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*

The abilities of the M3muscarinic acetylcholine receptor (mAChR) and Rac1 to regulate similar cellular responses, including cadherin-mediated adhesion, prompted us to investigate Rac1 regulation by M3 mAChR. We characterized changes in Rac1 induced by stimulating transfected M3 mAChR in Chinese hamster ovary cells stably expressing hemagglutinin (HA)-tagged wild-type or mutant Rac1. mAChR activation converts endogenous Rac1 to the GTP-bound form in cells expressing HA-Rac1 but not in cells expressing dominant negative HA-Rac1Asn-17 or constitutively active HA-Rac1Val-12. The competitive binding of endogenous IQGAP1 by HA-Rac1Val-12 may diminish the mAChR-mediated activation of endogenous Rac1. HA-Rac1 and HA-Rac1Val-12, but not HA-Rac1Asn-17, accumulate with IQGAP1 at cell junctions during mAChR-induced cell-cell compaction. Co-localization studies suggest that Rac1 can accumulate at junctions without IQGAP1, but IQGAP1 cannot accumulate at junctions without Rac1. mAChR activation also induces GTP-independent changes in Rac1 because mAChR activation redistributes HA-Rac1Asn-17, which does not bind GTP. Actin associates with complexes containing HA-Rac1 or HA-Rac1Val-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 M3 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 ox-ygen 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][8][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.
Potential participants in the M 3 mAChR-mediated activation of Rac1 include IQGAP1 and the Rho guanine nucleotide dissociation inhibitor RhoGDI. IQGAP1, which derives its name from the presence of several IQ domains and some sequence similarity to GTPase-activating proteins, binds multiple proteins in addition to Rac1, including Cdc42, calmodulin, E-cadherin, ␤-catenin, and actin (reviewed in Ref. 19). IQGAP1 may promote Rac1 activation by diminishing Rac1 GTPase activity and by preventing active Rac1 from interacting with negative regulators (reviewed in Ref. 20). In contrast, RhoGDI may impede Rac1 activation by diminishing the ability of Rac1-GDP to convert to Rac1-GTP (reviewed in Ref. 21). Thus, M 3 mAChR-mediated changes in the interactions of Rac1 with IQGAP1 or RhoGDI may contribute to Rac1 activation.
We are using Chinese hamster ovary (CHO) cells stably transfected with M 3 mAChR (CHO-m3 cells) to help define how M 3 mAChR may activate Rac1. M 3 mAChR-mediated responses in these cells resemble those induced by activating endogenous M 3 mAChR in other cell types. For example, M 3 mAChR activation affects actin/myosin interactions in CHO-m3 cells as it does in smooth muscle cells (reviewed in Ref. 14). Cadherin-mediated adhesion induced by M 3 mAChR activation in CHO-m3 cells (11) mimics almost exactly cadherin-mediated adhesion induced by activating endogenous M 3 mAChR in lung carcinoma cells (15). These similarities indicate that CHO-m3 cells are a reasonable model system to investigate how M 3 mAChR activation affects Rac1. The M 3 mAChR preferentially couples to heterotrimeric G proteins in the G q/11 family (reviewed in Ref. 12). Thus, the activation of Rac1 by other GPCR that preferentially couple to G q/11 proteins, such as receptors for angiotensin-II (1,2), thrombin, and histamine (reviewed in Ref. 9), may involve changes in Rac1 similar to those that are induced by M 3 mAChR activation.
We found that M 3 mAChR activation profoundly affects both endogenous Rac1 and hemagglutinin (HA)-tagged wild-type and mutant Rac1 proteins that were stably transfected in these CHO-m3 cells. The M 3 mAChR-dependent activation of Rac1 involves the conversion of Rac1 to the GTP-bound form, altered interactions between Rac1, IQGAP1, and actin, and translocation of Rac1 and IQGAP1 to cell junctions. These changes in Rac1 affect specific M 3 mAChR-dependent signaling pathways because expression of dominant negative HA-Rac1 Asn-17 inhibits the M 3 mAChR-dependent compaction of the cells and activation of JNK but not the activation of extracellular regulated kinase (ERK)-1 and ERK-2. These results help define the changes in Rac1 that allow it to participate in M 3 mAChR-dependent signaling.
Immunoprecipitation of 35 S-labeled Proteins and Enhanced Chemiluminescence (ECL)-Western Blotting-Cells were cultured for 16 h in either complete medium or labeling medium consisting of methionineand cysteine-free Dulbecco's modified essential medium, [ 35 S]methionine (10 Ci/ml), and 2% heat-inactivated fetal calf serum and then incubated in the absence or presence of 10 M carbachol. In the experiments shown in Fig. 1, cells were collected from the culture plates before they were lysed in ice-cold Nonidet P-40 (Nonidet P-40) lysis buffer (50 mM Tris-HCl, 120 mM NaCl, 2.5 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, pH 7.4) containing phosphatase inhibitors (22). In the experiments shown in Fig. 8, the cells were scraped from the culture plates in the presence of this lysis buffer. The lysates were centrifuged (13,000 ϫ g, 10 min, 4°C), and the resulting supernatants were immunoprecipitated as described previously (22). The immunoprecipitates were subjected to SDS-PAGE followed by either autoradiography or ECL-Western blotting as described previously (14,22). Peptide sequencing of immunoprecipitated IQGAP1 was performed using previously described techniques (22).
Co-precipitation of Proteins with the Glutathione S-Transferasetagged p21-binding Domain of the p21-activated Kinase (GST-PBD)-Assays were performed as described by Ren et al. (23) using a bacterial expression vector coding for GST-PBD (10), which was generously provided by Dr. Gary Bokoch (Scripps Research Institute). The cells were subjected to a Ϫ70°C freeze/thaw cycle, incubated with the indicated drugs (3 min, 37°C), and lysed in lysis buffer (23). The lysates were incubated with bacterially expressed GST-PBD followed by precipitation with glutathione-Sepharose 4B beads, as described previously (20). The precipitates were subjected to ECL-Western blotting using antibodies to Rac1 or HA. Densitometry of the samples in the ECL-Western blots was performed to quantify the relative amounts of endogenous Rac1 and HA-tagged Rac1 proteins that co-precipitated with GST-PBD. Optical density values higher than 2.4 may not accurately indicate the relative amounts of proteins in the samples because these values are above the maximum densitometry detection limit.
Immunofluorescence Assays-Immunofluorescence assays were conducted as described previously (22). Cells were cultured on glass coverslips and incubated with 10 M carbachol, fixed in formaldehyde (3%, 15 min, 4°C), and permeabilized in Triton X-100 (0.2%, 10 min, 25°C). The cells were incubated with mouse or rabbit antibody to HA or mouse antibody to IQGAP1. After washing, the cells were incubated with fluorescein isothiocyanate-or tetramethylrhodamine isothiocyanate-labeled anti-mouse IgG or fluorescein isothiocyanate-labeled anti-rabbit IgG. The cells were washed, mounted in phosphate-buffered saline containing 90% glycerol and 0.1% p-phenylenediamine, and examined using a Nikon Optiphot fluorescence microscope.
Phosphorylation of GST-c-Jun-Phosphorylation of c-Jun was assayed as described by Wylie et al. (12) using a bacterial expression vector for GST-tagged c-Jun (amino acids 5-89) (24), which was provided by Dr. Andrew Kraft (University of Colorado). Cells were incubated with 10 M carbachol and lysed (12). Bacterially expressed GSTc-Jun was incubated (1 h, 4°C) with the cleared lysates in the presence of glutathione-Sepharose 4B beads, precipitated, and incubated (20 min, 30°C) with kinase buffer (12) containing 100 Ci/ml [ 32 P]ATP (4.5 mCi/mmol). The samples were boiled in sample buffer for 5 min and subjected to SDS-PAGE followed by autoradiography. Densitometry of the samples in the autoradiographs was performed to quantify the levels of c-Jun phosphorylation.
Phosphorylation of ERK-1 and ERK-2-Cells were incubated with 10 M carbachol and lysed in Nonidet P-40 lysis buffer containing phos-phatase inhibitors as described above. The cleared supernatants were subjected to ECL-Western blotting using antibodies recognizing the phosphorylated and non-phosphorylated forms of ERK-1 and ERK-2. Densitometry of the samples in the ECL-Western blots was performed to quantify the levels of phosphorylated and non-phosphorylated ERK-1 and ERK-2. To examine the protein interactions of the HA-tagged wildtype and mutant Rac1 proteins, the HA-tagged GTPases were immunoprecipitated from 35 S-labeled cells and examined for co-precipitating proteins (Fig. 1B). For comparison HA-tagged wild-type RhoA, constitutively active RhoA Val-14 , and dominant negative RhoA Asn-19 were also immunoprecipitated from 35 Slabeled stably transfected CHO-m3 sublines that we established previously (14). IQGAP1 was identified as the 195-kDa protein that co-precipitates with HA-Rac1 Val-12 and to a lesser extent with HA-Rac1 (Fig. 1B). Enzymatic digestion and peptide sequencing of this 195-kDa protein yielded a peptide with the sequence LPYDVTPEQA, which corresponds to residues 1112-1121 of murine IQGAP1 (Swiss-Prot accession number Q9JKF1). The identification of this protein as IQGAP1 was further indicated by its reaction with IQGAP1 antibodies in ECL-Western blots of immunoprecipitated HA-Rac1 Val-12 (Fig.  1C). The inability of IQGAP1 to co-precipitate with HA-Rac1 Asn-17 or the HA-tagged RhoA proteins ( Fig. 1B) is consistent with the report that IQGAP1 preferentially associates with the GTP-bound form of Rac1 and does not associate with RhoA (25).

The Wild-type and Mutant Rac1 Proteins Have Different
Two proteins that co-precipitate with HA-Rac1 Val-12 and to a much lesser extent with HA-Rac1 have approximate relative molecular masses of 19 and 16 kDa (Fig. 1B). Several lines of evidence indicate that these two proteins are calmodulin. Calmodulin reportedly migrates on SDS gels with relative molecular masses of 21 and 15 kDa when the protein is Ca 2ϩ -free and -bound, respectively (reviewed in Ref. 26). Calmodulin also physically associates with IQGAP1, which has three binding sites for Ca 2ϩ -free calmodulin and one binding site for Ca 2ϩbound calmodulin (reviewed in Ref. 27). The ratio of IQGAP1/ 19-kDa protein/16-kDa protein is 1:0.57 Ϯ 0.09:0.23 Ϯ 0.05 in HA-Rac1 Val-12 immunoprecipitates (n ϭ 5 independent experiments). These proteins are present in a similar ratio of 1:0.47 Ϯ 0.08:0.24 Ϯ 0.05 in HA-Rac1 immunoprecipitates, even though much less IQGAP1 co-precipitates with HA-Rac1 than with HA-Rac1 Val-12 (n ϭ 5 independent experiments). These similar ratios support the possibility that the 19-and 16-kDa proteins are Ca 2ϩ -free and Ca 2ϩ -bound calmodulin, respectively, associated with IQGAP1. This possibility is further supported by the demonstration that calmodulin antibodies recognize the 19-kDa protein in immunoprecipitates of HA-Rac1 Val-12 (Fig.  1D, top panel). The lack of detectable reactivity of these antibodies with the 16-kDa protein may be because of an inability of the calmodulin antibodies to recognize Ca 2ϩ -bound calmodulin.
We hypothesized that Rac1 associates with IQGAP1 and RhoGDI in two mutually exclusive complexes. This hypothesis was tested by comparing immunoprecipitates of IQGAP1, RhoGDI, and HA-Rac1 Val-12 from m3CARac cells. Immunoprecipitates of RhoGDI contain neither calmodulin nor proteins that co-migrate with IQGAP1 (Fig. 1E). Conversely, immunoprecipitates of IQGAP1 contain calmodulin but not RhoGDI (Fig. 1E). These results indicate that HA-Rac1 Val-12 forms a complex with RhoGDI and forms another complex with IQ-GAP1 and calmodulin in m3CARac cells.
The Wild-type and Mutant HA-Rac1 Proteins Have Different Susceptibilities to Activation by mAChR and Different Effects on the Activity of Endogenous Rac1-The effects of M 3 mAChR activation on the activities of the HA-tagged Rac1 proteins were investigated by precipitating the GTP-bound forms of the proteins using GST-PBD and examining the precipitates by ECL-Western blotting using antibody to HA ( Fig. 2A, top panel, and Fig. 2B). We found that the association of HA-Rac1 with GST-PBD is increased by M 3 mAChR activation or by the nonspecific activation of GTPases with GTP␥S or guanosine 5Ј-(␤,␥-imido)triphosphate (GppNHp). In contrast, constitutively active HA-Rac1 Val-12 maximally associates with GST-PBD, and this association is not enhanced by mAChR activation or by exposure to GTP␥S or GppNHp. Dominant negative HA-Rac1 Asn-17 does not detectably associate with GST-PBD and cannot be induced to associate with GST-PBD by mAChR agonists or by GTP␥S or GppNHp ( Fig To determine whether expression of the mutant HA-tagged Rac1 proteins interferes with the activation of endogenous Rac1 by M 3 mAChR, GST-PBD was used to precipitate endogenous Rac1 from the different sublines. The precipitated endogenous Rac1, which migrates with a relative molecular mass of 25 kDa, was detected by ECL-Western blotting using antibodies to Rac1 ( Fig. 2A, bottom panel, and Fig. 2C). As expected, the association of endogenous Rac1 with GST-PBD is increased by mAChR activation in m3WTRac cells but not in m3DNRac cells (Fig. 2A, bottom panel, and Fig. 2C). Surprisingly, the association of endogenous Rac1 with GST-PBD is not increased by mAChR activation in m3CARac cells expressing constitutively active HA-Rac1 Val-12 ( Fig. 2A, bottom panel, and  Fig. 2C). This response is not due to a general depression of Rac1 activity in these cells because nonspecific activation of GTPases with either GTP␥S or GppNHp increases the association or Rac1 with GST-PBD in all of the sublines ( Fig. 2A,  bottom panel, and Fig. 2C).

Activation of M 3 mAChR Induces the Translocation of IQGAP1 and the Wild-type and Mutant HA-Rac1 Proteins-
The effects of M 3 mAChR activation on the intracellular distributions of IQGAP1 and the HA-tagged Rac1 proteins were examined by incubating the cells with carbachol for 0 -90 min and immunofluorescently staining the cells using antibodies to IQGAP1 and HA. Translocation of the proteins was consis-tently detectable within 10 min after exposure to carbachol, resulting in a redistribution that culminated 30 min after exposure. Subsequent translocation resulted in a new distribution of the proteins that was most noticeable 90 min after exposure to carbachol. Because differences in the distributions of the proteins were most obvious at 30 and 90 min after carbachol treatment, images of the intracellular localization of the proteins in control cells and in cells treated with carbachol In m3WTRac cells, HA-Rac1 is diffusely distributed throughout the cell (Fig. 3A), whereas IQGAP1 accumulates at spherical membrane protrusions at the cell surface (Fig. 3B). Exposure to carbachol for 30 min induces cell-cell compaction and results in the localization of HA-Rac1 at most cell junctions (Fig. 3C) and IQGAP1 at many cell junctions (Fig. 3D). Decom-paction of the cells occurs within 90 min of carbachol exposure, resulting in spread cells that exhibit a diffuse distribution of HA-Rac1 (Fig. 3E) and high concentrations of IQGAP1 at punctate membrane protrusions at the cell surface (Fig. 3F).
To determine whether the mAChR-mediated redistribution of IQGAP1 also occurs in cells expressing normal levels of Rac1, we examined the localization of IQGAP1 in m3Zeo-2 cells, which are CHO-m3 cells stably transfected with the empty pZeoSV2 vector (14). IQGAP1 is present in spherical membrane protrusions on m3Zeo-2 cells (Fig. 4A), and it accumulates at the junctions of these cells that are compacted because of exposure to carbachol for 30 min (Fig. 4B). IQGAP1 subsequently accumulates at punctate membrane protrusions on m3Zeo-2 cells that are decompacting after exposure to carbachol for 90 min (Fig. 4C). These results indicate that mAChR activation induces a similar redistribution of IQGAP1 in m3Zeo-2 cells and m3WTRac cells.
Both dominant negative HA-Rac1 Asn-17 and IQGAP1 localize in unique sites in m3DNRac cells (Fig. 5), which are more spread than m3WTRac cells or m3Zeo-2 cells. Activation of mAChR does not induce compaction of m3DNRac cells as it does in m3WTRac cells or m3Zeo-2 cells. However, dominant negative HA-Rac1 Asn-17 translocates from a juxtanuclear distribution (Fig. 5A) to a more diffuse distribution after treatment with carbachol for 30 min (Fig. 5C). Some relocalization of HA-Rac1 Asn-17 to the juxtanuclear region occurs after treatment with carbachol for 90 min (Fig. 5E). IQGAP1 does not accumulate in spherical membrane protrusions in m3DNRac cells (Fig. 5B) as much as it does in m3WTRac cells or m3Zeo-2 cells. However, these spherical membrane protrusions, exhib- iting high concentrations of IQGAP1, are sometimes visible on m3DNRac cells that are not completely spread (Fig. 5B). IQGAP1 does not translocate to junctions following M 3 mAChR activation in m3DNRac cells (Fig. 5D) but accumulates in punctate membrane protrusions in cells treated with carbachol for 90 min (Fig. 5F).
Both constitutively active HA-Rac1 Val-12 and IQGAP1 accumulate in prominent membrane ruffles on m3CARac cells (Fig.  6, A and B). Cell-cell compaction and translocation of HA-Rac1 Val-12 to cell junctions occurs within 30 min of carbachol exposure (Fig. 6C). In contrast, IQGAP1 is detectable at both cell junctions and membrane ruffles after 30 min of carbachol exposure (Fig. 6D). Neither HA-Rac1 Val-12 nor IQGAP1 are detectable at the junctions of cells that are decompacting after 90 min of carbachol exposure; HA-Rac1 Val-12 is diffusely distributed with some relocalization to membrane ruffles (Fig.  6E), whereas IQGAP1 is present at some membrane ruffles and at punctate membrane protrusions (Fig. 6F).
We consistently found that the junctional localization of HA-Rac1 Val-12 exceeds that of IQGAP1 in m3CARac cells exposed to carbachol for 30 min (Fig. 6, C and D). To test the possibility that HA-Rac1 Val-12 accumulates at some junctions in the absence of IQGAP1, HA-Rac1 Val-12 and IQGAP1 were detected simultaneously in m3CARac cells incubated with or without carbachol for 15 min (Fig. 7). Prior to mAChR activation, HA-Rac1 Val-12 and IQGAP1 co-localize on prominent membrane ruffles (Fig. 7, A and B). After 15 min of mAChR activation, HA-Rac1 Val-12 accumulates at junctions both in the presence and absence of IQGAP1 (Fig. 7, C and D). We did not observe any localization of IQGAP1 to junctions in the absence of HA-Rac1 Val-12 . Similar results were obtained when the co-localization of HA-Rac1 and IQGAP in m3WTRac cells was examined (data not shown).
Because the activation and intracellular localization of Rac1 is regulated by signaling cascades involving pertussis toxinsensitive G i proteins in some systems (28 -32), we investigated the effects of pertussis toxin on the carbachol-induced redistribution of the HA-tagged wild-type and mutant Rac1 proteins and IQGAP1 in the CHO-m3 sublines. Cells that were preincubated in the absence or presence of pertussis toxin (100 ng/ml, 20 h) exhibited similar carbachol-induced changes in the intracellular localization of the HA-tagged wild-type and mutant Rac1 proteins and IQGAP1 (data not shown). This apparent lack of G i participation in the M 3 mAChR-mediated regulation of Rac1 is perhaps not surprising because M 3 mAChR preferentially couple to G q/11 proteins rather than G i proteins (reviewed in Ref. 12). Our previous finding that CHO cell compaction is induced by activating transfected G q/11 -coupled M 1 or M 3 mAChR, but not by activating transfected G i/o -coupled M 2 mAChR (11), is consistent with the lack of G i participation in the mAChR-mediated regulation of Rac1 in this system.

Activation of M 3 mAChR Alters the Association of HA-Rac1 with Intracellular
Proteins-The effects of mAChR activation on the protein interactions of Rac1 were tested by immunoprecipitating HA-Rac1 and HA-Rac1 Val-12 from 35 S-labeled cells treated with carbachol for different times (Fig. 8). Somewhat surprisingly, mAChR activation did not induce detectable changes in the association of RhoGDI with HA-Rac1 (Fig. 8A) or HA-Rac1 Val-12 (Fig. 8C). However, mAChR activation increased the co-precipitation of HA-Rac1 with proteins that migrate in the region of IQGAP1 (Fig. 8B). Activation of mAChR also caused two proteins with relative molecular masses of 58 and 42 kDa to co-precipitate with HA-Rac1 (Fig.  8A) and to a greater extent with HA-Rac1 Val-12 (Fig. 8C). The 42-kDa protein was identified as actin by ECL-Western blotting (Fig. 8D), and the 58-kDa protein has not yet been identified. Actin and the 58-kDa protein co-precipitate with IQGAP1 (Fig. 8A), consistent with previous reports that actin associates with IQGAP1 (33).

Activation of M 3 mAChR Causes the Rac1-dependent Phosphorylation of c-Jun and the Rac1-independent Phosphorylation of ERK-1 and ERK-2-
A previous report that JNK and ERK-1 are activated by M 3 mAChR stimulation in CHO-m3 cells (12) prompted us to examine whether these mAChRinduced events are altered in m3DNRac or m3CARac cells. The mAChR-induced phosphorylation of c-Jun is significantly enhanced in m3CARac cells and significantly diminished in m3DNRac cells, when compared with the responses induced in m3WTRac and m3Zeo-2 cells (Fig. 9, A and B). In contrast, the mAChR-induced phosphorylation of ERK-1 and ERK-2 is similar in all of the sublines except m3CARac cells, which exhibit a more prolonged elevation of ERK-1 and ERK-2 phosphorylation following mAChR activation (Fig. 9, C-E). These results indicate that functional Rac1 is required for c-Jun phosphorylation, but not for ERK-1 or ERK-2 phosphorylation, induced by M 3 mAChR activation. DISCUSSION This study indicates that M 3 mAChR activation has multiple effects on Rac1 and IQGAP1. Activation of mAChR converts Rac1 to the GTP-bound state and induces the translocation of Rac1 and IQGAP1 to cell junctions. Prolonged M 3 mAChR activation causes actin to associate with protein complexes containing Rac1. By comparing these mAChR-induced effects in cells expressing wild-type Rac1, constitutively active Rac1 Val-12 , and dominant negative Rac1 Asn-17 , we developed a model depicting the mAChR-dependent changes in Rac1 and IQGAP1, as shown in Fig. 10.

Effects of M 3 mAChR Activation on the Association of Rac1 with GST-PBD-
The increased association of HA-Rac1 with GST-PBD in carbachol-treated cells indicates that M 3 mAChR activation increases the GTP-bound state of HA-Rac1. Greater association of HA-Rac1 with GST-PBD is induced by exposing cells to GTP␥S or GppNHp than to carbachol. Several factors may contribute to this finding. Neither GTP␥S nor GppNHp, which are non-hydrolyzable analogs of GTP, would be hydrolyzed readily to GDP during the isolation of HA-Rac1. Thus, the association of HA-Rac1 with GST-PBD may be enhanced or prolonged when the GTPase is bound to GTP␥S or GppNHp rather than to GTP.
An additional and complementary explanation for the modest association of GST-PBD with HA-Rac1 induced by mAChR agonists as compared with GTP␥S or GppNHp is that only a small proportion of HA-Rac1 in m3WTRac cells is converted to the GTP-bound form following mAChR activation. This is a reasonable possibility because it would be physiologically detrimental for the activity of all Rac1 molecules expressed in a cell to be regulated by only one receptor such as the M 3 mAChR. According to this model some HA-Rac1 molecules, such as those associated with RhoGDI, may be resistant to M 3 mAChR-mediated signals and may not readily convert to the GTP-bound form following mAChR activation. This model is consistent with our finding that although a detectable amount of HA-Rac1 translocates to cell junctions after mAChR activation, a significant amount of HA-Rac1 remains diffusely distributed throughout the cell after mAChR activation (Fig. 3C).
The inability of dominant negative HA-Rac1 Asn-17 to associate with GST-PBD either in the absence or presence of mAChR activation is consistent with the inability of HA-Rac1 Asn-17 to bind GTP. Replacing the threonine at amino acid 17 with asparagine is believed to cause Rac1 Asn-17 to associate with a GEF without being converted by the GEF to the GTP-bound state (reviewed in Ref. 22). Interactions with Rac1 Asn-17 may inhibit the GEF from associating with endogenous Rac1, resulting in the dominant negative function of Rac1 Asn-17 . Our previous studies indicate that the competitive binding of Smg-GDS by HA-RhoA Asn-19 contributes to the dominant negative function of HA-RhoA Asn-19 (14,22). In contrast, SmgGDS does not co-precipitate with HA-Rac1 Asn-17 (Fig. 1B), indicating that the dominant negative function of HA-Rac1 Asn-17 may not involve competitive binding of SmgGDS.
Surprisingly, constitutively active HA-Rac1 Val-12 expression diminishes the mAChR-mediated activation of endogenous Rac1 in m3CARac cells. Dumotier et al. (34) similarly found that some of the phenotypic alterations induced by dominant negative Rac1  in Dictyostelium are also induced by the expression of constitutively active Rac1 Val-12 . These investigators speculated that constitutively active Rac1 Val-12 may have a negative effect by competitively binding proteins, such as the IQGAP1-related protein DGAP1, which may be required for the normal functioning of endogenous Rac1 in Dictyostelium. Our results are consistent with this possibility. If HA-Rac1 Val-12 competitively binds all of the IQGAP1 that is available to interact with Rac1, then HA-Rac1 Val-12 could inhibit IQGAP1 from participating in the mAChR-mediated activation of Rac1. The potential involvement of IQGAP1 in the GPCR-dependent activation of GTPases is supported by a recent report that expression of mutant IQGAP1 inhibits the bradykinin-dependent activation of Cdc42, whereas expression of wild-type IQGAP1 enhances Cdc42 activity (19). These findings suggest that several of the reported negative effects of constitutively active Rac1 Val-12 (reviewed in Ref. 9) may be caused by the competitive binding of IQGAP1 by Rac1 Val-12 .
Effects of M 3 mAChR Activation on the Association of Rac1 with RhoGDI-Our observation that RhoGDI co-precipitates more readily with HA-Rac1 than with HA-Rac1 Val-12 is consistent with reports that RhoGDI preferentially associates with the GDP-bound form of Rho family members (reviewed in Ref. 21). Measurements of GTP␥S binding indicated that HA-Rac1 may be in the GDP-bound form more often than HA-Rac1 Val-12 . These different amounts of GDP-bound HA-Rac1 and HA-Rac1 Val-12 may explain the greater association of RhoGDI with HA-Rac1 than with HA-Rac1 Val-12 .
Based on previous reports (21,35,36), we expected that complexes of HA-Rac1/RhoGDI would dissociate when HA-Rac1 converts to the GTP-bound form and translocates to cell junctions following mAChR activation. Surprisingly, however, M 3 mAChR activation does not detectably alter the co-precipitation of RhoGDI with HA-Rac1 or HA-Rac1 Val-12 . Complexes of HA-Rac1/RhoGDI may not dissociate in carbachol-treated cells because HA-Rac1 that is complexed with RhoGDI may not readily convert to the GTP-bound form following mAChR activation. This proposition is supported by reports that interactions with RhoGDI inhibit the dissociation of GDP from Rho family members (reviewed in Ref. 21). Overexpression of RhoGDI also inhibits p21-associated kinase activation, consistent with RhoGDI sequestering Rac1 and inhibiting its activity (37). Furthermore, RhoGDI inhibits mAChR-mediated responses involving RhoA, suggesting that the binding of RhoA by RhoGDI blocks the mAChR-mediated activation of RhoA (36). These findings support the possibility that HA-Rac1 and HA-Rac1 Val-12 are most responsive to mAChR-mediated signals when the GTPases are complexed with proteins other than RhoGDI.
Effects of M 3 mAChR Activation on Rac1/IQGAP1 Complexes-Activation of mAChR increases the co-precipitation of HA-Rac1 with proteins that migrate in the region of IQGAP1 on SDS-PAGE gels (Fig. 8B), suggesting that mAChR activation increases the association of HA-Rac1 with IQGAP1. Our finding that mAChR activation causes HA-Rac1 to co-localize with IQGAP1 at some cell junctions is also consistent with mAChR activation causing Rac1 to associate with IQGAP1.
The co-precipitation of actin and an unidentified 58-kDa protein with either HA-Rac1 Val-12 or HA-Rac1 is probably caused by the interactions of these proteins with IQGAP1. A previous study found that actin incorporates into complexes of IQGAP1 and Cdc42 when actin polymerization is induced by phalloidin (33). We reported previously that stress fibers form within 90 min of mAChR activation in CHO-m3 cells (14), which are the parental cells of the lines used in this study. It is A, before mAChR activation, the GDP-bound form of Rac1 associates with RhoGDI in the cytoplasm, whereas IQGAP1 accumulates at the plasma membrane. The GTPbound form of Rac1, which may be present because of stimulatory signals other than mAChR activation, associates with IQGAP1/calmodulin complexes at membrane ruffles. B, within 30 min of stimulating M 3 mAChR, which activates G q/11 proteins, Rac1 is converted to the GTPbound form and accumulates at the junctions of cells that have undergone cell-cell compaction. Rac1-GTP apparently can accumulate at cell junctions either in the absence or presence of IQGAP1. However, IQ-GAP1 apparently accumulates at cell junctions only in the presence of Rac1-GTP. Our results indicate that mAChR activation must induce further changes in Rac1, in addition to its conversion to the GTP-bound form and association with IQGAP1, to cause Rac1 to accumulate at cell junctions. Asterisks represent these additional uncharacterized changes in Rac1-GTP induced by mAChR activation. The inability of mAChR activation to alter Rac1/RhoGDI complexes suggests that the majority of these complexes may be relatively insensitive to mAChR-mediated signals. C, prolonged mAChR activation results in the departure of Rac1 and IQGAP1 from cell junctions and the decompaction of the cells. Rac1 associates with actin and an unidentified 58-kDa protein in complexes of IQGAP1 and calmodulin. diograph from three independent experiments is shown. Densitometry of the autoradiographs was performed (B) to quantify the level of c-Jun phosphorylation induced by mAChR activation. Values shown are the means Ϯ 1 S.E. from three independent experiments. C, an equal number of cells from the indicated sublines were incubated with 10 M carbachol for the specified times and lysed. The lysates were immunoblotted with an antibody that recognizes the phosphorylated forms of ERK-1 and ERK-2 (lanes 1-7) and an antibody that recognizes ERK-1 and ERK-2 (lanes 8 -14). A representative immunoblot from three independent experiments is shown. Densitometry of the immunoblots was performed to quantify the phosphorylation of ERK-1 (D) and ERK-2 (E) induced by mAChR activation. Values shown are the means Ϯ 1 S.E. from three independent experiments. ever, we did not observe the immunolocalization of these complexes to stress fibers in cells treated with carbachol for 0 -90 min (data not shown). Alternatively, it is more likely that mAChR-mediated changes in cortical actin are associated with the incorporation of actin into IQGAP1 complexes. This possibility is consistent with the localization of IQGAP1 in membrane protrusions, where IQGAP1 might interact with cortical actin.
The mAChR-mediated changes in the shape of membrane protrusions exhibiting high levels of IQGAP1 may involve remodeling of cortical actin. Prior to mAChR activation, IQGAP1 accumulates in spherical membrane protrusions that are prominent on most m3Zeo-2 and m3WTRac cells. Although these structures may be unique to the CHO-m3 sublines examined in this study, published photomicrographs suggest that other cell types may have similar spherical membrane protrusions exhibiting high levels of IQGAP1 (see Fig. 7D in Ref. 8). The disappearance of these spherical membrane protrusions on cells that are spreading because of migration, HA-Rac1 Asn-17 expression, or mAChR activation may result from reorganization of the cortical actin cytoskeleton. Remodeling of cortical actin may also cause these surface structures to change from a spherical form to the more punctate form that is detectable on the cells after prolonged mAChR activation.
Effects of M 3 mAChR Activation on the Translocation of Rac1 and IQGAP1 to Cell Junctions-Several of our findings indicate that conversion to the GTP-bound form and/or association with IQGAP1 may promote the localization of Rac1 at cell junctions after mAChR activation. Activation of mAChR induces greater localization of constitutively active HA-Rac1 Val-12 than HA-Rac1 at cell junctions. Constitutively active HA-Rac1 Val-12 is in the GTP-bound form more often than HA-Rac1 and exhibits greater association with IQGAP1 than does HA-Rac1. Furthermore, dominant negative HA-Rac1 Asn-17 , which neither binds GTP nor associates with IQGAP1, does not localize at cell junctions after mAChR activation. Interestingly, IQGAP1 does not co-localize with HA-Rac1 or HA-Rac1 Val-12 at all cell junctions. This finding indicates that the mAChR-induced localization of Rac1 at cell junctions may depend more on the conversion of Rac1 to the GTP-bound form rather than the association of Rac1 with IQGAP1.
Intriguingly, our studies indicate that further mAChR-mediated changes in Rac1, in addition to conversion to the GTPbound form and potential interactions with IQGAP1, are required for the localization of Rac1 at cell junctions. If the GTP-bound state and association with IQGAP1 are the only requirements for the junctional localization of Rac1, then HA-Rac1 Val-12 would be expected to accumulate at cell junctions in the absence of mAChR activation. However, HA-Rac1 Val-12 only accumulates at cell junctions after mAChR activation. These findings support our model that mAChR activation promotes the junctional localization of Rac1 by converting Rac1 to the GTP-bound form (and potentially by enhancing IQGAP1 association) and by inducing additional uncharacterized GTP-independent changes in Rac1 (Fig. 10B).
The homophilic engagement of cadherins may provide additional signals that promote the localization of both Rac1 and IQGAP1 to cell junctions following mAChR activation. 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 HA-Rac1, HA-Rac1 Val-12 , and IQGAP1 induced by mAChR activation in the CHO-m3 sublines used in this study. Thus, signals arising from homophilic interactions between cadherins may promote the junctional localization of HA-Rac1, HA-Rac1 Val-12 , and IQGAP1 following mAChR activation.
Homophilic interactions of cadherins following mAChR activation may also promote Rac1 activation. The homophilic ligation of transfected C-or E-cadherin activates Rac1 within 30 -60 min in CHO cells, as indicated by the increased precipitation of Rac1 with GST-PBD (40,42). Rac1 activity also increases 30 min after E-cadherin-mediated adhesion of MDCKII cells is induced by the Ca 2ϩ switch technique (41). Although these studies suggest differences in the kinetics of Rac1 activation induced by cadherin ligation and by mAChR activation, both mAChR-and cadherin-mediated signals may promote the observed activation and junctional localization of HA-Rac1 and HA-Rac1 Val-12 in m3WTRac and m3CARac cells, respectively.
Although mAChR activation does not cause dominant negative HA-Rac1 Asn-17 to accumulate at cell junctions, it causes HA-Rac1 Asn-17 to translocate from the juxtanuclear region to a more diffuse distribution in m3DNRac cells. Because HA-Rac1 Asn-17 cannot be converted to the GTP-bound form, mAChR activation must induce a GTP-independent change in HA-Rac1 Asn-17 , which causes it to adopt a more diffuse distribution. Activation of mAChR may alter a protein that associates with HA-Rac1 Asn-17 , resulting in translocation of a protein complex containing HA-Rac1 Asn-17 . Alternatively, mAChR activation may release HA-Rac1 Asn-17 from its association with a GEF in the juxtanuclear region, resulting in the diffusion of HA-Rac1 Asn-17 from this area. HA-Rac1 Asn-17 may not accumulate at cell junctions after mAChR activation because HA-Rac1 Asn-17 cannot convert to the GTP-bound form, as discussed above.
The inability of IQGAP1 to accumulate at cell junctions in m3DNRac cells following mAChR activation suggests that the junctional localization of IQGAP1 requires active Rac1 at these junctions. Our observation that IQGAP1 localizes to junctions only in the presence of HA-Rac1 or HA-Rac1 Val-12 (Fig. 7) provides further evidence that active Rac1 must be present at junctions for IQGAP1 to accumulate in these regions.
Consequences of the mAChR-mediated Activation of Rac1-Our results provide evidence that Rac1 activation contributes to cadherin-mediated adhesion induced by M 3 mAChR activation, which was reported previously to occur in lung carcinoma cells (15) and CHO-m3 cells (11). The mAChR-mediated localization of HA-Rac1, HA-Rac1  , and IQGAP1 at the junctions of CHO-m3 cells occurs when cadherin-mediated adhesion is strongest (11), and the disappearance of these proteins from junctions occurs when cadherin-mediated adhesion is diminishing (11). The reduced mAChR-mediated compaction of m3DNRac cells expressing dominant negative HA-Rac1 Asn-17 is also consistent with the participation of Rac1 in cadherinmediated adhesion (reviewed in Refs. 7, 9, and 43).
Our findings are consistent with several aspects of a model recently presented by Fukata and Kaibuchi, which proposes that E-cadherin-mediated adhesion is strengthened by complexes of E-cadherin/␤-catenin/␣-catenin and diminished by complexes of E-cadherin/␤-catenin/IQGAP1 (reviewed in Ref. 43). According to this model, the presence of GTP-bound Rac1 at cell junctions causes IQGAP1 to dissociate from E-cadherin/ ␤-catenin/IQGAP1 complexes and form Rac1-GTP/IQGAP1 complexes instead. These events promote the formation of E-cadherin/␤-catenin/␣-catenin complexes, resulting in increased E-cadherin-mediated adhesion (reviewed in Ref. 43). In agreement with this model, we found that complexes of HA-Rac1/IQGAP1 and HA-Rac1 Val-12 /IQGAP1 accumulate at cell junctions during mAChR-induced cell-cell compaction. The absence of HA-Rac1 Asn-17 and IQGAP1 from cell junctions in m3DNRac cells is also consistent with this model.
Our results also indicate that Rac1 activation is required for the mAChR-dependent activation of JNK but not ERK-1 or ERK-2. It was reported previously that JNK is maximally active 40 min after exposing CHO-m3 cells to carbachol (12), similar to our findings. It is interesting that the mAChRmediated rise in JNK activity in CHO-m3 cells temporally correlates with the mAChR-mediated translocation of Rac1 to cell junctions. Whether this temporal correlation between JNK activation and Rac1 translocation to cell junctions is a causal or coincidental relationship remains to be determined. It is conceivable that Rac1 translocation stimulates JNK activity by affecting JNK signaling complexes located at the cell surface. This possibility is supported by studies indicating that JNK associates at the cell surface with complexes containing scaffolding proteins, cell surface receptors, and regulators of Rho family members (44 -46).
Conclusions-Our results indicate that Rac1 participates in M 3 mAChR-induced cell-cell compaction and JNK activation in CHO-m3 cells. The activation of Rac1 by M 3 mAChR involves the conversion of Rac1 to the GTP-bound form, the translocation of Rac1 and IQGAP1 to cell junctions, and the association of actin with Rac1 protein complexes. Each of these events may be affected by interactions between Rac1 and IQGAP1. The association of Rac1 with IQGAP1 may promote the conversion of Rac1 to the GTP-bound form or help maintain Rac1 in the GTP-bound form. Interactions between activated Rac1 and IQGAP1 may also promote the accumulation of these proteins at cell junctions. The interaction of IQGAP1 with a wide variety of proteins supports the possibility that IQGAP1 complexes bring together Rac1 and other proteins, such as actin, in several M 3 mAChR-mediated signaling pathways.
Our results also indicate that M 3 mAChR activation induces additional GTP-and IQGAP1-independent changes in Rac1. This conclusion is based on our finding that mAChR activation induces the redistribution of HA-Rac1 Asn-17 , which neither binds GTP nor associates with IQGAP1, as well as the translocation of HA-Rac1 Val-12 , which is already in the GTP-bound form and associated with IQGAP1 before mAChR activation. These events are probably caused by M 3 mAChR-dependent changes in proteins that are associated with HA-Rac1 Asn-17 or HA-Rac1 Val-12 . However, the identities of these Rac1-associated proteins and their responses to M 3 mAChR activation remain to be determined.