Titration-based normalization of antibody amount improves consistency of ChIP-seq experiments.

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts.

Glucocorticoids unmask silent non-coding genetic risk variants for common diseases.

Understanding the function of non-coding genomic sequence variants represents a challenge for biomedicine. Many diseases are products of gene-by-environment interactions with complex mechanisms. This study addresses these themes by mechanistic characterization of non-coding variants that influence gene expression only after drug or hormone exposure. Using glucocorticoid signaling as a model system, we integrated genomic, transcriptomic, and epigenomic approaches to unravel mechanisms by which variant function could be revealed by hormones or drugs. Specifically, we identified cis-regulatory elements and 3D interactions underlying ligand-dependent associations between variants and gene expression. One-quarter of the glucocorticoid-modulated variants that we identified had already been associated with clinical phenotypes. However, their affected genes were 'unmasked' only after glucocorticoid exposure and often with function relevant to the disease phenotypes. These diseases involved glucocorticoids as risk factors or therapeutic agents and included autoimmunity, metabolic and mood disorders, osteoporosis and cancer. For example, we identified a novel breast cancer risk gene, MAST4, with expression that was repressed by glucocorticoids in cells carrying the risk genotype, repression that correlated with MAST4 expression in breast cancer and treatment outcomes. These observations provide a mechanistic framework for understanding non-coding genetic variant-chemical environment interactions and their role in disease risk and drug response.

RSK2 drives cell motility by serine phosphorylation of LARG and activation of Rho GTPases.

Directed migration is essential for cell motility in many processes, including development and cancer cell invasion. RSKs (p90 ribosomal S6 kinases) have emerged as central regulators of cell migration; however, the mechanisms mediating RSK-dependent motility remain incompletely understood. We have identified a unique signaling mechanism by which RSK2 promotes cell motility through leukemia-associated RhoGEF (LARG)-dependent Rho GTPase activation. RSK2 directly interacts with LARG and nucleotide-bound Rho isoforms, but not Rac1 or Cdc42. We further show that epidermal growth factor or FBS stimulation induces association of endogenous RSK2 with LARG and LARG with RhoA. In response to these stimuli, RSK2 phosphorylates LARG at Ser1288 and thereby activates RhoA. Phosphorylation of RSK2 at threonine 577 is essential for activation of LARG-RhoA. Moreover, RSK2-mediated motility signaling depends on RhoA and -B, but not RhoC. These results establish a unique RSK2-dependent LARG-RhoA signaling module as a central organizer of directed cell migration and invasion.

Analysis of the Rit subfamily GTPase-mediated signaling and neuronal differentiation and survival.

The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from the pathways that are regulated by other Ras family GTPases. Rit proteins have been demonstrated to be essential regulators in neuronal differentiation and survival. Here, we describe the materials and methods utilized to characterize cellular signaling for the Rit subfamily of G-proteins in neuronal differentiation and survival.

Rit subfamily small GTPases: regulators in neuronal differentiation and survival.

Ras family small GTPases serve as binary molecular switches to regulate a broad array of cellular signaling cascades, playing essential roles in a vast range of normal physiological processes, with dysregulation of numerous Ras-superfamily G-protein-dependent regulatory cascades underlying the development of human disease. However, the physiological function for many "orphan" Ras-related GTPases remain poorly characterized, including members of the Rit subfamily GTPases. Rit is the founding member of a novel branch of the Ras subfamily, sharing close homology with the neuronally expressed Rin and Drosophila Ric GTPases. Here, we highlight recent studies using transgenic and knockout animal models which have begun to elucidate the physiological roles for the Rit subfamily, including emerging roles in the regulation of neuronal morphology and cellular survival signaling, and discuss new genetic data implicating Rit and Rin signaling in disorders such as cancer, Parkinson's disease, autism, and schizophrenia.

Putting the Rit in cellular resistance: Rit, p38 MAPK and oxidative stress.

Cells mobilize diverse signaling pathways to protect against stress-mediated injury. Ras family GTPases play critical roles in this process, controlling the activation and integration of multiple regulatory cascades. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also promote cell survival. However, this functional dichotomy is incompletely understood, particularly regulation of p38-dependent survival. Here, we discuss our recent evidence that the Rit GTPase associates with and is required for stress-mediated activation of a scaffolded p38-MK2-HSP27-Akt pro-survival signaling cascade. Drosophila lacking D-Ric, a Rit homologue, are susceptible to a variety of environmental stresses, while embryonic fibroblasts derived from Rit knockout mice display blunted stress-dependent signaling and decreased viability. Conversely, expression of constitutively active Rit triggers p38-Akt-dependent cell survival. Together, our studies establish Rit as the central regulator of an evolutionarily conserved, p38-dependent signaling cascade that functions as a critical survival mechanism in response to stress.

Rit-mediated stress resistance involves a p38-mitogen- and stress-activated protein kinase 1 (MSK1)-dependent cAMP response element-binding protein (CREB) activation cascade.

The cAMP response element (CRE)-binding protein (CREB) is a key regulatory factor of gene transcription, and plays an essential role in development of the central nervous system and for neuroprotection. Multiple signaling pathways have been shown to contribute to the regulation of CREB-dependent transcription, including both ERK and p38 mitogen-activated protein (MAP) kinases cascades. Recent studies have identified the Ras-related small G-protein, Rit, as a central regulator of a p38-MK2-HSP27 signaling cascade that functions as a critical survival mechanism for cells adapting to stress. Here, we examine the contribution of Rit-p38 signaling to the control of stress-dependent gene transcription. Using a pheochromocytoma cell model, we find that a novel Rit-p38-MSK1/2 pathway plays a critical role in stress-mediated CREB activation. RNAi-mediated Rit silencing, or inhibition of p38 or MSK1/2 kinases, was found to disrupt stress-mediated CREB-dependent transcription, resulting in increased cell death. Furthermore, ectopic expression of active Rit stimulates CREB-Ser133 phosphorylation, induces expression of the anti-apoptotic Bcl-2 and Bcl(XL) proteins, and promotes cell survival. These data indicate that the Rit-p38-MSK1/2 signaling pathway may have an important role in the stress-dependent regulation of CREB-dependent gene expression.

Rem-GTPase regulates cardiac myocyte L-type calcium current.

RATIONALE: The L-type calcium channels (LTCC) are critical for maintaining Ca(2+)-homeostasis. In heterologous expression studies, the RGK-class of Ras-related G-proteins regulates LTCC function; however, the physiological relevance of RGK-LTCC interactions is untested. OBJECTIVE: In this report we test the hypothesis that the RGK protein, Rem, modulates native Ca(2+) current (I(Ca,L)) via LTCC in murine cardiomyocytes. METHODS AND RESULTS: Rem knockout mice (Rem(-/-)) were engineered, and I(Ca,L) and Ca(2+) -handling properties were assessed. Rem(-/-) ventricular cardiomyocytes displayed increased I(Ca,L) density. I(Ca,L) activation was shifted positive on the voltage axis, and ß-adrenergic stimulation normalized this shift compared with wild-type I(Ca,L). Current kinetics, steady-state inactivation, and facilitation was unaffected by Rem(-/-) . Cell shortening was not significantly different. Increased I(Ca,L) density in the absence of frank phenotypic differences motivated us to explore putative compensatory mechanisms. Despite the larger I(Ca,L) density, Rem(-/-) cardiomyocyte Ca(2+) twitch transient amplitude was significantly less than that compared with wild type. Computer simulations and immunoblot analysis suggests that relative dephosphorylation of Rem(-/-) LTCC can account for the paradoxical decrease of Ca(2+) transients. CONCLUSIONS: This is the first demonstration that loss of an RGK protein influences I(Ca,L) in vivo in cardiac myocytes.

Ras family small GTPase-mediated neuroprotective signaling in stroke.

Selective neuronal cell death is one of the major causes of neuronal damage following stroke, and cerebral cells naturally mobilize diverse survival signaling pathways to protect against ischemia. Importantly, therapeutic strategies designed to improve endogenous anti-apoptotic signaling appear to hold great promise in stroke treatment. While a variety of complex mechanisms have been implicated in the pathogenesis of stroke, the overall mechanisms governing the balance between cell survival and death are not well-defined. Ras family small GTPases are activated following ischemic insults, and in turn, serve as intrinsic switches to regulate neuronal survival and regeneration. Their ability to integrate diverse intracellular signal transduction pathways makes them critical regulators and potential therapeutic targets for neuronal recovery after stroke. This article highlights the contribution of Ras family GTPases to neuroprotective signaling cascades, including mitogen-activated protein kinase (MAPK) family protein kinase- and AKT/PKB-dependent signaling pathways as well as the regulation of cAMP response element binding (CREB), Forkhead box O (FoxO) and hypoxiainducible factor 1(HIF1) transcription factors, in stroke.

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress.

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.

Src-dependent TrkA transactivation is required for pituitary adenylate cyclase-activating polypeptide 38-mediated Rit activation and neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent neuropeptide that possesses both neurotrophic and neurodevelopmental effects. Recently, the Rit GTPase was found to be activated by a novel Galpha/cAMP/exchange protein activated by cyclic AMP (Epac)-dependent signaling pathway and required for PACAP-dependent cAMP response element-binding protein activation and neuronal differentiation. However, Epac did not function as a Rit guanine nucleotide exchange factor (GEF), and the nature of the PACAP regulatory cascade remained unclear. Here, we show that PACAP-mediated Rit activation involves Src family kinase-dependent TrkA receptor transactivation. PACAP receptor (PACR1) stimulation triggered both G(i)alpha and G(s)alpha/cAMP/Epac regulatory cascades resulting in Src kinase activity, which in turn induced TrkA kinase tyrosine phosphorylation. Importantly, Src inhibition, or the lack of functional Trk receptors, was found to inhibit PACAP-mediated Rit activation, whereas constitutively active Src alone was sufficient to stimulate Rit-guanosine triphosphate levels. A single tyrosine (Y(499)) phosphorylation event was identified as critical to both PACAP-mediated transactivation and TrkA-dependent Rit activation. Accordingly, PACAP stimulation resulted in TrkA-dependent phosphorylation of both the Shc adaptor and son of sevenless (SOS)1/2 GEFs, and Rit activation was inhibited by RNA interference silencing of SOS1/2, implicating a TrkA/Shc/SOS signaling complex in Rit regulation. Together, these observations expand upon the nature of PACR1-mediated transactivation and identify TrkA-Rit signaling as a key contributor to PACAP-dependent neuronal differentiation.

Rit signaling contributes to interferon-gamma-induced dendritic retraction via p38 mitogen-activated protein kinase activation.

The proinflammatory cytokine interferon-gamma (IFNgamma) alters neuronal connectivity via selective regressive effects on dendrites but the signaling pathways that mediate this effect are poorly understood. We recently demonstrated that signaling by Rit, a member of the Ras family of GTPases, modulates dendritic growth in primary cultures of sympathetic and hippocampal neurons. In this study, we investigated a role for Rit signaling in IFNgamma-induced dendritic retraction. Expression of a dominant negative Rit mutant inhibited IFNgamma-induced dendritic retraction in cultured embryonic rat sympathetic and hippocampal neurons. In pheochromacytoma cells and hippocampal neurons, IFNgamma caused rapid Rit activation as indicated by increased GTP binding to Rit. Silencing of Rit by RNA interference suppressed IFNgamma-elicited activation of p38 MAPK in pheochromacytoma cells, and pharmacological inhibition of p38 MAPK significantly attenuated the dendrite-inhibiting effects of IFNgamma in cultured sympathetic and hippocampal neurons without altering signal transducer and activator of transcription 1 activation. These observations identify Rit as a downstream target of IFNgamma and suggest that a novel IFNgamma-Rit-p38 signaling pathway contributes to dendritic retraction and may, therefore, represent a potential therapeutic target in diseases with a significant neuroinflammatory component.

Pituitary adenylate cyclase-activating polypeptide 38-mediated Rin activation requires Src and contributes to the regulation of HSP27 signaling during neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) is a potent neuropeptide that acts through G-protein-coupled receptors. While it is well established that PACAP mediates both neurotrophic and neurodevelopmental effects, the signaling cascades that underlie these diverse actions remain incompletely characterized. Here we show that the Ras-related Rin GTP-binding protein, a GTPase that is expressed predominantly in neurons, is regulated by PACAP38 signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to PACAP38-mediated pheochromocytoma cell differentiation. Rin is activated following stimulation of both Gsalpha and Gialpha cascades but does not rely upon cyclic AMP (cAMP)-, Ca(2+)-, or Epac-dependent signaling pathways. Instead, Rin is activated in a Src kinase-dependent manner. Surprisingly, Rin knockdown significantly inhibits PACAP38-mediated neurite outgrowth, without affecting mitogen-activated protein kinase signaling cascades. Instead, Rin loss attenuates PACAP38-mediated HSP27 activation by disrupting a cAMP-protein kinase A cascade. RNA interference-mediated HSP27 silencing suppresses both PACAP38- and Rin-mediated neurite outgrowth, while expression of a constitutively active Rin mutant increases both HSP27 protein and phospho-HSP27 levels, supporting a role for Rin-HSP27 signaling in neuronal differentiation. Together, these observations identify an unsuspected role for Rin in neuronal PACAP signaling and establish a novel Galpha-Src-Rin-HSP27 signal transduction pathway as a critical element in PACAP38-mediated neuronal differentiation signaling.

Rit mutants confirm role of MEK/ERK signaling in neuronal differentiation and reveal novel Par6 interaction.

Rit is a novel member of the Ras superfamily of small GTP-binding proteins that regulates signaling pathways controlling cellular fate determination. Constitutively activated mutants of Rit induce terminal differentiation of pheochromocytoma (PC6) cells resulting in a sympathetic neuron-like phenotype characterized by the development of highly-branched neurites. Rit signaling has been found to activate several downstream pathways including MEK/ERK, p38 MAPK, Ral-specific guanine nucleotide exchange factors (GEFs), and Rit associates with the Par6 cell polarity machinery. In this study, a series of Rit effector loop mutants was generated to test the importance of these cellular targets to Rit-mediated neuronal differentiation. We find that Rit-mediated neuritogenesis is dependent upon MEK/ERK MAP kinase signaling but independent of RalGEF activation. In addition, in vivo binding studies identified a novel mechanism of Par6 interaction, suggesting that the cell polarity machinery may serve to spatially restrict Rit signaling.

The novel GTPase Rit differentially regulates axonal and dendritic growth.

The Rit GTPase is widely expressed in developing and adult nervous systems, and our previous data with pheochromocytoma cells implicate Rit signaling in NGF-induced neurite outgrowth. In this study, we investigated a role for Rit in neuronal morphogenesis. Expression of a dominant-negative (dn) Rit mutant in hippocampal neurons inhibited axonal growth but potentiated dendritic growth. Conversely, a constitutively active (ca) Rit mutant promoted axonal growth but inhibited dendritic growth. Dendritogenesis is regulated differently in sympathetic neurons versus hippocampal neurons in that sympathetic neurons require NGF and bone morphogenetic proteins (BMPs) to trigger dendritic growth. Despite these differences, dnRit potentiated and caRit blocked BMP7-induced dendritic growth in sympathetic neurons. Biochemical studies indicated that BMP7 treatments that caused dendritic growth also decreased Rit GTP loading. Additional studies demonstrate that caRit increased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and pharmacological inhibition of MEK1 (mitogen-activated protein kinase/ERK 1) blocked the axon-promoting and dendrite-inhibiting effects of caRit. These observations suggest that Rit is a convergence point for multiple signaling pathways and it functions to promote axonal growth but inhibit dendritic growth via activation of ERK1/2. Modulation of the activational status of Rit may therefore represent a generalized mechanism across divergent neuronal cell types for regulating axonal versus dendritic growth modes.

A novel cyclic AMP-dependent Epac-Rit signaling pathway contributes to PACAP38-mediated neuronal differentiation.

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G(s)alpha protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of G(s)alpha/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.

Analysis of Rit signaling and biological activity.

Rit (Ras-like expressed in many tissues) is the founding member of a novel subgroup within the larger Ras superfamily of small GTP-binding proteins. Although Rit shares more than 50% amino acid identity with Ras, it contains a unique effector domain in common with the closely related Rin and Drosophila Ric proteins and lacks the C-terminal lipidation motifs critical for the membrane association and biological activity of many Ras proteins. Interestingly, whereas Rit has only modest transforming ability when assayed in NIH 3T3 cells, Rit exhibits neuronal differentiation activities comparable to those of oncogenic mutants of Ras when assayed in PC12 and other neuronal cell lines. This cell-type specificity is explained in part by the ability of Rit to selectively activate the neuronal Raf isoform, B-Raf. Importantly, Rit seems to play a critical role in neurotrophin-mediated MAP kinase signaling, because Rit gene silencing significantly alters NGF-dependent MAP kinase signaling and neuronal differentiation. In this chapter, we discuss the reagents and methods used to characterize Rit-mediated signaling to MAP kinase-signaling pathways to determine the extracellular stimuli that regulate Rit activation and to characterize Rit-induced neuronal differentiation.

Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation.

In neuronal precursor cells, the magnitude and longevity of mitogen-activated protein (MAP) kinase cascade activation contribute to the nature of the cellular response, differentiation, or proliferation. However, the mechanisms by which neurotrophins promote prolonged MAP kinase signaling are not well understood. Here we defined the Rin GTPase as a novel component of the regulatory machinery contributing to the selective integration of MAP kinase signaling and neuronal development. Rin is expressed exclusively in neurons and is activated by neurotrophin signaling, and loss-of-function analysis demonstrates that Rin makes an essential contribution to nerve growth factor (NGF)-mediated neuronal differentiation. Most surprisingly, although Rin was unable to stimulate MAP kinase activity in NIH 3T3 cells, it potently activated isoform-specific p38alpha MAP kinase signaling and weakly stimulated ERK signaling in pheochromocytoma (PC6) cells. This cell-type specificity is explained in part by the finding that Rin binds and stimulates b-Raf but does not activate c-Raf. Accordingly, selective down-regulation of Rin in PC6 cells suppressed neurotrophin-elicited activation of b-Raf and p38, without obvious effects on NGF-induced ERK activation. Moreover, the ability of NGF to promote neurite outgrowth was inhibited by Rin knockdown. Together, these observations establish Rin as a neuronal specific regulator of neurotrophin signaling, required to couple NGF stimulation to sustain activation of p38 MAP kinase and b-Raf signaling cascades required for neuronal development.

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades.

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling.

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.