RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

Natural Products Discovered in a High-Throughput Screen Identified as Inhibitors of RGS17 and as Cytostatic and Cytotoxic Agents for Lung and Prostate Cancer Cell Lines.

Regulator of G Protein Signaling (RGS) 17 is an overexpressed promoter of cancer survival in lung and prostate tumors, the knockdown of which results in decreased tumor cell proliferation in vitro. Identification of drug-like molecules inhibiting this protein could ameliorate the RGS17's pro-tumorigenic effect. Using high-throughput screening, a chemical library containing natural products was interrogated for inhibition of the RGS17-Gαo interaction. Initial hits were verified in control and counter screens. Leads were characterized via biochemical, mass spectrometric, Western blot, microscopic, and cytotoxicity measures. Four known compounds (1-4) were identified with IC50 values ranging from high nanomolar to low micromolar. Three compounds were extensively characterized biologically, demonstrating cellular activity determined by confocal microscopy, and two compounds were assessed via ITC exhibiting high nanomolar to low micromolar dissociation constants. The compounds were found to have a cysteine-dependent mechanism of binding, verified through site-directed mutagenesis and cysteine reactivity assessment. Two compounds, sanguinarine (1) and celastrol (2), were found to be cytostatic against lung and prostate cancer cell lines and cytotoxic against prostate cancer cell lines in vitro, although the dependence of RGS17 on these phenomena remains elusive, a result that is perhaps not surprising given the multimodal cytostatic and cytotoxic activities of many natural products.

Dawn of a New RAMPage.

Receptor activity-modifying proteins (RAMPs) interact with G-protein-coupled receptors (GPCRs) to modify their functions, imparting significant implications upon their physiological and therapeutic potentials. Resurging interest in identifying RAMP-GPCR interactions has recently been fueled by coevolution studies and orthogonal technological screening platforms. These new studies reveal previously unrecognized RAMP-interacting GPCRs, many of which expand beyond Class B GPCRs. The consequences of these interactions on GPCR function and physiology lays the foundation for new molecular therapeutic targets, as evidenced by the recent success of erenumab. Here, we highlight recent papers that uncovered novel RAMP-GPCR interactions, human RAMP-GPCR disease-causing mutations, and RAMP-related human pathologies, paving the way for a new era of RAMP-targeted drug development.

The Orphan G-Protein Coupled Receptor 182 Is a Negative Regulator of Definitive Hematopoiesis through Leukotriene B4 Signaling.

The G protein-coupled receptor 182 (GPR182) is an orphan GPCR, the expression of which is enriched in embryonic endothelial cells (ECs). However, the physiological role and molecular mechanism of action of GPR182 are unknown. Here, we show that GPR182 negatively regulates definitive hematopoiesis in zebrafish and mice. In zebrafish, gpr182 expression is enriched in the hemogenic endothelium (HE), and gpr182 -/- display an increased expression of HE and hematopoietic stem cell (HSC) marker genes. Notably, we find an increased number of myeloid cells in gpr182 -/- compared to wild-type. Further, by time-lapse imaging of zebrafish embryos during the endothelial-to-hematopoietic transition, we find that HE/HSC cell numbers are increased in gpr182 -/- compared to wild-type. GPR182 -/- mice also exhibit an increased number of myeloid cells compared to wild-type, indicating a conserved role for GPR182 in myelopoiesis. Using cell-based small molecule screening and transcriptomic analyses, we further find that GPR182 regulates the leukotriene B4 (LTB4) biosynthesis pathway. Taken together, these data indicate that GPR182 is a negative regulator of definitive hematopoiesis in zebrafish and mice, and provide further evidence for LTB4 signaling in HSC biology.

hCALCRL mutation causes autosomal recessive nonimmune hydrops fetalis with lymphatic dysplasia.

We report the first case of nonimmune hydrops fetalis (NIHF) associated with a recessive, in-frame deletion of V205 in the G protein-coupled receptor, Calcitonin Receptor-Like Receptor (hCALCRL). Homozygosity results in fetal demise from hydrops fetalis, while heterozygosity in females is associated with spontaneous miscarriage and subfertility. Using molecular dynamic modeling and in vitro biochemical assays, we show that the hCLR(V205del) mutant results in misfolding of the first extracellular loop, reducing association with its requisite receptor chaperone, receptor activity modifying protein (RAMP), translocation to the plasma membrane and signaling. Using three independent genetic mouse models we establish that the adrenomedullin-CLR-RAMP2 axis is both necessary and sufficient for driving lymphatic vascular proliferation. Genetic ablation of either lymphatic endothelial Calcrl or nonendothelial Ramp2 leads to severe NIHF with embryonic demise and placental pathologies, similar to that observed in humans. Our results highlight a novel candidate gene for human congenital NIHF and provide structure-function insights of this signaling axis for human physiology.

A high throughput screen for RGS proteins using steady state monitoring of free phosphate formation.

G-protein coupled receptors are a diverse group that are the target of over 50% of marketed drugs. Activation of these receptors results in the exchange of bound GDP for GTP in the Gα subunit of the heterotrimeric G-protein. The Gα subunit dissociates from the ß/γ subunits and both proceed to affect downstream signaling targets. The signal terminates by the hydrolysis of GTP to GDP and is temporally regulated by Regulators of G-protein Signaling (RGS) proteins that act as GTPase Activating Proteins (GAPs). This makes RGS proteins potentially desirable targets for "tuning" the effects of current therapies as well as developing novel pharmacotherapies. Current methods for evaluating RGS activity depend on laborious and/or expensive techniques. In this study we developed a simple and inexpensive assay for the steady state analysis of RGS protein GAP activity, using RGS4, RGS8 and RGS17 as models. Additionally, we report the use of RGS4 as a model for high throughput assay development. After initial setup, this assay can be conducted in a highly parallel fashion with a read time of less than 8 minutes for a 1536-well plate. The assay exhibited a robust Z-factor of 0.6 in a 1536-well plate. We conducted a pilot screen for inhibitors using a small, 2320 compound library. From this screen, 13 compounds were identified as compounds for further analysis. The successful development of this assay for high-throughput screening provides a low cost, high speed, simple method for assessing RGS protein activity.

RGS17: an emerging therapeutic target for lung and prostate cancers.

Ligands for G-protein-coupled receptors (GPCRs) represent approximately 50% of currently marketed drugs. RGS proteins modulate heterotrimeric G proteins and, thus, GPCR signaling, by accelerating the intrinsic GTPase activity of the Gα subunit. Given the prevalence of GPCR targeted therapeutics and the role RGS proteins play in G protein signaling, some RGS proteins are emerging as targets in their own right. One such RGS protein is RGS17. Increased RGS17 expression in some prostate and lung cancers has been demonstrated to support cancer progression, while reduced expression of RGS17 can lead to development of chemotherapeutic resistance in ovarian cancer. High-throughput screening is a powerful tool for lead compound identification, and utilization of high-throughput technologies has led to the discovery of several RGS inhibitors, thus far. As screening technologies advance, the identification of novel lead compounds the subsequent development of targeted therapeutics appears promising.

RABL6A, a novel RAB-like protein, controls centrosome amplification and chromosome instability in primary fibroblasts.

RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53-/- MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis.

Development of a novel high-throughput screen and identification of small-molecule inhibitors of the Gα-RGS17 protein-protein interaction using AlphaScreen.

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gα(o)-RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gα(o)-RGS17 protein-protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC(50) <10 µM in dose-response experiments. Four exhibited IC(50) values <6 µM while inhibiting the Gα(o)-RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.