PMID: 18948008;PMCID: PMC2590931;Abstract:
Cells' ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown [1]. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects [2]. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2 [2-5]. To investigate the role of Ras in directional sensing, we studied the effect of its misregulation by using cells with disrupted RasGAP activity. We identified an ortholog of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. We show that disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, we show that tight regulation of Ras is important for gradient sensing. Together, our findings suggest that Ras is part of the cell's compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing. © 2008 Elsevier Ltd. All rights reserved.
PMID: 12900404;Abstract:
A large number of G protein-coupled receptors are palmitoylated on cysteine residues located in their carboxyl tail, but the general role of this post-translational modification remains poorly understood. Here we show that preventing palmitoylation of the V2 vasopressin receptor, by site-directed mutagenesis of cysteines 341 and 342, significantly delayed and decreased both agonist-promoted receptor endocytosis and mitogen-activated protein kinase activation. Pharmacological blockade of receptor endocytosis is without effect on the vasopressin-stimulated mitogen-activated protein kinase activity, excluding the possibility that the reduced kinase activation mediated by the palmitoylation-less mutant could result from altered receptor endocytosis. In contrast, two dominant negative mutants of β-arrestin which inhibit receptor endocytosis also attenuated vasopressin-stimulated mitogen-activated protein kinase activity, suggesting that the scaffolding protein, β-arrestin, represents the common link among receptor palmitoylation, endocytosis, and kinase activation. Co-immunoprecipitation and bioluminescence resonance energy transfer experiments confirmed that inhibiting receptor palmitoylation considerably reduced the vasopressin-stimulated recruitment of β-arrestin to the receptor. Interestingly, the changes in β-arrestin recruitment kinetics were similar to those observed for vasopressin-stimulated receptor endocytosis and mitogen-activated protein kinase activation. Taken together the results indicate that palmitoylation enhances the recruitment of β-arrestin to the activated V2 vasopressin receptor thus facilitating processes requiring the scaffolding action of β-arrestin.
PMID: 17675294;Abstract:
The most widely studied pathway underlying agonist-promoted internalization of G protein-coupled receptors (GPCRs) involves β-arrestin and clathrin-coated pits. However, both β-arrestin- and clathrin-independent processes have also been reported. Classically, the endocytic routes are characterized using pharmacological inhibitors and various dominant negative mutants, resulting sometimes in conflicting results and interpretational difficulties. Here, taking advantage of the fact that β-arrestin binding to the β2 subunit of the clathrin adaptor AP-2 (β2-adaptin) is needed for the β-arrestin-mediated targeting of GPCRs to clathrin-coated pits, we developed a bioluminescence resonance energy transfer-based approach directly assessing the molecular steps involved in the endocytosis of GPCRs in living cells. For 10 of the 12 receptors tested, including some that were previously suggested to internalize via clathrin-independent pathways, agonist stimulation promoted β-arrestin 1 and 2 interaction with β2-adaptin, indicating a β-arrestin-and clathrin-dependent endocytic process. Detailed analyses of β-arrestin interactions with both the receptor and β2-adaptin also allowed us to demonstrate that recruitment of β-arrestins to the receptor and the ensuing conformational changes are the leading events preceding AP-2 engagement and subsequent clathrin-mediated endocytosis. Among the receptors tested, only the endothelin A and B receptors failed to promote interaction between β-arrestins and β2-adaptin. However, both receptors recruited β-arrestins upon agonist stimulation, suggesting a β-arrestin- dependent but clathrin-independent route of internalization for these two receptors. In addition to providing a new tool to dissect the molecular events involved in GPCR endocytosis, the bioluminescence resonance energy transfer-based β-arrestin/β2-adaptin interaction assay represents a novel biosensor to assess receptor activation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
PMID: 22215733;PMCID: PMC3928814;Abstract:
Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein - coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase - activating protein acting as a global inhibitor.
PMID: 21145496;PMCID: PMC3033560;Abstract:
During cell migration, chemoattractant-induced signaling pathways determine the direction of movement by controlling the spatiotemporal dynamics of cytoskeletal components. In this issue of Developmental Cell, Liu et al. report that the target of rapamycin complex 2 (TORC2) controls cell polarity and chemotaxis through regulation of both F-actin and myosin II in migrating neutrophils. © 2010 Elsevier Inc.
