, GenBank: NP_726877.2 and <i>DAR2</i>, GenBank: NP_524544.1) and human (GALR1, NP_001471.2). The amino acid position is indicated on the left. Conserved residues are in red, and conservative changes in blue. Vertical bars (|) indicate locations of putative introns; grey bars indicate putative trans-membrane regions (TM1–TM7). Amino acids that are characteristic of class A GPCRs are indicated by *, open diamonds (◊) indicate putative N-linked glycolysation sites, # indicate cysteine residues for disulfide bridge (between TM2—TM3 and TM4 –TM5) or palmitoylation (intracellular domain), and dots (●) indicate conserved putative phosphorylation sites for PKA/C. Sequences are based on transcripts (cDNA).</p

Gene expression of allatostatins.

<p>PCR was performed on cDNA samples obtained from different brain regions of adult worker bees: optic lobes (yellow in the bee brain illustration), ventral area (including antennal lobes in blue) and dorsal area (including mushroom bodies in red) of the central brain. (A) Electrophoresis on agarose gel of PCR fragments of <i>Actin</i> (used as a positive control 162 nt), <i>Apime-AstA</i> (120 bp), <i>Apime-AstC</i> (121 bp) and <i>Apime-AstCC</i> (105 bp, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146248#pone.0146248.s002" target="_blank">S1 Table</a> for primer sequences, original agarose gels are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146248#pone.0146248.s001" target="_blank">S1 Fig</a>). (B) qPCR was performed on dissected dorsal brain region from pollen foragers (n = 9, each sample run in triplicate) for the three allatostatin genes. Expression levels are normalized and relative to the geometric mean of <i>Am18S</i> and <i>elf1α</i>.</p

Targeting a G‑Protein-Coupled Receptor Overexpressed in Endocrine Tumors by Magnetic Nanoparticles To Induce Cell Death

Nanotherapy using targeted magnetic nanoparticles grafted with peptidic ligands of receptors overexpressed in cancers is a promising therapeutic strategy. However, nanoconjugation of peptides can dramatically affect their properties with respect to receptor recognition, mechanism of internalization, intracellular trafficking, and fate. Furthermore, investigations are needed to better understand the mechanism whereby application of an alternating magnetic field to cells containing targeted nanoparticles induces cell death. Here, we designed a nanoplatform (termed MG-IONP-DY647) composed of an iron oxide nanocrystal decorated with a ligand of a G-protein coupled receptor, the cholecystokinin-2 receptor (CCK2R) that is overexpressed in several malignant cancers. MG-IONP-DY647 did not stimulate inflammasome of Raw 264.7 macrophages. They recognized cells expressing CCK2R with a high specificity, subsequently internalized <i>via</i> a mechanism involving recruitment of β-arrestins, clathrin-coated pits, and dynamin and were directed to lysosomes. Binding and internalization of MG-IONP-DY647 were dependent on the density of the ligand at the nanoparticle surface and were slowed down relative to free ligand. Trafficking of CCK2R internalized with the nanoparticles was slightly modified relative to CCK2R internalized in response to free ligand. Application of an alternating magnetic field to cells containing MG-IONP-DY647 induced apoptosis and cell death through a lysosomal death pathway, demonstrating that cell death is triggered even though nanoparticles of low thermal power are internalized in minute amounts by the cells. Together with pioneer findings using iron oxide nanoparticles targeting tumoral cells expressing epidermal growth factor receptor, these data represent a solid basis for future studies aiming at establishing the proof-of-concept of nanotherapy of cancers using ligand-grafted magnetic nanoparticles specifically internalized <i>via</i> cell surface receptors

Distinct CCK‑2 Receptor Conformations Associated with β‑Arrestin‑2 Recruitment or Phospholipase‑C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Nearly 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or β-arrestin-dependent signaling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2R^G) or recruiting β-arrestin2 (CCK2R^β) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150013X) acted as a high affinity competitive antagonist on CCK2R^G but was nearly inefficient as inhibitor of CCK2R^β. Several structural elements on both GV150013X and in CCK2R binding cavity, which hinder binding of GV150013X only to the CCK2R^β were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulfur–aromatic interaction was shown to be crucial for selective stabilization of the CCK2R^β state. These data establish structural evidence for distinct conformations of a 7TMR associated with β-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs