Elucidating agonist-induced signaling patterns of human G protein-coupled receptor GPR17 and uncovering pranlukast as a biased mixed agonist-antagonist at GPR17

The progress of human genome sequencing has revealed the existence of several hundred orphan G protein-coupled receptors (GPCRs), whose endogenous ligands are not yet identified, thus their deorphanization and characterization is fundamental in order to clarify their physiological and pathological role as well as their relevance as new drug targets. Recently, the orphan GPCR GPR17 that is phylogenetically and structurally related to the known P2Y and CysLT receptors has been identified as a dual uracil nucleotide/cysteinyl-leukotriene receptor. In spite of this, these deorphanization efforts could not be verified yet by independent laboratories, thus this classification remains a controversial matter and GPR17 most likely still represents an orphan GPCR. Additionally, a subsequent study revealed a ligand-independent regulatory role for GPR17 suppressing CysLT1 receptor function via GPCR-GPCR interactions. By means of a high throughput pharmacogenomic approach our group has identified a small molecule agonist for GPR17 that is used as pharmacological tool for characterization of ligand-dependent behaviors triggered by this receptor. As a consequence, in the present thesis, evidence is provided that GPR17 does not lack the common features of GPCR signaling. Upon agonist challenge GPR17 induces signaling via promiscuous G protein-coupling (Gαi/o, Gαq and Gαs) in an agonist-concentration-dependent manner, as determined by means of traditional second messenger assays (cAMP and IP1) and by the label-free dynamic mass redistribution (DMR) technology in two different cellular backgrounds (CHO and HEK293 cells) engineered to stably express the short isoform of human GPR17. The use of ELISA and immunofluorescence techniques revealed that activation of GPR17 with the agonist is also linked with a time-dependent reduction of cell surface expression. Furthermore, GPR17 recruits β-arrestin2 upon ligand-stimulation in a G protein-dependent and –independent manner as monitored by use of bioluminescence resonance energy transfer (BRET2) analyses. The GPR17-mediated signaling can be efficiently abrogated by the CysLT1 antagonist pranlukast in a non-competitive mode of action, but not by montelukast, zafirlukast and MK571, as investigated by use of DMR analyses, traditional second messenger assays and BRET2 approach. Additionally, evidence is provided that pranlukast acts as a mixed agonist/antagonist at GPR17, differentially modulating individual signaling pathways. Furthermore, it is demonstrated that β-arrestin2, known as scaffolding protein involved in desensitization and trafficking processes of GPCRs, exhibits the capability to fine-tune G protein signaling specifity of GPR17 via shifting the preference to the most preferred G protein subunit (here Gαi/o), a phenomenon that has not been described before.Aufdeckung der Agonist-induzierten Signalwege des humanen G Protein-gekoppelten Rezeptors GPR17 und Charakterisierung von Pranlukast als signalwegspezifischer gemischter Agonist- AntagonistDie Sequenzierungsfortschritte des menschlichen Erbgutes offenbarten die Existenz von mehreren hundert orphanen G Protein-gekoppelten Rezeptoren (GPCRs), dessen endogene Liganden noch nicht identifiziert wurden. Ihre Deorphanisierung sowie Charakterisierung sind elementar um die physiologische und pathologische Rolle aufzuklären und sie können als neue potenzielle Arzneimittel Targets betrachtet werden. Kürzlich ist der orphane GPCR GPR17, der phylogenetisch und strukturell mit P2Y und CysLT Rezeptoren verwandt ist, als ein Uracil Nukleotid/Cysteinyl-Leukotrien Rezeptor identifiziert worden. Diese Deorphanisierung konnte jedoch noch nicht durch unabhängige Labore verifiziert werden, somit bleibt diese Klassifikation eine umstrittene Postulierung und GPR17 muss weiterhin als orphaner GPCR betrachtet werden. Weiterhin zeigte eine nachfolgende Studie eine Ligand-unabhängige Funktion für GPR17 über GPCR-GPCR Wechselwirkungen als negativer Regulator der CysLT1 Rezeptor-vermittelten Signalwegsaktivierung. Dennoch hat unsere Arbeitsgruppe einen synthetischen GPR17-Agonisten identifiziert, der als pharmakologisches Werkzeug für die Charakterisierung von Ligand-induzierten Rezeptor-nachgeschalteten Ereignissen verwendet wird. Entgegen der bisher postulierten Thesen wird in der gegenwärtigen Arbeit gezeigt, dass GPR17 allgemeine Eigenschaften der GPCR-Signaltransduktion aufweist. Agonist-Stimulierung des GPR17 induziert Signaltransduktion über Kopplung mit promiskuitiven G Proteinen (Gαi/o, Gαq und Gαs) in einer konzentrationsabhängigen Weise, wie mittels der klassischen funktionellen GPCR Analysen (cAMP und IP1) und durch die Technologie der dynamischen Massenumverteilung (DMR) gezeigt werden konnte, unter Verwendung von zwei verschiedenen rekombinanten Zelllinien (CHO und HEK293), welche die kurze Isoform des humanen GPR17 stabil exprimieren. Der Gebrauch von ELISA und Immunfluoreszenz Techniken offenbarte, dass die Aktivierung von GPR17 mit dem Agonisten auch mit einer zeitabhängigen Verminderung des Zelloberflächenexpression verbunden ist. Außerdem wird unter Verwendung der Biolumineszenz Resonanz Energietransfer (BRET2) Methode gezeigt, dass GPR17 β-Arrestin2 nach Ligand-Stimulierung rekrutiert, und zwar in einer G Proteinabhängigen als auch -unabhängigen Weise. Die GPR17-vermittelte Signaltransduktion kann vom CysLT1 Antagonisten Pranlukast in einer nicht-kompetitiven Weise gehemmt werden, aber nicht durch Montelukast, Zafirlukast und MK571. Weiterhin wird gezeigt, dass Pranlukast als ein gemischter Agonist/Antagonist am GPR17 wirkt, der partial und funktionell selektiv die Gαi/overmittelte Signalwegstransduktion aktiviert. Weiterhin wird dargelegt, dass β-Arrestin2, bekannt als Gerüst-Protein, das an der Desensibilisierung und Internalisierung von GPCRs beteiligt ist, die Fähigkeit besitzt die gemischte G Protein-Kopplung von GPR17 zu modulieren, und zwar über die Verschiebung der Kopplungspräferenzen zur bevorzugtesten G Protein-Untereinheit (hier Gαi/o). Dies stellt ein Phänomen dar, das zuvor so nicht beschrieben wurde

A Cell-Permeable Inhibitor to Trap Gαq Proteins in the Empty Pocket Conformation

In spite of the crucial role of heterotrimeric G proteins as molecular switches transmitting signals from G protein-coupled receptors, their selective manipulation with small molecule, cell-permeable inhibitors still remains an unmet challenge. Here, we report that the small molecule BIM-46187, previously classified as pan-G protein inhibitor, preferentially silences Gαq signaling in a cellular context-dependent manner. Investigations into its mode of action reveal that BIM traps Gαq in the empty pocket conformation by permitting GDP exit but interdicting GTP entry, a molecular mechanism not yet assigned to any other small molecule Gα inhibitor to date. Our data show that Gα proteins may be “frozen” pharmacologically in an intermediate conformation along their activation pathway and propose a pharmacological strategy to specifically silence Gα subclasses with cell-permeable inhibitors

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms

Discovery of a subnanomolar and selective spirocyclic agonist of the glucocorticoid receptor

International audienc

Pullulanase and Starch Synthase III Are Associated with Formation of Vitreous Endosperm in Quality Protein Maize.

The opaque-2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called "Quality Protein Maize" (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, we focused on analysis of two starch biosynthetic enzymes that may influence kernel vitreousness. Analysis of recombinant inbred lines derived from a cross of W64Ao2 and K0326Y revealed that pullulanase activity had significant positive correlation with kernel vitreousness. We also found that decreased Starch Synthase III abundance may decrease the pullulanase activity and average glucan chain length given the same Zpu1 genotype. Therefore, Starch Synthase III could indirectly influence the kernel vitreousness by affecting pullulanase activity and coordinating with pullulanase to alter the glucan chain length distribution of amylopectin, resulting in different starch structural properties. The glucan chain length distribution had strong positive correlation with the polydispersity index of glucan chains, which was positively associated with the kernel vitreousness based on nonlinear regression analysis. Therefore, we propose that pullulanase and Starch Synthase III are two important factors responsible for the formation of the vitreous phenotype of QPM endosperms

Thermal properties of starch.

<p>(A-C) Comparison of starch thermal properties, including onset temperature, maximum temperature and enthalpy, between W64A<i>o2</i> and K0326Y. Significant level indicated by asterisks at p<0.05 by two-tailed <i>t</i> test (D-F) Comparison of the starch thermal properties among RILs. For parental lines, each column represents mean thermal property values of three independent ears of corresponding lines; and for RILs, each column represents mean thermal property values of all RILs of the corresponding genotype with three independent ears for each individual RIL. The letters above each column represent statistically significant differences among the lines for <i>p</i><0.05 by pairwise two-tailed t-test. Columns sharing the same letter are not significantly different from one another. The error bars represent standard error.</p

Pullulanase activity and SSIII abundance in parental lines and RILs.

<p>(A) Pullulanase activity of W64A+, W64A<i>o2</i>,K0326Y and RILs with four Zpu1-SSIII genotypes. Pullulanase activity was measured by the spectrophotometer at 490 nm absorbance (y-axis). For the first three columns, each represents mean pullulanase activity of three independent ears of the corresponding lines; and for the remaining four columns, each represents mean pullulanase activity of all RILs with the corresponding genotype from three independent ears of each individual RIL. The letters above each column represent statistically significant differences among the lines with <i>p</i><0.05 by pairwise two-tailed t-test. Columns with the same letter are not significantly different from one another. The error bars represent standard error. (B) Positive correlation between pullulanase activity and kernel vitreousness. The significance level of the correlation was tested by ANOVA of the slope at <i>p</i><0.05. Each data point represents mean pullulanase activity of three independent ears of each individual RIL. (C) SSIII abundance and SSIII activity of W64A+, W64A<i>o2</i>, K0326Y and <i>du</i>1M4. SSIII abundance was tested by SDS-PAGE followed by Western blot (Images of the whole blots and SDS-PAGE gels are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s004" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s006" target="_blank">S6</a> Figs). SSIII activity was tested by native PAGE followed by a zymogram for the RILs. (Image of whole zymogram gel is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s004" target="_blank">S4 Fig</a>) (D) Western blot of SSIII among RILs homologous for W64Ao2 (W) or QPM (Q)–derived <i>Zpu1</i> or <i>SSIII</i> alleles. The order of samples corresponded to their kernel vitreousness, from most opaque to most vitreous (left to right; images of whole blots and SDS-PAGE gels are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s005" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s007" target="_blank">S7</a> Figs).</p

Sequence analysis of maize Zpu1 and <i>SSIII</i> genes.

<p>(A) Multiple alignments of Zpu1 gene sequences between W64A+, W64A<i>o2</i> and K0326Y. The highlighted positions showed four single nucleotide polymorphisms in K0326Y. (B) Multiple alignments of translated amino acid sequences of pullulanase. One amino acid difference (highlighted) was found in K0326Y due to the substitution of nucleotide from A to C at position 2864. (C) Restriction analysis of a 3’ end fragment (position 2632–2965) of the Zpu1 gene. The gel bands represent the size of fragments before (uncut) and after digestion (dig) with BslI. (D) Multiple alignments of <i>SSIII</i> gene sequences between W64A+, W64A<i>o2</i> and K0326Y. Hash marks on each sequence showed nucleotide polymorphisms. Prior studies identified three regions of the nucleotide sequences: N-terminal region (base 1–2304), homology region (base 2305–3679), and catalytic region (base 3680–5025) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.ref028" target="_blank">28</a>]. Full nucleotide sequence alignments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s001" target="_blank">S1 Fig</a>. (E) Multiple alignments of translated amino acid sequences of SSIII, and hash marks on each sequence showed amino acid changes. Sequence annotation showed three domains of the amino acid sequences: N-terminal domain (amino acid 1–768) and homology domain (amino acid 769–1226), and catalytic domain (amino acid 1227–1674). Full protein sequence alignments are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130856#pone.0130856.s002" target="_blank">S2 Fig</a>.</p

Average glucan chain length and polydispersity index (PDI).

<p>(A) Comparison of the average glucan chain length among RILs. Each column contains multiple lines homozygous for the parental alleles (W64A<i>o2</i> or QPM) of <i>Zpu1</i> or <i>SSIII</i>. Each column represents mean glucan chain length of all RILs of the corresponding genotype with three independent ears of each individual RIL. (B) Positive correlation between average glucan chain length and PDI. The correlations are significant, tested by ANOVA of the slope at <i>p</i><0.05. Each data point represents mean glucan chain length of three independent ears of each individual RIL. (C) Comparison of PDI between parental lines (W64A+, W64A<i>o2</i> and K0326Y). Each column represents the mean PDI value of three independent ears of corresponding lines. The letters above each column of (A) and (C) represent statistically significant differences among the lines for <i>p</i><0.05 by each pair t test. Columns sharing the same letter are not significantly different from one another. The error bars represent corresponding standard error. (D) Nonlinear regression analysis between PDI and kernel vitreousness among RILs, test statistics are listed below. The relationship was significantly positive, because 0 was excluded from the 95% confidence interval of Growth Rate, indicating that the Growth Rate was greater than 0 at <i>p</i>-value = 0.05 level. Each data point represents mean PDI of three independent ears of each individual RIL. (E-G) Glucan chain length distribution between opaque lines (dash curves) and vitreous lines (solid curves).</p