AGS3 and Gαi3 Are Concomitantly Upregulated as Part of the Spindle Orientation Complex during Differentiation of Human Neural Progenitor Cells.

Adult neurogenesis is modulated by many Gi-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gαi subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (ß-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gαi3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gαi3 in ENStem-A cells, and this specificity for Gαi3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gαi subunits. Moreover, the binding of Gαi3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gαi3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis.

Regulator of G protein signaling 20 enhances cancer cell aggregation, migration, invasion and adhesion.

Several RGS (regulator of G protein signaling) proteins are known to be upregulated in a variety of tumors but their roles in modulating tumorigenesis remain undefined. Since the expression of RGS20 is elevated in metastatic melanoma and breast tumors, we examined the effects of RGS20 overexpression and knockdown on the cell mobility and adhesive properties of different human cancer cell lines, including cervical cancer HeLa, breast adenocarcinoma MDA-MB-231, and non-small cell lung carcinoma H1299 and A549 cells. Expression of RGS20 enhanced cell aggregation, migration, invasion and adhesion as determined by hanging drop aggregation, wound healing, transwell chamber migration and invasion assays. Conversely, shRNA-mediated knockdown of endogenous RGS20 impaired these responses. In addition, RGS20 elevated the expression of vimentin (a mesenchymal cell marker) but down-regulated the expression of E-cadherin, two indicators commonly associated with metastasis. These results suggest that the expression of RGS20 may promote metastasis of tumor cells.

RGS19 inhibits Ras signaling through Nm23H1/2-mediated phosphorylation of the kinase suppressor of Ras.

Besides serving as signal terminators for G protein pathways, several regulators of G protein signaling (RGS) can also modulate cell proliferation. RGS19 has previously been shown to enhance Akt signaling despite impaired Ras signaling. The present study examines the mechanism by which RGS19 inhibits Ras signaling. In HEK293 cells stably expressing RGS19, serum-induced Ras activation and phosphorylations of Raf/MEK/ERK were significantly inhibited, while cells expressing RGS2, 4, 7, 8, 10, or 20 did not exhibit this inhibitory phenotype. Conversely, siRNA-mediated knockdown of RGS19 enabled partial recovery of serum-induced ERK phosphorylation. Interestingly, two isoforms of the tumor metastasis suppressor Nm23 (H1 and H2) were upregulated in 293/RGS19 cells. As a nucleoside diphosphate kinase, Nm23H1 can phosphorylate the kinase suppressor of Ras (KSR). Elevated levels of phosphorylated KSR were indeed detected in the nuclear fractions of 293/RGS19 cells. Co-immunoprecipitation assays revealed that Nm23H1/2 can form complexes with RGS19, Ras, or KSR. siRNA-mediated knockdown of Nm23H1/2 allowed 293/RGS19 cells to partially recover their ERK responses to serum treatment, while overexpression of Nm23H1/2 in HEK293 cells suppressed the serum-induced ERK response. This study demonstrates that expression of RGS19 can suppress Ras-mediated signaling via upregulation of Nm23.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

Interleukin-6 autocrine signaling mediates melatonin MT(1/2) receptor-induced STAT3 Tyr(705) phosphorylation.

Melatonin receptors have previously been shown to elicit cellular signaling through the hematopoietic-specific G protein, G(16) . In the present study, we show that this functional coupling elicited biphasic stimulatory phosphorylation on STAT3 in recombinant MT(1) /Gα(16) cells and native Jurkat T cells (endogenously expressing MT(1) and Gα(16) ), with maximal Ser(727) phosphorylation occurring at 15min, while marked Tyr(705) phosphorylation became detectable only upon agonist treatment for 4 hr or more. By employing signal transducer and activator of transcription 3 (STAT3) phosphorylation-resistant mutants (STAT3-Y705F and STAT3-S727A), we further showed that the receptor-mediated STAT3 phosphorylations at Ser(727) and Tyr(705) were independent of each other. Results obtained from fractionation of 2-IMT-induced cells revealed that the Ser(727) and Tyr(705) phosphorylations were spatially distinct, with the former mainly situated in mitochondria and cytosol, while the latter was predominantly located in the nucleus. Further experiments revealed that the agonist-induced STAT3 phosphorylation at Tyr(705) was significantly suppressed by pretreatment with cycloheximide (a ribosome inhibitor), suggesting that de novo protein synthesis might play a critical role for this response. Using conditioned media obtained from 2-IMT-treated MT(1) /Gα(16) cells, multiplex immunoassays revealed that prolonged agonist treatment led to elevated productions of IL-6, GM-CSF and CXCL-8. Antibody against IL-6, but not those for GM-CSF and CXCL-8, effectively abolished the agonist-induced STAT3 Tyr(705) phosphorylation, suggesting the involvement of IL-6 in melatonin receptor-mediated STAT3 activation. Our results demonstrate that melatonin receptor/Gα(16) coupling is capable of triggering the production of cytokines including IL-6, and this autocrine loop may account for the subsequent STAT3 phosphorylation at Tyr(705) .

Elevated expression of RGS19 impairs the responsiveness of stress-activated protein kinases to serum.

Regulators of G protein signaling (RGS proteins) serve as GTPase activating proteins for the signal transducing Gα subunits. RGS19, also known as Gα-interacting protein (GAIP), has been shown to subserve other functions such as the regulation of macroautophagy and growth factor signaling. We have recently demonstrated that the expression of RGS19 in human embryonic kidney (HEK) 293 cells resulted in the disruption of serum-induced mitogenic response along the classical Ras/Raf/MEK/ERK pathway. Here, we further examined the effect of RGS19 expression on the stress-activated protein kinases (SAPKs). Both c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) became non-responsive to serum in 293/RGS19 cells, yet the two SAPKs responded to UV irradiation or osmotic stress induced by sorbitol. Kinases upstream of JNK and p38 MAPK, including MKK3/6, MKK4, and MLK3, also failed to respond to serum stimulation in 293/RGS19 cells. Serum-induced activation of the small GTPases Rac1 and Cdc42 was similarly suppressed in these cells. Our results indicate that elevated expression of RGS19 can severely disrupt the regulation of MAPKs by small GTPases.

Galpha16 activates Ras by forming a complex with tetratricopeptide repeat 1 (TPR1) and Son of Sevenless (SOS).

Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.

CCR1-mediated activation of Nuclear Factor-kappaB in THP-1 monocytic cells involves Pertussis Toxin-insensitive Galpha(14) and Galpha(16) signaling cascades.

Agonists of CC chemokine receptor CCR1 contribute to the pathogenesis of autoimmune and other inflammatory diseases, possibly via the regulation of the transcription factor NF-kappaB. CCR1 and CCR2b have been demonstrated to use PTX-insensitive Galpha(14) and Galpha(16) to stimulate PLCbeta in cotransfected cells, and Galpha(14) and Galpha(16) are capable of activating NF-kappaB. The coexpression of Galpha(14), Galpha(16), and CCR1 in human monocytic THP-1 cells suggests that CCR1 may use Galpha(14) or Galpha(16) to induce NF-kappaB activation. Here, we demonstrated that a CCR1 agonist, Lkn-1, stimulated NF-kappaB phosphorylation via PTX-insensitive G proteins in THP-1 cells. Lkn-1 also mediated IKK/NF-kappaB phosphorylations in HEK293 cells overexpressing CCR1 and Galpha(14/16). Using various kinase inhibitors, Raf-1, MEK1/2, PLCbeta, PKC, CaM, CaMKII, and c-Src were found to participate in Lkn-1-stimulated IKK/NF-kappaB phosphorylations in THP-1 and transfected HEK293 cells. Although c-Jun N-terminal kinase and p38 MAPK were activated by Lkn-1, they were not required in Lkn-1-induced IKK phosphorylation. The ability of CCR1 to signal through Galpha(14/16) thus provides a linkage for chemokines to regulate NF-kappaB-dependent responses.

Differential involvement of Galpha16 in CC chemokine-induced stimulation of phospholipase Cbeta, ERK, and chemotaxis.

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.

Differential chemokine activation of CC chemokine receptor 1-regulated pathways: ligand selective activation of Galpha 14-coupled pathways.

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.