Activation of Transfected M1or M3Muscarinic Acetylcholine Receptors Induces Cell–Cell Adhesion of Chinese Hamster Ovary Cells Expressing Endogenous Cadherins

Shafer, Shulamith H.; Puhl, Henry L.; Phelps, Scott H.; Williams, Carol L.

doi:10.1006/excr.1998.4385

Cited by 18 publications

(14 citation statements)

References 42 publications

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“…This extensively characterized cell line (15, 20 -22) was used because we are investigating the effects of muscarinic acetylcholine receptor activation on the observed Rac1-and RhoA-dependent responses, which will be the subject of a future report. In the absence of agonist for the muscarinic acetylcholine receptor, which is the case in this study, the CHO-m3 cells do not exhibit any detectable differences from parental CHO cells (20,21). The cells were transfected by suspension in Ham's F-12 medium (6 ϫ 10 6 cells/200 l of medium) containing 8 g of the indicated cDNA, and electroporation by a single electric pulse (200 V, 50 ms) using a BTX Electro Square Porator (Genetronics, Inc., San Diego, CA).…”

Section: Methodsmentioning

confidence: 53%

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Lanning

Ruiz-Velasco

Williams

2003

Journal of Biological Chemistry

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131

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The armadillo protein SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), such as Rac1 and RhoA. Because the PBR resembles a nuclear localization signal (NLS) sequence, we investigated the nuclear transport of SmgGDS with Rac1 or RhoA. We show that the Rac1 PBR has significant NLS activity when it is fused to green fluorescent protein (GFP) or in the context of full-length Rac1. In contrast, the RhoA PBR has very poor NLS activity when it is fused to GFP or in the context of full-length RhoA. The nuclear accumulation of both Rac1 and SmgGDS is enhanced by Rac1 activation and diminished by mutation of the Rac1 PBR. Conversely, SmgGDS nuclear accumulation is diminished by interactions with RhoA. An SmgGDS nuclear export signal sequence that we identified promotes SmgGDS nuclear export. These results suggest that SmgGDS⅐ Rac1 complexes accumulate in the nucleus because the Rac1 PBR has NLS activity and because Rac1 supplies the appropriate GTP-dependent signal. In contrast, SmgGDS⅐RhoA complexes accumulate in the cytoplasm because the RhoA PBR does not have NLS activity. This model may be applicable to other armadillo proteins in addition to SmgGDS, because we demonstrate that activated Rac1 and RhoA also provide stimulatory and inhibitory signals, respectively, for the nuclear accumulation of p120 catenin. These results indicate that small GTPases with a PBR can regulate the nuclear transport of armadillo proteins.Armadillo (ARM) 1 family proteins that contain multiple copies of the ϳ42-amino acid (aa) ARM motif include SmgGDS, p120 catenin (p120ctn), ␤-catenin, plakoglobin, APC, karyopherin ␣ (also known as importin ␣), and several other proteins (reviewed in Refs. 1-3). Nucleocytoplasmic shuttling by many ARM proteins allows them to regulate events in different cellular compartments, including gene transcription and cell adhesion (reviewed in Refs. 2-7). ARM proteins enter the nucleus by different mechanisms (reviewed in Refs. 2-7). Karyopherin ␣ enters the nucleus when it associates with proteins containing a nuclear localization signal (NLS) sequence consisting of a series of adjacent lysines or arginines (reviewed in Ref. 3). The NLS sequence is believed to anchor within the long surface groove formed by the multiple ARM repeats of karyopherin ␣, promoting the nuclear import of both the NLS-containing protein and karyopherin ␣ (3, 5). APC possesses two NLS sequences and may enter the nucleus by associating with karyopherin ␣ or related proteins (4, 7). The mechanisms by which other ARM proteins enter the nucleus are less clear, because some of these proteins neither possess classic NLS sequences, nor have they been reported to associate with NLS-containing proteins.Several ARM proteins interact with the Rho family of small GTPases (8 -15) or with guanine nucleotide exchange factors (GEFs) for these GTPases (16,17). SmgGDS promotes guanine nucleotide exchange by small GTPases containing a C-terminal polybasic region (PBR), which is a series of adjacent l...

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Section: Methodsmentioning

confidence: 53%

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Lanning

Ruiz-Velasco

Williams

2003

Journal of Biological Chemistry

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131

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“…This role of PKC was different from the previously reported role of PKC in cell-cell adhesion. It has been reported that activation of PKC with the use of PMA could induce cell-cell adhesion in epithelial cells that were defective in cell-cell adhesion even under a high calcium environment (Williams et al, 1993;Shafer et al, 1999). This PMA-induced cell-cell adhesion could be observed in NHEKs grown in a low calcium environment.…”

Section: Loss Of Nocodazole-induced Cell-cell Adhesion In Advanced DImentioning

confidence: 86%

“…However, the underlying molecular regulation for cell-cell adhesion seems to be complicated and diverse. For example, endogenous nonfunctional E-cadherin in Chinese hamster ovary cells can be activated by stimulation of ectopically expressed muscarinic acetylcholine receptor through protein kinase C (PKC) activation (Shafer et al, 1999). On the other hand, adhesive activity of Colo205 tumor cells can be activated with staurosporine, which is a serine kinase inhibitor (Aono et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

Kee

Steinert

2001

MBoC

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The association of the cytoskeleton with the cadherin-catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the Ecadherin-catenin-actin complex to the cell periphery. This was accompanied by actin polymerization. Also, it was found that microtubule disruption-induced cell-cell adhesion was significantly reduced in more advanced differentiated keratinocytes. For example, when NHEK cells cultured under high calcium (1.2 mM) for 8 d and then in low calcium for 1 d were treated with nocodazole, there was no induction of cell-cell adhesion. Also long-term treatment of a phorbol ester for 48 h inhibited nocodazole-induced cell-cell adhesion of NHEK. Furthermore, this nocodazole-induced cell-cell adhesion could be observed in squamous cancer cell lines (A431 and SCC-5, -9, and -25) under low calcium condition, but not in the keratinocyte cell lines derived from normal epidermis (HaCaT, RHEK). On the other hand, HaCaT cells continuously cultivated in low calcium media regained a less differentiated phenotype such as decreased expression of cytokeratin 10, and increased K5; these changes were accompanied with inducibility of cell-cell adhesion by nocodazole. Together, our results suggest that microtubule disruption can induce the cell-cell adhesion via activation of endogenous E-cadherin in non-or early differentiating keratinocytes. However, this is no longer possible in advanced terminally differentiating keratinocytes, possibly due to irreversible changes effected by cell envelope barrier formation.

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“…Rac1 and IQGAP1 are recruited to cell junctions when E-cadherin adhesive activity is experimentally increased in several different cell types (38 -41). We reported previously that several forms of cadherins, including E-cadherin, are expressed by CHO-m3 cells (11). Transient cadherin-mediated adhesion induced by mAChR activation in CHO-m3 cells (11) temporally correlates with the transient junctional localization of HARac1, HA-Rac1…”

Section: Effects Of M 3 Machr Activation On the Association Of Rac1mentioning

confidence: 99%

“…Activation of M 3 mAChR induces several cellular responses that may involve Rac1 including JNK activation (12), reactive oxygen species generation (13), smooth muscle contraction (reviewed in Ref. 14), and cadherinmediated adhesion (11,15). Many of these responses have important physiological effects.…”

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confidence: 99%

The Activation of Rac1 by M3 Muscarinic Acetylcholine Receptors Involves the Translocation of Rac1 and IQGAP1 to Cell Junctions and Changes in the Composition of Protein Complexes Containing Rac1, IQGAP1, and Actin

Ruiz-Velasco

Lanning

Williams

2002

Journal of Biological Chemistry

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Val-12 after prolonged mAChR activation. We also demonstrate that Rac1 participates in mAChR-induced cell-cell compaction and c-Jun phosphorylation. These results indicate that M 3 mAChR activation converts Rac1 to the GTP-bound form, alters interactions between Rac1, IQGAP1, and actin, and causes the junctional accumulation of Rac1 and IQGAP1.The small GTPase Rac1 is emerging as an important participant in a variety of signaling pathways. Activation of Rac1 contributes to many responses in smooth muscle cells including c-Jun NH 2 -terminal kinase (JNK) 1 activation (1), reactive oxygen species generation (2), and contraction (3). The involvement of Rac1 in neuronal growth cone remodeling and neurite outgrowth implicates Rac1 as an intriguing regulator of axonal pathfinding and synaptogenesis (4 -6). The participation of Rac1 in the cadherin-mediated adhesion of epithelial and endothelial cells indicates that Rac1 signaling cascades may also regulate wound healing and vascular permeability (reviewed in Refs. 7-9). In many instances, activation of heterotrimeric G proteincoupled receptors (GPCR) initiates these Rac1-dependent cellular responses (1, 2, 5, 9). Activation of GPCR stimulates the conversion of Rac from the GDP-bound form to the GTP-bound form (1, 10). In addition to this event, GPCR activation undoubtedly induces additional uncharacterized changes in Rac1, such as altered interactions with protein partners or translocation to unique intracellular sites that promote the participation of Rac1 in these different signaling pathways. However, little is known regarding the concurrent changes in GTP binding activity, protein interactions, and subcellular localization of Rac1, which are induced by GPCR activation.The M 3 muscarinic acetylcholine receptor (mAChR) is a likely candidate to regulate Rac1 activity in a variety of cell types. The M 3 mAChR and the closely related M 1 mAChR are GPCR that are expressed in a wide variety of cells including smooth muscle cells, neurons, and epithelial and endothelial cells (reviewed in Ref. 11). Activation of M 3 mAChR induces several cellular responses that may involve Rac1 including JNK activation (12), reactive oxygen species generation (13), smooth muscle contraction (reviewed in Ref. 14), and cadherinmediated adhesion (11,15). Many of these responses have important physiological effects. For example, smooth muscle contraction induced by M 3 mAChR activation significantly alters pulmonary and cardiovascular function (reviewed in Refs. 16 and 17). The induction of E-cadherin-mediated adhesion by M 3 mAChR activation in lung carcinoma cells may diminish metastatic potential (reviewed in Refs. 11 and 15). The M 3 mAChR-mediated activation of JNK (12) may play an important role in AP-1-mediated transcription in a variety of cell types (reviewed in Ref. 18). The probability that these M 3 mAChR-dependent functions involve Rac1 provides a strong rationale for investigating how M 3 mAChR activation alters Rac1. * This work was supported by Grant R01 HL63921 from the NHLBI...

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Activation of Transfected M1or M3Muscarinic Acetylcholine Receptors Induces Cell–Cell Adhesion of Chinese Hamster Ovary Cells Expressing Endogenous Cadherins

Cited by 18 publications

References 42 publications

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Novel Mechanism of the Co-regulation of Nuclear Transport of SmgGDS and Rac1

Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

The Activation of Rac1 by M3 Muscarinic Acetylcholine Receptors Involves the Translocation of Rac1 and IQGAP1 to Cell Junctions and Changes in the Composition of Protein Complexes Containing Rac1, IQGAP1, and Actin

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