ROLE OF RECEPTORS COUPLED TO G PROTEINS IN CELL DIVISION: THE EXAMPLE OF 5-HT1A RECEPTORS TRANSFECTED IN NIH-3T3 CELLS

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.

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Potent transforming activity of the small GTP-binding protein Rit in NIH 3T3 cells: evidence for a role of a p38γ-dependent signaling pathway

FEBS Letters ◽

10.1016/s0014-5793(01)03264-1 ◽

2001 ◽

Vol 511 (1-3) ◽

pp. 15-20 ◽

Cited By ~ 28

Author(s):

Kaoru Sakabe ◽

Hidemi Teramoto ◽

Muriel Zohar ◽

Babak Behbahani ◽

Hiroshi Miyazaki ◽

...

Keyword(s):

Signaling Pathway ◽

Binding Protein ◽

3T3 Cells ◽

Gtp Binding Protein ◽

Gtp Binding ◽

Transforming Activity ◽

Nih 3T3 ◽

Dependent Signaling Pathway ◽

Nih 3T3 Cells

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FoxF1 is Required for Ciliogenesis and Distribution of Sonic Hedgehog Signaling Components in Cilium

Current Molecular Medicine ◽

10.2174/1566524019666190405115420 ◽

2019 ◽

Vol 19 (5) ◽

pp. 326-334

Author(s):

Lu Huang ◽

Marco Tjakra ◽

Desha Luo ◽

Lin Wen ◽

Daoxi Lei ◽

...

Keyword(s):

Sonic Hedgehog ◽

Hedgehog Signaling ◽

3T3 Cells ◽

Shh Signaling ◽

Nih 3T3 ◽

Signaling Components ◽

Nih 3T3 Cells

Background: In vertebrates, cilium is crucial for Hedgehog signaling transduction. Forkhead box transcriptional factor FoxF1 is reported to be associated with Sonic Hedgehog (Shh) signaling in many cases. However, the role of FoxF1 in cilium remains unknown. Here, we showed an essential role of FoxF1 in the regulation of ciliogenesis and in the distribution of Shh signaling components in cilium. Methods: NIH/3T3 cells were serum starved for 24h to induce cilium. Meanwhile, shRNA was used to knockdown the FoxF1 expression in the cells and CRISPR/Cas9 was used to generate the FoxF1 zebrafish mutant. The mRNA and protein expression of indicated genes were detected by the qRT-PCR and western blot, respectively. Immunofluorescence staining was performed to detect the cilium and Shh components distribution. Results: FoxF1 knockdown decreased the cilium length in NIH/3T3 cells. Meanwhile, the disruption of FoxF1 function inhibited the expression of cilium-related genes and caused an abnormal distribution of Shh components in the cilium. Furthermore, homozygous FoxF1 mutants exhibited defective development of pronephric cilium in early zebrafish embryos. Conclusion: Together, our data illustrated that FoxF1 is required for ciliogenesis in vitro and in vivo and for the proper localization of Shh signaling components in cilium.

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Inhibition of Ca2+ inflow causes an abrupt cessation of growth-factor-induced repetitive free Ca2+ transients in single NIH-3T3 cells

Biochemical Journal ◽

10.1042/bj2780849 ◽

1991 ◽

Vol 278 (3) ◽

pp. 849-855 ◽

Cited By ~ 12

Author(s):

A J Polverino ◽

B P Hughes ◽

G J Barritt

Keyword(s):

Calf Serum ◽

Cell Types ◽

Transient Increase ◽

Foetal Calf Serum ◽

3T3 Cells ◽

3T3 Fibroblasts ◽

Carbonyl Cyanide ◽

Nih 3T3 ◽

Nih 3T3 Cells

In single NIH-3T3 fibroblasts loaded with fura-2, bombesin induced one of three patterns of increase in the concentration of intracellular free Ca2+ [( Ca2+]i): a single transient increase, a sustained increase, or repetitive transient increases in [Ca2+]i. Foetal-calf serum and ATP also gave these three patterns of response, although a lower proportion of cells gave repetitive Ca2+ transients in response to ATP. An increase in the concentration of bombesin from 1 to 25 nM increased the proportion of cells which exhibited repetitive Ca2+ transients. At 25 nM-bombesin, the proportion of cells which exhibited repetitive Ca2+ transients increased as the extracellular Ca2+ (Ca2+o) concentration was increased from 1 to 5 mM. Removal of Ca2+o by addition of EGTA, or inhibition of Ca2+ inflow by treatment of cells incubated in the presence of Ca2+o with verapamil or an activator of protein kinase C, abruptly terminated repetitive Ca2+ transients, with only one transient observed after the cessation of Ca2+ inflow. Repetitive Ca2+ transients were not observed in cells incubated in the absence of Ca2+o and in the presence of EGTA. Addition of Ca2+o to cells previously incubated in the presence of EGTA caused a resumption of repetitive Ca2+ transients. Addition of thapsigargin alone induced a large transient increase in [Ca2+]i, whereas much smaller transient increases in [Ca2+]i were induced in about 30% of cells tested by caffeine or carbonyl cyanide m-chlorophenylhydrazone (CCCP) plus oligomycin. Thapsigargin or the combination of CCCP plus oligomycin completely inhibited bombesin-induced repetitive Ca2+ transients, whereas caffeine had no effect. It is concluded from the studies of the role of Ca2+o that NIH-3T3 cells differ from other cell types in the anatomical or chemical links between extracellular Ca2+ and the intracellular stores involved in the generation of Ca2+ transients, whereas the results of the experiments with inhibitors indicate that the generation of repetitive Ca2+ transients in NIH-3T3 cells is unlikely to involve Ca(2+)-induced Ca2+ release from caffeine-sensitive stores.

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Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4687 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4687-4693 ◽

Cited By ~ 115

Author(s):

G Kalinec ◽

A J Nazarali ◽

S Hermouet ◽

N Xu ◽

J S Gutkind

Keyword(s):

Adenylyl Cyclase ◽

G Proteins ◽

Alpha Subunit ◽

Endocrine Tumors ◽

3T3 Cells ◽

Activating Mutations ◽

Alpha Subunits ◽

G Alpha Q ◽

Nih 3T3 ◽

Nih 3T3 Cells

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.

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Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells

Biochemistry and Biophysics Reports ◽

10.1016/j.bbrep.2016.08.025 ◽

2016 ◽

Vol 8 ◽

pp. 162-167

Author(s):

Rebecca A. Hill ◽

Wu Xu ◽

Masami Yoshimura

Keyword(s):

Gene Expression ◽

Adenylyl Cyclase ◽

3T3 Cells ◽

Regulated Gene Expression ◽

Nih 3T3 ◽

Nih 3T3 Cells

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RACK1, a Receptor for Activated C Kinase and a Homolog of the β Subunit of G Proteins, Inhibits Activity of Src Tyrosine Kinases and Growth of NIH 3T3 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.18.6.3245 ◽

1998 ◽

Vol 18 (6) ◽

pp. 3245-3256 ◽

Cited By ~ 186

Author(s):

Betty Y. Chang ◽

Karen B. Conroy ◽

Eric M. Machleder ◽

Christine A. Cartwright

Keyword(s):

G Proteins ◽

Tyrosine Kinases ◽

Sh2 Domain ◽

Dependent Manner ◽

Β Subunit ◽

3T3 Cells ◽

Concentration Dependent Manner ◽

Human Lung Fibroblast ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT To isolate and characterize proteins that interact with the unique domain and SH3 and SH2 domains of Src and potentially regulate Src activity, we used the yeast two-hybrid assay to screen a human lung fibroblast cDNA library. We identified RACK1, a receptor for activated C kinase and a homolog of the β subunit of G proteins, as a Src-binding protein. Using GST-Src fusion proteins, we determined that RACK1 binds to the SH2 domain of Src. Coimmunoprecipitation of Src and RACK1 was demonstrated with NIH 3T3 cells. Purified GST-RACK1 inhibited the in vitro kinase activity of Src in a concentration-dependent manner. GST-RACK1 (2 μM) inhibited the activities of purified Src and Lck tyrosine kinases by 40 to 50% but did not inhibit the activities of three serine/threonine kinases that we tested. Tyrosine phosphorylation on many cellular proteins decreased in 293T cells that transiently overexpressed RACK1. Src activity and cell growth rates decreased by 40 to 50% in NIH 3T3 cells that stably overexpressed RACK1. Flow cytometric analyses revealed that RACK1-overexpressing cells do not show an increased rate of necrosis or apoptosis but do spend significantly more time in G0/G1 than do wild-type cells. Prolongation of G0/G1 could account for the increased doubling time of RACK1-overexpressing cells. We suggest that RACK1 exerts its effect on the NIH 3T3 cell cycle in part by inhibiting Src activity.

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