The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
David P Siderovski - One of the best experts on this subject based on the ideXlab platform.
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Role of RGS12 in the differential regulation of kappa opioid receptor-dependent signaling and behavior
Neuropsychopharmacology, 2019Co-Authors: Joshua D Gross, David P Siderovski, Adam B. Schroer, Shane W Kaski, Karl T. Schmidt, Elizabeth S. Cogan, Kristen M. Boyt, Kim Wix, Zoe A. Mcelligott, Vincent SetolaAbstract:Kappa opioid receptor (KOR) agonists show promise in ameliorating disorders, such as addiction and chronic pain, but are limited by dysphoric and aversive side effects. Clinically beneficial effects of KOR agonists (e.g., analgesia) are predominantly mediated by heterotrimeric G protein signaling, whereas β-arrestin signaling is considered central to their detrimental side effects (e.g., dysphoria/aversion). Here we show that Regulator of G protein Signaling-12 (RGS12), via independent signaling mechanisms, simultaneously attenuates G protein signaling and augments β-arrestin signaling downstream of KOR, exhibiting considerable selectivity in its actions for KOR over other opioid receptors. We previously reported that RGS12-null mice exhibit increased dopamine transporter-mediated dopamine (DA) uptake in the ventral (vSTR), but not dorsal striatum (dSTR), as well as reduced psychostimulant-induced hyperlocomotion; in the current study, we found that these phenotypes are reversed following KOR antagonism. Fast-scan cyclic voltammetry studies of dopamine (DA) release and reuptake suggest that striatal disruptions to KOR-dependent DAergic neurotransmission in RGS12-null mice are restricted to the nucleus accumbens. In both ventral striatal tissue and transfected cells, RGS12 and KOR are seen to interact within a protein complex. Ventral striatal-specific increases in KOR levels and KOR-induced G protein activation are seen in RGS12-null mice, as well as enhanced sensitivity to KOR agonist-induced hypolocomotion and analgesia—G protein signaling-dependent behaviors; a ventral striatal-specific increase in KOR levels was also observed in β-arrestin-2-deficient mice, highlighting the importance of β-arrestin signaling to establishing steady-state KOR levels in this particular brain region. Conversely, RGS12-null mice exhibited attenuated KOR-induced conditioned place aversion (considered a β-arrestin signaling-dependent behavior), consistent with the augmented KOR-mediated β-arrestin signaling seen upon RGS12 over-expression. Collectively, our findings highlight a role for RGS12 as a novel, differential regulator of both G protein-dependent and -independent signaling downstream of KOR activation.
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A role for Regulator of G protein Signaling-12 (RGS12) in the balance between myoblast proliferation and differentiation.
PloS one, 2019Co-Authors: Adam B. Schroer, Melinda D. Willard, Emily A. Oestreich, Junaith S. Mohamed, Vincent Setola, David P SiderovskiAbstract:Regulators of G Protein Signaling (RGS proteins) inhibit G protein-coupled receptor (GPCR) signaling by accelerating the GTP hydrolysis rate of activated Gα subunits. Some RGS proteins exert additional signal modulatory functions, and RGS12 is one such protein, with five additional, functional domains: a PDZ domain, a phosphotyrosine-binding domain, two Ras-binding domains, and a Gα·GDP-binding GoLoco motif. RGS12 expression is temporospatially regulated in developing mouse embryos, with notable expression in somites and developing skeletal muscle. We therefore examined whether RGS12 is involved in the skeletal muscle myogenic program. In the adult mouse, RGS12 is expressed in the tibialis anterior (TA) muscle, and its expression is increased early after cardiotoxin-induced injury, suggesting a role in muscle regeneration. Consistent with a potential role in coordinating myogenic signals, RGS12 is also expressed in primary myoblasts; as these cells undergo differentiation and fusion into myotubes, RGS12 protein abundance is reduced. Myoblasts isolated from mice lacking RGS12 expression have an impaired ability to differentiate into myotubes ex vivo, suggesting that RGS12 may play a role as a modulator/switch for differentiation. We also assessed the muscle regenerative capacity of mice conditionally deficient in skeletal muscle RGS12 expression (via Pax7-driven Cre recombinase expression), following cardiotoxin-induced damage to the TA muscle. Eight days post-damage, mice lacking RGS12 in skeletal muscle had attenuated repair of muscle fibers. However, when mice lacking skeletal muscle expression of RGS12 were cross-bred with mdx mice (a model of human Duchenne muscular dystrophy), no increase in muscle degeneration was observed over time. These data support the hypothesis that RGS12 plays a role in coordinating signals during the myogenic program in select circumstances, but loss of the protein may be compensated for within model syndromes of prolonged bouts of muscle damage and repair.
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Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants.
Journal of psychopharmacology (Oxford England), 2018Co-Authors: Joshua D Gross, David P Siderovski, Adam B. Schroer, Shane W Kaski, Kimberley A Wix, Vincent SetolaAbstract:Regulators of G protein signaling are proteins that accelerate the termination of effector stimulation after G protein-coupled receptor activation. Many regulators of G protein signaling proteins are highly expressed in the brain and therefore considered potential drug discovery targets for central nervous system pathologies; for example, here we show that RGS12 is highly expressed in microdissected mouse ventral striatum. Given a role for the ventral striatum in psychostimulant-induced locomotor activity, we tested whether RGS12 genetic ablation affected behavioral responses to amphetamine and cocaine. RGS12 loss significantly decreased hyperlocomotion to lower doses of both amphetamine and cocaine; however, other outcomes of administration (sensitization and conditioned place preference) were unaffected, suggesting that RGS12 does not function in support of the rewarding properties of these psychostimulants. To test whether observed response changes upon RGS12 loss were caused by changes to dopamine transporter expression and/or function, we prepared crude membranes from the brains of wild-type and RGS12-null mice and measured dopamine transporter-selective [3H]WIN 35428 binding, revealing an increase in dopamine transporter levels in the ventral-but not dorsal-striatum of RGS12-null mice. To address dopamine transporter function, we prepared striatal synaptosomes and measured [3H]dopamine uptake. Consistent with increased [3H]WIN 35428 binding, dopamine transporter-specific [3H]dopamine uptake in RGS12-null ventral striatal synaptosomes was found to be increased. Decreased amphetamine-induced locomotor activity and increased [3H]WIN 35428 binding were recapitulated with an independent RGS12-null mouse strain. Thus, we propose that RGS12 regulates dopamine transporter expression and function in the ventral striatum, affecting amphetamine- and cocaine-induced increases in dopamine levels that specifically elicit acute hyperlocomotor responses.
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A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction
Combinatorial chemistry & high throughput screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P SiderovskiAbstract:The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
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selective role for RGS12 as a ras raf mek scaffold in nerve growth factor mediated differentiation
The EMBO Journal, 2007Co-Authors: Melinda D. Willard, Francis S. Willard, Steven D. Cappell, William D. Snider, David P SiderovskiAbstract:Regulator of G‐protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G‐protein α subunits and thus inhibit signaling by many G protein‐coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase‐accelerating activity. RGS12 contains a tandem repeat of Ras‐binding domains but, to date, the role of this protein in Ras‐mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H‐Ras, B‐Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF‐mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA‐mediated knockdown of RGS12 expression also inhibits NGF‐induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor‐selective, role in coordinating Ras‐dependent signals that are required for promoting and/or maintaining neuronal differentiation.
Shuying Yang - One of the best experts on this subject based on the ideXlab platform.
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RGS12 is a novel tumor suppressor in osteosarcoma that inhibits YAP-TEAD1-Ezrin signaling
Oncogene, 2021Co-Authors: Min Liu, Shuting Yang, Ashley M. Fuller, T. S. Karin Eisinger-mathason, Shuying YangAbstract:Osteosarcoma (OS) is the most common primary malignancy of the bone that predominantly affects children and adolescents. Hippo pathway is a crucial regulator of organ size and tumorigenesis. However, how Hippo pathway regulates the occurrence of osteosarcoma is largely unknown. Here, we reported the regulator of G protein signaling protein 12 (RGS12) is a novel Hippo pathway regulator and tumor suppressor of osteosarcoma. Depletion of RGS12 promotes osteosarcoma progression and lung metastasis in an orthotopic xenograft mouse model. Our data showed that the knockdown of RGS12 upregulates Ezrin expression through promoting the GNA12/13-RhoA-YAP pathway. Moreover, RGS12 negatively regulates the transcriptional activity of YAP/TEAD1 complex through its PDZ domain function to inhibit the expression and function of the osteosarcoma marker Ezrin. PDZ domain peptides of RGS12 can inhibit the development of intratibial tumor and lung metastases. Collectively, this study identifies that the RGS12 is a novel tumor suppressor in osteosarcoma through inhibiting YAP-TEAD1-Ezrin signaling pathway and provides a proof of principle that targeting RGS12 may be a therapeutic strategy for osteosarcoma.
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RGS12 Represses Oral Cancer via the Phosphorylation and SUMOylation of PTEN.
Journal of dental research, 2020Co-Authors: Gongsheng Yuan, Shuying Yang, D Zhang, Shuting YangAbstract:Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer characterized by aggressive local invasion and metastasis. The pathogenesis of OSCC is mainly due to the accumulation of genetic alterations in epithelial cells, but the underlying mechanism for its development remains unclear. Here, we found that the expression level of regulator of G protein signaling 12 (RGS12) was significantly reduced in human OSCC. To understand the role and mechanism of RGS12 in OSCC, we generated a novel RGS12 global knockout (CMVCre/+; RGS12fl/fl) mouse model by crossing RGS12fl/fl mice with CMV-Cre transgenic mice and then further induced the mice to develop OSCC by using 4-nitroquinoline 1-oxide (4NQO). Deletion of RGS12 exhibited aggressive OSCC in the tongue compared with the control RGS12fl/fl mice. Knockdown of RGS12 in OSCC cells significantly increased cell proliferation and migration. Mechanistically, we found that RGS12 associated with phosphatase and tension homolog (PTEN) via the PDZ domain to upregulate the phosphorylation and SUMOylation of PTEN and then correspondingly inactivated the AKT/mTOR signaling pathway. To test the potential therapeutic effect of RGS12 on OSCC, we overexpressed RGS12 in OSCC cells and found a significant inhibition of cancer cell proliferation and migration. Moreover, subcutaneous inoculation of RGS12-overexpressed OSCC cells in NOD scid mice showed a significant reduction in tumor formation. Our findings reveal that RGS12 is an essential tumor suppressor and highlights RGS12 as a potential therapeutic target and prognostic biomarker of OSCC.
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RGS12 is required for the maintenance of mitochondrial function during skeletal development
Cell Discovery, 2020Co-Authors: Gongsheng Yuan, Shuting Yang, Min Liu, Shuying YangAbstract:Mitochondrial morphology and function are crucial for tissue homeostasis, such as for skeletal development, but the cellular and molecular mechanisms remain unclear. Here, we provide evidence that regulator of G-protein signaling 12 (RGS12) is present in the mitochondria of primary chondrocytes and cartilage tissues. Deletion of RGS12 in type II collagen-positive cells led to a significant decrease in mitochondrial number, membrane potential, and oxidative phosphorylation function. Mechanistically, RGS12 promoted the function of ATP5A as an enhancer of tyrosine phosphorylation. Mice with RGS12 deficiency in the chondrocyte lineage showed serious body retardation, decreased bone mass, and chondrocyte apoptosis due to the defective activity of ATP synthase. To our knowledge, this is the first report that RGS12 is required for maintaining the function of mitochondria, which may allow it to orchestrate responses to cellular homeostasis.
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RGS12 Is a Novel Critical NF-κB Activator in Inflammatory Arthritis.
iScience, 2020Co-Authors: Gongsheng Yuan, Shuting Yang, Merry Jo Oursler, Lianping Xing, Shuying YangAbstract:Summary Rheumatoid arthritis (RA) is the most common inflammatory disease, which currently lacks effective treatment. Here, we discovered that the Regulator of G Protein Signaling 12 (RGS12) plays a key role in regulating inflammation. Transcriptional and protein analysis revealed that RGS12 was upregulated in human and mouse RA macrophages. Deletion of RGS12 in myeloid lineage or globally inhibits the development of collagen-induced arthritis including joint swelling and bone destruction. Mechanistically, RGS12 associates with NF-κB(p65) to activate its phosphorylation and nuclear translocation through PTB domain, and NF-κB(p65) regulates RGS12 expression in a transcriptional manner. The nuclear translocation ability of NF-κB(p65) and RGS12 can both be enhanced by cyclooxygenase-2 (COX2). Furthermore, ablation of RGS12 via RNA interference significantly blocks the inflammatory process in vivo and in vitro. These results demonstrate that RGS12 plays a critical role in the pathogenesis of inflammatory arthritis.
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Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species.
eLife, 2019Co-Authors: Megan M. Jones, Merry Jo Oursler, Shuting Yang, Shuying YangAbstract:Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that RGS12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted RGS12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed RGS12 in RAW264.7 cells. RGS12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. RGS12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of RGS12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in RGS12-deficient cells. Conversely, RGS12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of RGS12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.
Bryan E. Snow - One of the best experts on this subject based on the ideXlab platform.
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gtpase activating specificity of RGS12 and binding specificity of an alternatively spliced pdz psd 95 dlg zo 1 domain
Journal of Biological Chemistry, 1998Co-Authors: Bryan E. Snow, Randy A Hall, Andrejs M Krumins, Denis Bouchard, Stephen Chung, Joan Mangion, Alfred G Gilman, Robert J Lefkowitz, David P SiderovskiAbstract:Abstract Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein α subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3′ end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3′ end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class α subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3′ exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.
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GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD-95/Dlg/ZO-1) domain.
The Journal of biological chemistry, 1998Co-Authors: Bryan E. Snow, Randy A Hall, Andrejs M Krumins, Denis Bouchard, Stephen Chung, Joan Mangion, Alfred G Gilman, Robert J Lefkowitz, David P SiderovskiAbstract:Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. Here, we describe the GTPase specificity of RGS12 and identify four alternatively spliced forms of human RGS12 mRNA. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms, of 3.1 and 3.7 kb, which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization.
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Molecular cloning and expression analysis of rat RGS12 and Rgs14
Biochemical and biophysical research communications, 1997Co-Authors: Bryan E. Snow, Laarni Antonio, Sid Suggs, Howard B. Gutstein, David P SiderovskiAbstract:Abstract We report the cloning of two novel rat regulators of G-protein signaling (RGS) cDNAs using a degenerate PCR strategy. The rRGS12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. We have also identified the human orthologue of rRGS12 by alignment of cosmid sequences in the database which map the human RGS12 gene to chromosome 4p16.3. Furthermore, we identified human ESTs with high homology to rRgs14 which map to human chromosome 5qter. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRGS12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. We have also identified conserved regions in RGS12 which are similar to protein domains found in mouse rhophilin and coiled-coil proteins suggesting possible interactions with ras-like G-proteins.
Michael X. Zhu - One of the best experts on this subject based on the ideXlab platform.
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Regulator of G-protein signalling and GoLoco proteins suppress TRPC4 channel function via acting at Gαi/o
The Biochemical journal, 2016Co-Authors: Jaepyo Jeon, Dhananjay P. Thakur, Jin Bin Tian, Michael X. ZhuAbstract:Transient receptor potential canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions. Previously, TRPC4 was shown to be activated by the Gi/o subgroup of heterotrimeric G-proteins involving Gαi/o, rather than Gβγ, subunits. Because the lifetime and availability of Gα-GTP are regulated by regulators of G-protein signalling (RGS) and Gαi/o-Loco (GoLoco) domain-containing proteins via their GTPase-activating protein (GAP) and guanine-nucleotide-dissociation inhibitor (GDI) functions respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch-clamp recordings, we show that both RGS and GoLoco proteins [RGS4, RGS6, RGS12, RGS14, LGN or activator of G-protein signalling 3 (AGS3)] suppress receptor-mediated TRPC4 activation without causing detectable basal current or altering surface expression of the channel protein. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco protein AGS3. In addition, RGS, but not GoLoco, proteins accelerate desensitization of receptor-activation evoked TRPC4 currents. The inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS12 and RGS14, which contain both RGS and GoLoco domains. Our data support the notion that the Gα, but not Gβγ, arm of the Gi/o signalling is involved in TRPC4 activation and unveil new roles for RGS and GoLoco domain proteins in fine-tuning TRPC4 activities. The versatile and diverse functions of RGS and GoLoco proteins in regulating G-protein signalling may underlie the complexity of receptor-operated TRPC4 activation in various cell types under different conditions.
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Regulator of G Protein Signaling (RGS) and Goloco Proteins Suppress TRPC4 Channel Function via Acting at GAI/O
Biophysical Journal, 2016Co-Authors: Jaepyo Jeon, Dhananjay P. Thakur, Jin Bin Tian, Michael X. ZhuAbstract:Transient Receptor Potential Canonical 4 (TRPC4) forms non-selective cation channels implicated in the regulation of diverse physiological functions, including smooth muscle contractility, synaptic transmission, and neurodegeneration. Unique among TRPC subfamily members, TRPC4 activation is dependent on Gi/o subgroup of heterotrimeric G proteins in addition to stimulation of phospholipase C pathway. Previously, the Gi/o, rather than Gbetgamma, subunits were suggested to mediate Gi/o-induced TRPC4 activation. Because the lifetime and availability of Galpha-GTP are regulated by Regulators of G protein signaling (RGS) and GoLoco domain-containing proteins via their GTPase-activating protein (GAP) and guanosine nucleotide dissociation inhibition (GDI) functions, respectively, we tested how RGS and GoLoco domain proteins affect TRPC4 currents activated via Gi/o-coupled receptors. Using whole-cell patch clamp recordings, we show that both RGS (RGS4, RGS6, RGS12, or RGS14) and GoLoco domain proteins (LGN, AGS3, RGS12, or RGS14) suppress receptor-mediated TRPC4 activation without eliciting any basal current. The inhibitory effects are dependent on the GAP and GoLoco domains and facilitated by enhancing membrane targeting of the GoLoco domains of AGS3. In addition, the Gi/o-specific RGS proteins, but not the GoLoco domain proteins, accelerate desensitization of receptor activation-evoked TRPC4 currents. We found that the inhibitory effects of RGS and GoLoco domains are additive and are most prominent with RGS proteins that contain both the RGS and GoLoco domains, i.e. RGS12 and RGS14. Our data support the notion that the Galpha, but not Gbetagamma, arm of the Gi/o signaling is involved in TRPC4 activation and indicate that acting through Gi/o via GAP and GDI functions, RGS and GoLoco domain proteins can fine tune TRPC4 activity and thereby allow delicate regulation of cellular functions in nervous and other systems.
Francis S. Willard - One of the best experts on this subject based on the ideXlab platform.
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Regulator of G-protein signaling 14 (RGS14) is a selective H-Ras effector.
PloS one, 2009Co-Authors: Francis S. Willard, Randall J Kimple, Adam J. Kimple, Melinda D. Willard, Meera Soundararajan, Emily A. Oestreich, Nathaniel A. Sowa, Declan A. Doyle, Channing J. DerAbstract:Regulator of G-protein signaling (RGS) proteins have been well-described as accelerators of Galpha-mediated GTP hydrolysis ("GTPase-accelerating proteins" or GAPs). However, RGS proteins with complex domain architectures are now known to regulate much more than Galpha GTPase activity. RGS14 contains tandem Ras-binding domains that have been reported to bind to Rap- but not Ras GTPases in vitro, leading to the suggestion that RGS14 is a Rap-specific effector. However, more recent data from mammals and Drosophila imply that, in vivo, RGS14 may instead be an effector of Ras. Full-length and truncated forms of purified RGS14 protein were found to bind indiscriminately in vitro to both Rap- and Ras-family GTPases, consistent with prior literature reports. In stark contrast, however, we found that in a cellular context RGS14 selectively binds to activated H-Ras and not to Rap isoforms. Co-transfection / co-immunoprecipitation experiments demonstrated the ability of full-length RGS14 to assemble a multiprotein complex with components of the ERK MAPK pathway in a manner dependent on activated H-Ras. Small interfering RNA-mediated knockdown of RGS14 inhibited both nerve growth factor- and basic fibrobast growth factor-mediated neuronal differentiation of PC12 cells, a process which is known to be dependent on Ras-ERK signaling. In cells, RGS14 facilitates the formation of a selective Ras.GTP-Raf-MEK-ERK multiprotein complex to promote sustained ERK activation and regulate H-Ras-dependent neuritogenesis. This cellular function for RGS14 is similar but distinct from that recently described for its closely-related paralogue, RGS12, which shares the tandem Ras-binding domain architecture with RGS14.
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A high throughput fluorescence polarization assay for inhibitors of the GoLoco motif/G-alpha interaction
Combinatorial chemistry & high throughput screening, 2008Co-Authors: Adam J. Kimple, Francis S. Willard, Adam Yasgar, Mark A. Hughes, Ajit Jadhav, Robin E. Muller, Christopher P. Austin, James Inglese, Gordon C. Ibeanu, David P SiderovskiAbstract:The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.
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selective role for RGS12 as a ras raf mek scaffold in nerve growth factor mediated differentiation
The EMBO Journal, 2007Co-Authors: Melinda D. Willard, Francis S. Willard, Steven D. Cappell, William D. Snider, David P SiderovskiAbstract:Regulator of G‐protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G‐protein α subunits and thus inhibit signaling by many G protein‐coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase‐accelerating activity. RGS12 contains a tandem repeat of Ras‐binding domains but, to date, the role of this protein in Ras‐mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H‐Ras, B‐Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF‐mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA‐mediated knockdown of RGS12 expression also inhibits NGF‐induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor‐selective, role in coordinating Ras‐dependent signals that are required for promoting and/or maintaining neuronal differentiation.
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Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor‐mediated differentiation
The EMBO journal, 2007Co-Authors: Melinda D. Willard, Francis S. Willard, Steven D. Cappell, William D. Snider, David P SiderovskiAbstract:Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.
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the effect of RGS12 on pdgfβ receptor signalling to p42 p44 mitogen activated protein kinase in mammalian cells
Cellular Signalling, 2006Co-Authors: Balwinder Sambi, David P Siderovski, Melinda D. Hains, Catherine M. Waters, Michelle Connell, Francis S. Willard, Adam J. Kimple, Susan Pyne, Nigel J. PyneAbstract:We have previously shown that the PDGFβ receptor uses a classical GPCR-mediated pathway in order to induce efficient activation of p42/p44 MAPK in response to PDGF. We therefore, considered the possibility that GTPase accelerating proteins (RGS proteins), which regulate GPCR signalling, modulate PDGFβ receptor-mediated signal transmission. Several lines of evidence were obtained to support functional interaction between the PDGFβ receptor and RGS12 in HEK 293 and airway smooth muscle cells. Firstly, the over-expression of the RGS12 PDZ/PTB domain N-terminus or RGS12 PTB domain reduced the PDGF-induced activation of p42/p44 MAPK. Secondly, the RGS12 PDZ/PTB domain N-terminus and RGS12 PDZ domain can form a complex with the PDGFβ receptor. Therefore, the results presented here provide the first evidence to support the concept that the PDZ/PTB domain N-terminus and/or the PTB domain of RGS12 may modulate PDGFβ receptor signalling. In airway smooth muscle cells, over-expressed recombinant RGS12 and the isolated PDZ/PTB domain N-terminus co-localised with PDGFβ receptor in cytoplasmic vesicles. To provide additional evidence for a role of the PDZ/PTB domain N-terminus, we used RGS14. RGS14 has the same C-terminal domain architecture of an RGS box, tandem Ras-binding domains (RBDs) and GoLoco motif as RGS12, but lacks the PDZ/PTB domain N-terminus. In this regard, RGS14 exhibited a different sub-cellular distribution compared with RGS12, being diffusely distributed in ASM cells. These findings suggest that RGS12 via its PDZ/PTB domain N-terminus may regulate trafficking of the PDGFβ receptor in ASM cells.