Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.

Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

A new inhibitor of the ß-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ß-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ß-arrestin recruitment to the receptor and ß-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between ß-arrestin and the ß2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/ß-arrestin complexes. This selective ß-arrestin/ß2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical ß2-adrenergic (ß2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect ß-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and ß2AR, supporting the concept of ß-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

Quantifying biased signaling in GPCRs using BRET-based biosensors.

There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and ß-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or ß-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.

The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αßγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Pepducin targeting the C-X-C chemokine receptor type 4 acts as a biased agonist favoring activation of the inhibitory G protein.

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 ß-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 ß-arrestins, ATI-2341 leads to the engagement of the Gi subtypes but not G13 or the ß-arrestins. Calculation of the transduction ratio for each pathway revealed a strong negative bias of ATI-2341 toward G13 and ß-arrestins, revealing functional selectivity for the Gi pathways. The negative bias toward ß-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.

A synthetic biology approach reveals a CXCR4-G13-Rho signaling axis driving transendothelial migration of metastatic breast cancer cells.

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors (GPCRs) to metastasize to regional lymph nodes or distant organs. Indeed, the migration toward SDF-1 (stromal cell-derived factor 1) of tumor cells bearing CXCR4 [chemokine (C-X-C motif) receptor 4] has been implicated in the lymphatic and organ-specific metastasis of various human malignancies. Here, we used chimeric G proteins and GPCRs activated solely by artificial ligands to selectively activate the signaling pathways downstream of specific G proteins and showed that CXCR4-mediated chemotaxis and transendothelial migration of metastatic basal-like breast cancer cells required activation of Gα(13), a member of the Gα(12/13) G protein family, and of the small guanosine triphosphatase Rho. Multiple complementary experimental strategies, including synthetic biology approaches, indicated that signaling-selective inhibition of the CXCR4-Gα(13)-Rho axis prevents the metastatic spread of basal-like breast cancer cells.

Multimerization of Staufen1 in live cells.

Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.

Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.

The chemokine receptor CXCR4 is a widely expressed G protein-coupled receptor that has been implicated in a number of diseases including human immunodeficiency virus, cancer, and WHIM syndrome, with the latter two involving dysregulation of CXCR4 signaling. To better understand the role of phosphorylation in regulating CXCR4 signaling, tandem mass spectrometry and phospho-specific antibodies were used to identify sites of agonist-promoted phosphorylation. These studies demonstrated that Ser-321, Ser-324, Ser-325, Ser-330, Ser-339, and two sites between Ser-346 and Ser-352 were phosphorylated in HEK293 cells. We show that Ser-324/5 was rapidly phosphorylated by protein kinase C and G protein-coupled receptor kinase 6 (GRK6) upon CXCL12 treatment, whereas Ser-339 was specifically and rapidly phosphorylated by GRK6. Ser-330 was also phosphorylated by GRK6, albeit with slower kinetics. Similar results were observed in human astroglia cells, where endogenous CXCR4 was rapidly phosphorylated on Ser-324/5 by protein kinase C after CXCL12 treatment, whereas Ser-330 was slowly phosphorylated. Analysis of CXCR4 signaling in HEK293 cells revealed that calcium mobilization was primarily negatively regulated by GRK2, GRK6, and arrestin3, whereas GRK3, GRK6, and arrestin2 played a primary role in positively regulating ERK1/2 activation. In contrast, GRK2 appeared to play a negative role in ERK1/2 activation. Finally, we show that arrestin association with CXCR4 is primarily driven by the phosphorylation of far C-terminal residues on the receptor. These studies reveal that site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases resulting in both positive and negative modulation of CXCR4 signaling.

Neurosphere-derived neural cells show region-specific behaviour in vitro.

Neurosphere cultures provide a useful model to study neural stem/progenitor cells (NSC/NPCs). The degree to which neurospheres (NS) retain their regional identity in vitro has, however, been questioned. Here, NS obtained from mouse embryonic cortex, striatum or spinal cord were compared after differentiation. Neurons from cortical NS formed well ordered clusters containing astrocytes, those from striatal NS formed an external ring at the borderof the astrocyte layer, whereas those from spinal cord NS spread radially like the astrocytes. Such in-vitro neural behaviour was region-specific and persisted in clonal conditions, providing evidence of the maintenance of positional cues in NS cultures.