The G Protein Gα13 Is Required for Growth Factor-Induced Cell Migration

Shan, Dandan; Chen, Lin; Wang, Dawei; Ya, Tan; Gu, Jiaying; Huang, Xin‐Yun

doi:10.1016/j.devcel.2006.03.014

Cited by 71 publications

(84 citation statements)

References 73 publications

(97 reference statements)

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“…Protein Purification-GST-tagged Ric-8A, His 6 -tagged G␣ 13/i-5 , and His 6 -tagged G␣ i1 proteins were purified from Escherichia coli as described previously (27). BL21 (DE3) E. coli cells harboring pET28a-G␣ 13/i-5 plasmids were grown to A 600 ϭ 0.5-0.6 in LB medium at 37°C and induced with 30 M isopropyl 1-thio-␤-D-galactopyranoside at 30°C overnight (36).…”

Section: Methodsmentioning

confidence: 99%

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Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Is Critical for Growth Factor Receptor-induced Actin Cytoskeletal Reorganization

Wang

Guo

Xing

et al. 2011

Journal of Biological Chemistry

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Heterotrimeric G proteins are critical transducers of cellular signaling. In addition to their classic roles in relaying signals from G protein-coupled receptors (GPCRs), heterotrimeric G proteins also mediate physiological functions from non-GPCRs. Previously, we have shown that G␣ 13 , a member of the heterotrimeric G proteins, is essential for growth factor receptor-induced actin cytoskeletal reorganization such as dynamic dorsal ruffle turnover and cell migration. These G␣ 13 -mediated dorsal ruffle turnover and cell migration by growth factors acting on their receptor tyrosine kinases (RTKs) are independent of GPCRs. However, the mechanism by which RTKs signal to G␣ 13 is not known. Here, we show that cholinesterase-8A (Ric-8A), a nonreceptor guanine nucleotide exchange factor for some heterotrimeric G proteins, is critical for coupling RTKs to G␣ 13 . Down-regulation of Ric-8A protein levels in cells by RNA interference slowed down platelet-derived growth factor (PDGF)-induced dorsal ruffle turnover and inhibited PDGF-initiated cell migration. PDGF was able to increase the activity of Ric-8A in cells. Furthermore, purified Ric-8A proteins interact directly with purified G␣ 13 protein in a nucleotide-dependent manner. Deficiency of Ric-8A prevented the translocation of G␣ 13 to the cell cortex. Hence, Ric-8A is critical for growth factor receptorinduced actin cytoskeletal reorganization.Heterotrimeric G proteins are essential for the transmembrane signaling by G protein-coupled receptors (GPCRs).2 A structurally diverse repertoire of ligands activates GPCRs to elicit their physiological functions (1). Ligand-bound GPCRs function as guanine nucleotide exchange factors (GEFs) catalyzing the exchange of GDP bound on the G␣ subunit with GTP in the presence of G␤␥. This leads to the dissociation of the G␣ subunit from the G␤␥ dimer to form two functional units (G␣ and G␤␥) (2). Both G␣ and G␤␥ subunits signal to various cellular pathways. Based on the sequence and functional homologies, G proteins are grouped into four families: G s , G i , G q , and G 12 (3). Among these four subfamilies of G proteins, the physiological function of the G 12 subfamily is less well understood. In this family, there are two members, G 12 and G 13 . G␣ 12 knock-out mice appeared normal (4). G␣ 13 knock-out mice displayed embryonic lethality (ϳE9.5) (5). The molecular basis that underlies the vascular defect observed in G␣ 13 Ϫ/Ϫ mouse embryos has not been defined.In addition to their classic roles in GPCR signaling, heterotrimeric G proteins have been genetically demonstrated to play important roles in GPCR-independent signaling (6). The best examples are in the mitotic spindle positioning and orientation (in the establishment of cell polarity) during asymmetric division in Caenorhabditis elegans embryos and in Drosophila neuroblasts (7-11). In these processes, G␣ i/o -GDP binds to a protein with the tetratricopeptide-GoLoco domain (such as GPR-1/2 in C. elegans and Pins in Drosophila) and disrupts intramolecular tetratricopeptide-...

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Section: Methodsmentioning

confidence: 99%

“…Recently, we have discovered that G 13 is essential for growth factor-induced cell migration, independent of GPCRs (27). This represents a novel cellular signal aspect and physiological function of heterotrimeric G proteins (28).…”

mentioning

confidence: 99%

Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Is Critical for Growth Factor Receptor-induced Actin Cytoskeletal Reorganization

Wang

Guo

Xing

et al. 2011

Journal of Biological Chemistry

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“…In G␣ 12 G␣ 13 knock-out MEF cells, neither G protein-coupled receptors for thrombin or lysophosphatidic acid nor receptor tyrosine kinases for PDGF or epidermal growth factor induced migration of these cells (18,19,23). To visualize directly the effect of G␣ 12 and G␣ 13 on actin cytoskeletal reorganization, we utilized live cell imaging techniques to compare the dynamics of actin cytoskeletal reorganization in wild-type and G␣ 12 G␣ 13 knock-out MEF cells.…”

Section: Resultsmentioning

confidence: 99%

“…Cell Culture and Transfection-G␣ 12 Ϫ/Ϫ G␣ 13 Ϫ/Ϫ MEF cells were provided by J. Gu and M. Simon (California Institute of Technology) and have been previously described (18,19). Cells were grown in DMEM containing 10% fetal bovine serum and penicillin/streptomycin.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR products of chicken Src constitutively active mutant SrcE378G and SrcY527F cDNAs were subcloned into pcDNA3.1/hyg/myc. HA-Rac1G12V and HA-Rac1T14N were obtained from Guthrie Research Institute.Cell Culture and Transfection-G␣ 12 Ϫ/Ϫ G␣ 13 Ϫ/Ϫ MEF cells were provided by J. Gu and M. Simon (California Institute of Technology) and have been previously described (18,19). Cells were grown in DMEM containing 10% fetal bovine serum and penicillin/streptomycin.…”

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G Proteins G12 and G13 Control the Dynamic Turnover of Growth Factor-induced Dorsal Ruffles

Wang¹,

Ya²,

Kreitzer³

et al. 2006

Journal of Biological Chemistry

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Growth factors induce massive actin cytoskeletal remodeling in cells. These reorganization events underlie various cellular responses such as cell migration and morphological changes. One major form of actin reorganization is the formation and disassembly of dorsal ruffles (also named waves, dorsal rings, or circular ruffles). Dorsal ruffles are involved in physiological functions including cell migration, invasion, macropinocytosis, plasma membrane recycling, and others. Growth factors initiate rapid formation (within 5 min) of circular membrane ruffles, and these ruffles move along the dorsal side of the cells, constrict, close, and eventually disassemble (ϳ20 min). Considerable attention has been devoted to the mechanism by which growth factors induce the formation of dorsal ruffles. However, little is known of the mechanism by which these ruffles are disassembled. Here we have shown that G proteins G 12 and G 13 control the rate of disassembly of dorsal ruffles. In G␣ 12 ؊/؊ G␣ 13 ؊/؊ fibroblast cells, dorsal ruffles induced by growth factor treatment remain visible substantially longer (ϳ60 min) than in wild-type cells, whereas the rate of formation of these ruffles was the same with or without G␣ 12 and G␣ 13 . Thus, G␣ 12 /G␣ 13 critically regulate dorsal ruffle turnover.The earliest ultrastructural changes of cells treated with growth factors are the intensive bursts of ruffling of the dorsal surface plasma membranes as seen under the phase-contrast microscope (1-6). The physiological functions of dorsal ruffles, including macropinocytosis, cell migration, and invasion, are continually expanding (7-10). It has been suggested that one major function of dorsal ruffles is to reorganize the actin cytoskeleton to prepare a static cell for motility (11).In serum-starved fibroblasts, platelet-derived growth factor (PDGF) 2 induces at least two types of membrane ruffles, peripheral membrane ruffles (or lamellipodia) and dorsal ruffles (12). In addition to fibroblast cells, dorsal ruffle formation in response to growth factors has been reported in glial cells, endothelial cells, hippocampal neurons, kidney epithelial cells, and tumor cells (1,2,11,13,14). In addition, other stimuli can induce dorsal ruffles such as activation of IgE receptors in mast cells (15). Dorsal ruffles are dynamic structures. They form and disassemble rapidly (11,16,17 EXPERIMENTAL PROCEDURESReagents-Anti-HA monoclonal antibody and anti-Myc monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc. PDGF-BB was purchased from Oncogene. Texas Red Phalloidin and Alexa Fluor 488 F(abЈ) 2 fragment of goat anti-mouse IgG(HϩL) were purchased from Molecular Probes. ROCK inhibitor Y-27632 and Src family inhibitor PP2 were purchased from BD Biosciences. The PCR products of chicken Src constitutively active mutant SrcE378G and SrcY527F cDNAs were subcloned into pcDNA3.1/hyg/myc. HA-Rac1G12V and HA-Rac1T14N were obtained from Guthrie Research Institute.Cell Culture and Transfection-G␣ 12 Ϫ/Ϫ G␣ 13 Ϫ/Ϫ MEF cells were provided by J. Gu and...

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Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

Baruthio¹,

Quadroni

Rüegg³

et al. 2008

Proteomics

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The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.

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The G Protein Gα13 Is Required for Growth Factor-Induced Cell Migration

Cited by 71 publications

References 73 publications

Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Is Critical for Growth Factor Receptor-induced Actin Cytoskeletal Reorganization

Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Is Critical for Growth Factor Receptor-induced Actin Cytoskeletal Reorganization

G Proteins G12 and G13 Control the Dynamic Turnover of Growth Factor-induced Dorsal Ruffles

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage

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