Gα12 Inhibits α2β1 Integrin–mediated Madin-Darby Canine Kidney Cell Attachment and Migration on Collagen-I and Blocks Tubulogenesis

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that Gα12 inhibits interaction of MDCK cells with collagen-I, the major ligand for α2β1 integrin. Activating Gα12 (QL point mutation or stimulating endogenous Gα12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with Gα12-regulated attachment to collagen-I, Gα12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in Gα12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. Gα12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of α2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated Gα12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, Gα12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for Gα12–integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.     

Modification of Heterotrimeric G-Proteins in Swiss 3T3 Cells Stimulated with Pasteurella multocida Toxin

Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s). The Pasteurella multocida toxin (PMT) mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G_q, G₁₂ and G_i. PMT has been shown by others to lead to the deamidation of recombinant Gα_i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G_q and G_i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα₁₁, a member of the G_q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα₁₃ was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G_s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα_i, but not Gα_q. We also show that PMT inhibits the GTPase activity of G_q.     

PLEKHG2 Promotes Heterotrimeric G Protein βγ-Stimulated Lymphocyte Migration via Rac and Cdc42 Activation and Actin Polymerization

PLEKHG2 is a Dbl family Rho guanine nucleotide exchange factor (RhoGEF) whose gene was originally identified as being upregulated in a leukemia mouse model and was later shown to be activated by heterotrimeric G protein βγ (Gβγ) subunits. However, its function and activation mechanisms remain elusive. Here we show that, compared to its expression in primary human T cells, its expression is upregulated in several leukemia cell lines, including Jurkat T cells. Downregulation of PLEKHG2 in Jurkat T cells by small interfering RNAs (siRNAs) specifically inhibited Gβγ-stimulated Rac and Cdc42, but not RhoA, activation. Consequently, suppressing PLEKHG2 expression blocked actin polymerization and SDF1α-stimulated lymphocyte migration. Additional studies indicate that Gβγ likely activates PLEKHG2, in part by binding the N terminus of PLEKHG2 to release an autoinhibition imposed by its C terminus, which interacts with a region encompassing the catalytic Dbl homology (DH) domain. As a result, overexpressing either the N terminus or the C terminus of PLEKHG2 blocked Gβγ-stimulated Rac and Cdc42 activation and prevented Jurkat T cells from forming membrane protrusions and migrating. Together, our studies have provided the first evidence for the endogenous function of PLEKHG2, which may serve as a key Gβγ-stimulated RhoGEF that regulates lymphocyte chemotaxis via Rac and Cdc42 activation and actin polymerization.     

Growth-Inhibitory Functions of a Mutated Gelsolin (His321) in NIH/3T3 Mouse Fibroblasts

We have previously established a murine flat revertant cell line R1 from an activated H-ras transformant EJ-NIH/3T3 by subjecting it to ethyl methanesulfonate. From the R1 cells, we cloned a mutated gelsolin gene His321 and have shown the inhibitory activity of His321 against EJ-NIH/3T3 tumors. Our present experiments were conducted to find out whether the His321 gene has any effects on untransformed NIH/3T3 fibroblasts. Rhodamine-phalloidin staining revealed that two NIH/3T3 clones expressing His321 (NIH/λ2S-3 and NIH/λ2S-6) form organized actin stress fibers as two clones transfected with the vector alone (NIH/neo-3 and NIH/neo-5). We also found that in a liquid medium, NIH/λ2S-3 and NIH/λ2S-6 grew more slowly than NIH/neo-3 and NIH/neo-5 and that the doubling times of the former were about 10 h slower than those of the latter. To investigate the effects of His321 on the signal transduction pathway necessary for cell growth, we stimulated the cell lines by prostaglandin E1 (PGE1), a platelet-derived growth factor (PDGF), or the epidermal growth factor (EGF). Although stimulation by PGE1 increased intercellular cyclic AMP in R1 cells, it did not do so in NIH/λ2S-3 and NIH/λ2S-6 cells. On the other hand, stimulation by PDGF or EGF induced far less DNA synthesis in NIH/λ2S-3 and NIH/λ2S-6 than in NIH/neo-3 and NIH/neo5. These results suggest that through the effects on the signal transduction pathway of PDGF and/or EGF His321-mutated gelsolin inhibits the growth of NIH/3T3.     

A Function for Phosphatidylinositol 3-Kinase β (p85α-p110β) in Fibroblasts during Mitogenesis: Requirement for Insulin- and Lysophosphatidic Acid-Mediated Signal Transduction

We have previously shown that phosphatidylinositol 3-kinase α (PI 3-Kα) (p85α-p110α) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185–9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kβ (p85α-p110β) during mitogenesis. By using antibodies specific to p110β we showed that PI 3-Kβ is expressed in NIH 3T3 cells. PI 3-Kβ and PI 3-Kα have common features: PI 3-Kβ is tightly associated with a protein serine kinase that phosphorylates p85α, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kβ was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kα was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110β into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kβ plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kα and PI 3-Kβ, suggesting that Ras uses other effectors for DNA synthesis.     

Activation of Microtubule Dynamics Increases Neuronal Growth via the Nerve Growth Factor (NGF)- and Gαs-mediated Signaling Pathways

Signals that activate the G protein Gα_s and promote neuronal differentiation evoke Gα_s internalization in rat pheochromocytoma (PC12) cells. These agents also significantly increase Gα_s association with microtubules, resulting in an increase in microtubule dynamics because of the activation of tubulin GTPase by Gα_s. To determine the function of Gα_s/microtubule association in neuronal development, we used real-time trafficking of a GFP-Gα_s fusion protein. GFP-Gα_s concentrates at the distal end of the neurites in differentiated living PC12 cells as well as in cultured hippocampal neurons. Gα_s translocates to specialized membrane compartments at tips of growing neurites. A dominant-negative Gα chimera that interferes with Gα_s binding to tubulin and activation of tubulin GTPase attenuates neurite elongation and neurite number both in PC12 cells and primary hippocampal neurons. This effect is greatest on differentiation induced by activated Gα_s. Together, these data suggest that activated Gα_s translocates from the plasma membrane and, through interaction with tubulin/microtubules in the cytosol, is important for neurite formation, development, and outgrowth. Characterization of neuronal G protein dynamics and their contribution to microtubule dynamics is important for understanding the molecular mechanisms by which G protein-coupled receptor signaling orchestrates neuronal growth and differentiation.     

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-α in the primary cilium

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α and the Na⁺/H⁺ exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737^orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5′-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA–mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737^orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.     

A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

Background

Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells.

Methodology/Principal Findings

In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects.

Conclusions

3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

Prostate tumor cells infected with a recombinant influenza virus expressing a truncated NS1 protein activate cytolytic CD8+ cells to recognize noninfected tumor cells

Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.     

Differentiation of Human T Cells Alters Their Repertoire of G Protein α-Subunits

Because T cell differentiation leads to an expanded repertoire of chemokine receptors, a subgroup of G protein-coupled receptors, we hypothesized that the repertoire of G proteins might be altered in parallel. We analyzed the abundance of mRNA and/or protein of six G protein α-subunits in human CD4⁺ and CD8⁺ T cell subsets from blood. Although most G protein α-subunits were similarly expressed in all subsets, the abundance of Gα_o, a protein not previously described in hematopoietic cells, was much higher in memory versus naive cells. Consistent with these data, activation of naive CD4⁺ T cells in vitro significantly increased the abundance of Gα_o in cells stimulated under nonpolarizing or T_H17 (but not T_H1 or T_H2)-polarizing conditions. In functional studies, the use of a chimeric G protein α-subunit, Gα_qo5, demonstrated that chemokine receptors could couple to Gα_o-containing G proteins. We also found that Gα_i1, another α-subunit not described previously in leukocytes, was expressed in naive T cells but virtually absent from memory subsets. Corresponding to their patterns of expression, siRNA-mediated knockdown of Gα_o in memory (but not naive) and Gα_i1 in naive (but not memory) CD4⁺ T cells inhibited chemokine-dependent migration. Moreover, although even in Gα_o- and Gα_i1-expressing cells mRNAs of these α-subunits were much less abundant than Gα_i2 or Gα_i3, knockdown of any of these subunits impaired chemokine receptor-mediated migration similarly. Together, our data reveal a change in the repertoire of Gα_i/o subunits during T cell differentiation and suggest functional equivalence among Gα_i/o subunits irrespective of their relative abundance.     

αB-Crystallin Interacts with Cytoplasmic Intermediate Filament Bundles during Mitosis

The small heat-shock protein αB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of αB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of αB-crystallins on the intermediate filament proteins. Thus, the intracellular state of αB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that αB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of αB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, αB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of αB-crystallin showed that αB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, αB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of αB-crystallin protein was due to an increased level of αB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and αB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of αB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis αB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.     

Assembly and Function of the Regulator of G protein Signaling 14 (RGS14)·H-Ras Signaling Complex in Live Cells Are Regulated by Gαi1 and Gαi-linked G Protein-coupled Receptors

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα_i/o-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα_i1/3-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα_i1-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα_i1-GDP but not active Gα_i1-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα_i1 complex in brain lysates, and co-expression of RGS14 and Gα_i1 in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα_i-linked α_2A-adrenergic receptor induces a conformational change in the Gα_i1·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα_i1-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.     

Dissociated GαGTP and Gβγ Protein Subunits Are the Major Activated Form of Heterotrimeric Gi/o Proteins

Although most heterotrimeric G proteins are thought to dissociate into Gα and Gβγ subunits upon activation, the evidence in the Gi/o family has long been inconsistent and contradictory. The Gi/o protein family mediates inhibition of cAMP production and regulates the activity of ion channels. On the basis of experimental evidence, both heterotrimer dissociation and rearrangement have been postulated as crucial steps of Gi/o protein activation and signal transduction. We have now investigated the process of Gi/o activation in living cells directly by two-photon polarization microscopy and indirectly by observations of G protein-coupled receptor kinase-derived polypeptides. Our observations of existing fluorescently labeled and non-modified Gαi/o constructs indicate that the molecular mechanism of Gαi/o activation is affected by the presence and localization of the fluorescent label. All investigated non-labeled, non-modified Gi/o complexes dissociate extensively upon activation. The dissociated subunits can activate downstream effectors and are thus likely to be the major activated Gi/o form. Constructs of Gαi/o subunits fluorescently labeled at the N terminus (GAP43-CFP-Gαi/o) seem to faithfully reproduce the behavior of the non-modified Gαi/o subunits. Gαi constructs labeled within the helical domain (Gαi-L91-YFP) largely do not dissociate upon activation, yet still activate downstream effectors, suggesting that the dissociation seen in non-modified Gαi/o proteins is not required for downstream signaling. Our results appear to reconcile disparate published data and settle a long running dispute.     

Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway

The effect of a noncanonical Wnt, Wnt11, on canonical Wnt signaling stimulated by Wnt1 and activated forms of LRP5 (low density lipoprotein receptor-related protein-5), Dishevelled1 (Dvl1), and beta-catenin was examined in NIH3T3 cells and P19 embryonic carcinoma cells. Wnt11 repressed Wnt1-mediated activation of LEF-1 reporter activity in both cell lines. However, Wnt11 was unable to inhibit canonical signaling activated by LRP5, Dvl1, or beta-catenin in NIH3T3 cells, although it could in P19 cells. In addition, Wnt11-mediated inhibition of canonical signaling in NIH3T3 cells is ligand-specific; Wnt11 could effectively repress canonical signaling activated by Wnt1, Wnt3, or Wnt3a but not by Wnt7a or Wnt7b. Co-culture experiments with NIH3T3 cells showed that the co-expression of Wnt11 with Wnt1 was not an essential requirement for the inhibition, suggesting receptor competition as a possible mechanism. Moreover, in both cell types, elevation of intracellular Ca(2+) levels, which can result from Wnt11 treatment, led to the inhibition of canonical signaling. This result suggests that Wnt11 might not be able to signal in NIH3T3. Furthermore, P19 cells were found to express both endogenous canonical Wnts and Wnt11. Knockdown of Wnt11 expression using siRNA resulted in increased LEF-1 reporter activity, thus indicating that Wnt11-mediated suppression of canonical signaling exists in vivo.