A critical role for endocytosis in Wnt signaling

BACKGROUND: The Wnt signaling pathway regulates many processes during embryonic development, including axis specification, organogenesis, angiogenesis, and stem cell proliferation. Wnt signaling has also been implicated in a number of cancers, bone density maintenance, and neurological conditions during adulthood. While numerous Wnts, their cognate receptors of the Frizzled and Arrow/LRP5/6 families and downstream pathway components have been identified, little is known about the initial events occurring directly after receptor activation. RESULTS: We show here that Wnt proteins are rapidly endocytosed by a clathrin- and dynamin-mediated process. While endocytosis has traditionally been considered a principal mechanism for receptor down-regulation and termination of signaling pathways, we demonstrate that interfering with clathrin-mediated endocytosis actually blocks Wnt signaling at the level of β-catenin accumulation and target gene expression. CONCLUSION: A necessary component of Wnt signaling occurs in a subcellular compartment distinct from the plasma membrane. Moreover, as internalized Wnts transit partially through the transferrin recycling pathway, it is possible that a "signaling endosome" serves as a nexus for activated Wnt pathway components

SDF1-Induced Antagonism of Axonal Repulsion Requires Multiple G-Protein Coupled Signaling Components That Work in Parallel

SDF1 reduces the responsiveness of axonal growth cones to repellent guidance cues in a pertussis-toxin-sensitive, cAMP-dependent manner. Here, we show that SDF1's antirepellent effect can be blocked in embryonic chick dorsal root ganglia (DRGs) by expression of peptides or proteins inhibiting either Gαi, Gαq, or Gβγ. SDF1 antirepellent activity is also blocked by pharmacological inhibition of PLC, a common effector protein for Gαq. We also show that SDF1 antirepellent activity can be mimicked by overexpression of constitutively active Gαi, Gαq, or Gαs. These results suggest a model in which multiple G protein components cooperate to produce the cAMP levels required for SDF1 antirepellent activity

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

Impedance Responses Reveal β2-Adrenergic Receptor Signaling Pluridimensionality and Allow Classification of Ligands with Distinct Signaling Profiles

The discovery that drugs targeting a single G protein-coupled receptor (GPCR) can differentially modulate distinct subsets of the receptor signaling repertoire has created a challenge for drug discovery at these important therapeutic targets. Here, we demonstrate that a single label-free assay based on cellular impedance provides a real-time integration of multiple signaling events engaged upon GPCR activation. Stimulation of the β2-adrenergic receptor (β2AR) in living cells with the prototypical agonist isoproterenol generated a complex, multi-featured impedance response over time. Selective pharmacological inhibition of specific arms of the β2AR signaling network revealed the differential contribution of Gs-, Gi- and Gβγ-dependent signaling events, including activation of the canonical cAMP and ERK1/2 pathways, to specific components of the impedance response. Further dissection revealed the essential role of intracellular Ca2+ in the impedance response and led to the discovery of a novel β2AR-promoted Ca2+ mobilization event. Recognizing that impedance responses provide an integrative assessment of ligand activity, we screened a collection of β-adrenergic ligands to determine if differences in the signaling repertoire engaged by compounds would lead to distinct impedance signatures. An unsupervised clustering analysis of the impedance responses revealed the existence of 5 distinct compound classes, revealing a richer signaling texture than previously recognized for this receptor. Taken together, these data indicate that the pluridimensionality of GPCR signaling can be captured using integrative approaches to provide a comprehensive readout of drug activity

Expression of G protein-coupled receptors and related proteins in HEK293, AtT20, BV2, and N18 cell lines as revealed by microarray analysis

<p>Abstract</p> <p>Background</p> <p>G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret.</p> <p>Results</p> <p>To bridge this gap, we have used microarrays to measure the mRNA levels of a comprehensive profile of non-chemosensory GPCRs and over a hundred GPCR signaling related gene products in four cell lines frequently used for GPCR research: HEK293, AtT20, BV2, and N18.</p> <p>Conclusions</p> <p>This study provides researchers an easily accessible mRNA profile of the endogenous signaling repertoire that these four cell lines possess. This will assist in choosing the most appropriate cell line for studying GPCRs and related signaling proteins. It also provides a better understanding of the potential interactions between GPCRs and those signaling proteins.</p

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3′-5′-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:βγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades

Lysophosphatidic acid and sphingosine-1-phosphate promote morphogenesis and block invasion of prostate cancer cells in three-dimensional organotypic models

Normal prostate and some malignant prostate cancer (PrCa) cell lines undergo acinar differentiation and form spheroids in three-dimensional (3-D) organotypic culture. Acini formed by PC-3 and PC-3M, less pronounced also in other PrCa cell lines, spontaneously undergo an invasive switch, leading to the disintegration of epithelial structures and the basal lamina, and formation of invadopodia. This demonstrates the highly dynamic nature of epithelial plasticity, balancing epithelial-to-mesenchymal transition against metastable acinar differentiation. This study assessed the role of lipid metabolites on epithelial maturation. PC-3 cells completely failed to form acinar structures in delipidated serum. Adding back lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) rescued acinar morphogenesis and repressed invasion effectively. Blocking LPA receptor 1 (LPAR1) functions by siRNA (small interference RNA) or the specific LPAR1 inhibitor Ki16425 promoted invasion, while silencing of other G-protein-coupled receptors responsive to LPA or S1P mainly caused growth arrest or had no effects. The G-proteins Gα12/13 and Gαi were identified as key mediators of LPA signalling via stimulation of RhoA and Rho kinases ROCK1 and 2, activating Rac1, while inhibition of adenylate cyclase and accumulation of cAMP may be secondary. Interfering with these pathways specifically impeded epithelial polarization in transformed cells. In contrast, blocking the same pathways in non-transformed, normal cells promoted differentiation. We conclude that LPA and LPAR1 effectively promote epithelial maturation and block invasion of PrCa cells in 3-D culture. The analysis of clinical transcriptome data confirmed reduced expression of LPAR1 in a subset of PrCa's. Our study demonstrates a metastasis-suppressor function for LPAR1 and Gα12/13 signalling, regulating cell motility and invasion versus epithelial maturation