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

Abstract

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...

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

“…RhoGDI preferentially co-precipitates with HA-Rac1, whereas IQGAP1⅐ calmodulin complexes preferentially co-precipitate with HA-CA-Rac1 (Fig. 4a, lanes 1 and 3), consistent with our previous findings (22). These proteins do not co-precipitate with HARac1(PBRQ) or HA-CA-Rac1(PBRQ) (Fig.…”

“…To co-localize GFP-tagged proteins with HA-tagged proteins or with endogenous p120ctn, the cells were fixed with 3% formaldehyde in phosphate-buffered saline (PBS) (15 min, 4°C) and incubated with 50 mM ammonium chloride in PBS to quench formaldehyde fluorescence (10 min, 25°C). The fixed cells were permeabilized with acetone (10 s, 4°C) or with 0.2% Triton X-100 (TX-100) in PBS (10 min, 25°C), as described previously (15,22). After incubating with PBS containing 1% bovine serum albumin (30 min, 25°C), the cells were incubated with mouse antibody to HA (Covance) or to p120ctn (BD Transduction Laboratories, San Diego, CA) (1 h, 25°C), followed by incubation with TRITC-anti-mouse IgG (1 h, 25°C).…”

“…After incubating with PBS containing 1% bovine serum albumin (30 min, 25°C), the cells were incubated with mouse antibody to HA (Covance) or to p120ctn (BD Transduction Laboratories, San Diego, CA) (1 h, 25°C), followed by incubation with TRITC-anti-mouse IgG (1 h, 25°C). The cells were mounted in PBS containing 90% glycerol and 0.1% p-phenylenediamine and examined using a Nikon Optiphot fluorescence microscope, as previously described (22). Digital images of the cells were collected using a Kodak DC 290 zoom digital camera and Adobe Photoshop software.…”

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

“…In this case, cholinergic activation of stably transfected M 3 muscarinic receptors in Chinese hamster ovary cells induced the translocation of IQGAP1 and Rac1 to cell junctions (38). These findings were interpreted in the context of cell adhesion, and it was suggested that muscarinic receptor activation might enhance cell adhesion by increasing the level of GTP-bound Rac1, which would then bind to IQGAP1, causing the dissociation of this inhibitory protein from E-cadherin complexes (38).…”

“…In this case, cholinergic activation of stably transfected M 3 muscarinic receptors in Chinese hamster ovary cells induced the translocation of IQGAP1 and Rac1 to cell junctions (38). These findings were interpreted in the context of cell adhesion, and it was suggested that muscarinic receptor activation might enhance cell adhesion by increasing the level of GTP-bound Rac1, which would then bind to IQGAP1, causing the dissociation of this inhibitory protein from E-cadherin complexes (38). In the case of gastric epithelial cells, the rapid translocation of IQGAP1 to the apical pole of chief and mucous neck cells is temporally more in keeping with the known exocytotic response of these cells to cholinergic stimulation rather than cell adhesion, which is likely to be altered more slowly as these cells migrate downward through the glands (21).…”