Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622
Submitted 3 March 2003 ; accepted in final form 2 July 2003
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ABSTRACT |
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actin; cholecystokinin; Rho; Rho-kinase; stress fibers
CCK is known to induce changes in the distribution of actin in both pancreatic acinar cells and Chinese hamster ovary (CHO) cells bearing CCK receptors (29, 37, 38). High concentrations of CCK are known to redistribute actin away from the normal prominent apical subluminal bands in acinar cells, and this also occurs in several models of experimental pancreatitis (20, 43). Remodeling of the apical actin filamentous network may participate in physiological secretion, because altering the G-/F-actin equilibrium has been shown to inhibit amylase secretion (26). Regulation of the actin cytoskeleton, in many cell types, involves the Rho family of small GTP-binding proteins, which is made up of three members, Rho, Rac, and Cdc42. In their active, GTP-bound form, these proteins have been shown to be responsible for a wide range of biological actions including transcriptional regulation, vesicular trafficking, and cytoskeletal rearrangement (15, 19, 32). Activation of these proteins in fibroblast cells causes different and distinct cytoskeletal remodeling. RhoA causes the formation of actin stress fibers and focal adhesion complexes, whereas Rac and Cdc42 induce lamellipodia and filopodia, respectively (15, 33). Lysophosphatidic acid (LPA), the active component of serum, induces stress fibers in fibroblasts through the activation of RhoA, and generation of these stress fibers is blocked by Clostridium botulinum C3 transferase, which irreversibly ADP-ribosylates and inactivates Rho (28, 33). Several downstream effectors of Rho have recently been identified including Rho-kinase, protein kinase N (PKN), mDia, rhotekin, and citron (1, 3). Although not all of these effectors have known functions, it was shown recently that Rho-kinase is responsible for increased phosphorylation of the myosin light chain (MLC), leading to stress fiber formation in fibroblasts or contraction in smooth muscle (2, 23). The activity of Rho-kinase is inhibited by the pharmacological reagent Y-27632 (2, 46).
A number of G protein-coupled receptors that act by diverse G protein-mediated pathways have been shown to activate Rho (36, 40). Although evidence exists for participation of Gq and Gi, most studies suggest that G12/13 is the primary G protein coupling receptors to Rho activation. Constitutively active G12 and G
13 induce stress fibers in a Rho-dependent manner in a variety of cell types (4, 12, 13, 21). Rho is activated by a family of guanine nucleotide exchange factors (GEFs) termed RhoGEFs. Several RhoGEFs can physically associate with G
12 and G
13 through an amino-terminal regulator of G protein signaling (RGS) domain as first shown for p115 RhoGEF (7, 8, 24). Moreover, the association with G
13 increases the RhoGEF activity of p115 RhoGEF.
Because of the difficulty of transfecting differentiated pancreatic acinar cells, we have studied the role of Rho in mediating CCK-induced changes in the actin cytoskeleton by using as a model cell line NIH3T3 cells permanently transfected with CCK-A receptors. With these cells, we show that CCK activates RhoA and induces actin stress fibers that can be mimicked by constitutively active RhoV14 and inhibited by C3 exoenzyme, dominant-negative RhoN19, or the Rho-kinase inhibitor Y-27632. To probe the mechanism by which CCK activates Rho we used constitutively active heterotrimeric G protein -subunits, inhibitor minigene constructs coding for the carboxy terminus of the
-subunits, which act as inhibitors of receptor-G protein interaction, and plasmids coding for RGS domains. These studies indicate that CCK-A receptors activate Rho through G
12/13 and, most specifically, G
13.
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METHODS |
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Cell transfection, actin staining, and quantitation. NIH3T3 cells stably transfected with the rat CCK-A receptor were provided by Stephen Wank (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) and were previously described (42). They were maintained in DMEM supplemented with 10% FBS and 250 µg/ml geneticin (Life Technologies, Gaithersburg, MD) in a humidified 5% CO2 atmosphere at 37°C. For morphological studies, cells were grown on 22-mm fibronectin-coated coverslips (BioCoat, Becton Dickinson Labware, Bedford, MA) placed in 35-mm dishes. Transient cotransfections were performed with 0.5 µg of pEGFP-N1 (Clontech, Palo Alto, CA) and the indicated plasmids to a total of 2 µg of DNA by using Lipofectamine Plus (Life Technologies) according to the manufacturer's instructions. Expression of Rho plasmids was monitored by Western blot with monoclonal antibody 12CA5 to the HA tag (Roche, Indianapolis, IN). Twenty-four or forty-eight hours after transfection, cells were serum starved for six hours, treated with CCK, LPA, or serum, fixed with 4% paraformaldehyde for ten minutes, and permeabilized in 0.1% Triton X-100, and filamentous actin was stained with rhodamine-phalloidin diluted 1:400 (Molecular Probes, Eugene OR) following the protocol of Piotrowicz et al. (30). We found that when cells were starved for longer than 6 h, the basal level of stress fibers was increased. Fluorescent images were captured with a SPOT digital camera (Diagnostic Instruments, Dexter, MI) with the same capture settings. Quantitation of actin stress fiber fluorescence was performed with Scion Image Beta 4.0.2. The original TIF files were first converted to inverted index files, the intracellular areas of cells were specified, and the mean intracellular rhodamine fluorescence intensity was determined. Green fluorescent protein (GFP)-transfected cells were used as control, and the ratio of cells transfected with various constructs minus background to GFP-transfected minus background was calculated. At least three independent experiments were analyzed, each of which contained at least 10 and usually >20 individual cells. Statistic analysis was performed by ANOVA.
Rhotekin pull-down assay. GST-rhotekin was purified from bacterial culture with a modified method of Ren et al. (31). After induction by isopropylthiogalactoside (IPTG), cells were lysed with a French press and GST-rhotekin was purified with glutathione agarose. Recombinant protein was isolated from the beads by incubation with reduced glutathione, followed by dialysis against buffer containing 25 mM Tris·HCl, pH 7.5, 2 mM DTT, 1 mM MgCl2, and 2.5% glycerol. To assay active Rho, NIH3T3-CCKAR cells were grown on 10-cm plates, serum starved, and then treated with either LPA (1 µM) or CCK (1 nM). They were then scraped into 300 µl of lysis buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2) containing 30 µg of GST-rhotekin and centrifuged to remove cellular debris. The supernatant was then mixed with 400 µl of binding buffer (25 mM Tris·HCl, pH 7.5, 30 mM MgCl2, 40 mM NaCl, 1 mM DTT, 0.5% NP-40) and 15 µl of a 50% slurry of glutathione agarose beads and rotated at 4°C for 1 h. The beads were then centrifuged, washed, and resuspended in SDS sample buffer, and eluted proteins were separated on a 12% SDS-PAGE gel. After transfer to nitro-cellulose membranes, immunoblotting was performed with RhoA polyclonal antibody (SC-179, Santa Cruz Biotechnology, Santa Cruz, CA) and detected with Femto Western blotting reagent (Pierce, Rockford, IL).
Luciferase reporter assay. NIH3T3-CCKAR cells were counted, and 150,000 were seeded in 35-mm wells the day before transfection. Cells were transfected with 0.1 ug/well SRE-luciferase plasmid, which includes the c-fos promoter minus the ternary complex factor-binding site (49) in combinations with other plasmids by using Lipofectamine Plus as described in Cell transfection, actin staining, and quantitation. Twenty-four hours after transfection, cells were starved for twelve hours before stimulation with different agonists for five hours. Cells were lysed in 300 µl of reporter lysis buffer (0.2% Triton X-100, 100 mM K2HPO4, pH 7.8, 10 mM DTT) for 15 min at room temperature. Luciferase activity was measured with beetle luciferin as a substrate, and raw data were collected with a Berthold luminometer. Six replicate wells were used for each control group and three for all experimental groups. The fold increase was calculated in each experiment, and results from five experiments were used to calculate means and SE.
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RESULTS |
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CCK-induced stress fibers are RhoA dependent. To test whether RhoA is involved in the changes in actin fibers induced by CCK, we used constitutively active RhoV14 and dominant-negative RhoN19 constructs and a plasmid coding for the catalytic portion of C3 transferase. Transient transfection of constitutively active RhoV14 induced stress fiber formation in a dose-dependent manner in all transfected cells, and some cells rounded up (Fig. 2B). Transfection of dominant-negative RhoN19 followed by CCK stimulation blocked stress fiber induction in the transfected cells compared with adjacent nontransfected cells (Fig. 2A). In contrast, transfection of dominant-negative RacN19 did not block the CCK-induced stress fiber formation, indicating the specific involvement of Rho (data not shown). C3 transferase, which ribosylates and inactivates Rho, decreased the number of remaining stress fibers in serum-starved cells (Fig. 3A) and blocked CCK-induced stress fiber formation (Fig. 3B). Together, our data indicate that CCK-induced stress fiber formation is Rho dependent.
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CCK-induced stress fibers are Rho-kinase dependent. Many downstream effectors of Rho have been discovered such as Rho kinase, PKN, and rhotekin (3). Among these effectors, Rho-kinase (also known as ROCK II or ROCK-) has been closely related to stress fiber formation (1, 44). To examine whether the stress fiber formation observed in response to CCK was Rhokinase dependent, we pretreated the cells with the specific Rho-kinase inhibitor Y-27632 followed by stimulation with CCK or LPA. Addition of Y-27632 prevented the assembly of CCK- or LPA-induced stress fibers, suggesting that the CCK induction of stress fibers was under the control of a Rho-kinase-dependent pathway (Fig. 4). Addition of Y-27632 to CCK-treated cells already expressing stress fibers also brought about their disappearance (data not shown).
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CCK activates RhoA. To further establish the relationship between CCK stimulation and Rho activation, endogenous active RhoA was measured in control and CCK- or LPA-treated cells with a pull-down assay that captured the active GTP-bound form of the GTPase (31). Both CCK and LPA activated Rho, with the effect of LPA peaking at 3 min and the effect of CCK peaking at 5 min (Fig. 5) with a decrease after 10 min (not shown). The maximal increase of active Rho was 2.13 ± 0.10- and 2.20 ± 0.13-fold for LPA and CCK stimulation, respectively. To further confirm that CCK activates Rho, a more easily quantifiable, indirect assay was performed. This assay used coexpression of a reporter plasmid containing the luciferase gene under the control of a c-fos SRE but lacking the ternary complex factor-binding site to avoid the complication of MAP kinase activation of SRE. Transcriptional stimulation by this element in response to activation of RhoA is well documented (8, 45). CCK dose-dependently activated the reporter expression over the concentration range of 100 pM to 100 nM, with a maximal increase of 12-fold. This compares with a 10-fold increase induced by 10 µM LPA and by 10% serum (Fig. 6A). Active phorbol ester minimally activated luciferase expression, as expected (data not shown). Cotransfection of a plasmid expressing the botulinum C3 exoenzyme was used to block signaling by RhoA and, as expected, blocked induction of luciferase activity by RhoV14 (data not shown). Activation by CCK was almost totally blocked by the C3 exoenzyme and largely blocked by dominant-negative RhoN19 (Fig. 6B). Thus data from two assays supported the hypothesis that CCK activates RhoA.
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Active G12 or G
13 but not G
q, mimics CCK in inducing stress fibers. Although it is well known that the CCK-A receptor couples to G
q to activate PLC and thereby mobilize Ca2+ and activate PKC, it is unknown whether G
q also mediates cytoskeletal remodeling or whether the CCK-A receptor is able to activate G
12/13. Therefore, to elucidate which G protein is involved in the CCK-stimulated stress fiber formation, we first transfected the cells with constitutively active G
12(Q229L), G
13(Q226L), and G
q(Q209L) for 24 h, serum starved for 6 h, and stained with rhodaminephalloidin. G
13QL-transfected cells showed intense stress fibers, and 90% of cells rounded up (Fig. 7D). G
12QL-transfected cells showed moderate stress fibers (Fig. 7C), whereas G
qQL-transfected cells had minimal stress fiber formation (Fig. 7B) compared with the pEGFP plasmid-transfected controls, which had few stress fibers (Fig. 7A). Because of the ability of G
12QL and G
13QL to intensely induce stress fibers and cause cells to round up, the amount of plasmid used for transfection was reduced stepwise. As little as 0.125 µg of G
12QL and G
13QL still induced stress fibers, whereas as much as 2 µg of G
qQL plasmid had no effect. The combination of 0.125 µg of plasmid coding for each of G
12QL and G
13QL induced a greater amount of stress fibers and cell rounding than did 0.125 µg of either alone.
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p115 RhoGEF RGS, but not RGS2 or RGS4, suppresses CCK-induced stress fibers. RGS are small proteins that can serve as a GTPase-activating protein (GAP) for the heterotrimeric G proteins. A number of RhoGEFs contain a homologous sequence termed a RGS domain. p115 RhoGEF has been shown to bridge G12/13 to Rho through its RGS domain and at the same time to act as a GAP for G
12/13 and a GEF for Rho (24). Recently, isolated RGS domains from two other RhoGEFs were shown to act as dominant negatives to interfere with Rho signaling (5). We therefore constructed a GFP-tagged p115 RhoGEF RGS domain expression plasmid. RGS2 and RGS4 were used as controls because they have been shown to interact with G
q, leading to an increase in its GTPase activity and thereby dampening signaling by agonists that interact with receptors that couple to Gq (17, 18). As shown in Fig. 8, p115 RhoGEF RGS domain inhibited the CCK-induced increase in stress fibers from 2.49 ± 0.08- to 1.35 ± 0.15-fold whereas RGS2 and RGS4 had minimal effects that were not significant. We also tested these RGS domains on G
12QL- and G
13QL-induced stress fibers. p115 RhoGEF RGS domain totally abolished G
13QL-induced stress fibers and moderately inhibited the effect of G
12QL, whereas RGS2 and RGS4 had no effect (data not shown).
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Carboxy-terminal minigenes inhibit CCK-induced stress fibers. G protein -subunits bind to G protein-coupled receptors through their carboxy termini. The carboxy-terminal 11-amino acid peptides have therefore been used as inhibitors of their respective G proteins (10, 11). To further establish the relationship between CCK and specific G proteins, we analyzed the effects of transfecting minigenes coding for carboxy-terminal peptides on CCK-induced stress fibers. Cells were transfected with specific minigenes for 48 h and then stimulated with CCK. The carboxy-terminal minigene of G
13 totally blocked the CCK-induced increase in stress fibers from 2.31 ± 0.17- to 1.07 ± 0.16-fold (Fig. 9, D and E), and the minigene of G
12 partially blocked (Fig. 9, C and E) and that of G
q had no effect to decrease actin stress fibers induced by CCK (Fig. 9, B and E). In contrast, the carboxy-terminal minigene of G
13 had no effect to block stress fibers induced by RhoV14 (data not shown).
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DISCUSSION |
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The CCK receptor is well known to activate Gq/11. Although other heterotrimeric G proteins including Gs and Gi have also been identified by covalent labeling with azido GTP, the identity and function of all the labeled G proteins was not clear (39). Murthy et al. (27) recently showed that CCK activates Gq, G12, and G13 in rabbit intestinal smooth muscle by 35S-labeled guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding. Previous studies showed Gq/11 to be required for calcium signaling in response to CCK in both cultured cell models and pancreatic acini (52). Activation of phospholipase D by CCK in smooth muscle cells was mediated by G13 (27). In most cells, Rho activation has been shown to depend on G12/13, two related heterotrimeric G proteins whose
-subunits
12 and
13 are distinct from
q family members (4, 12, 40). Our studies indicate that CCK-A receptors inserted into NIH3T3 cells also activate RhoA through G12/13 rather than Gq. This was shown first by mimicking the effect of CCK with constitutively active G
12/13 and not with G
q (Fig. 7). More definitively, two separate approaches targeted at G
12/13 inhibited CCK-induced actin stress fibers as a readout for Rho activation. These include inhibition by a p115 RhoGEF RGS domain known to specifically bind G
12/13 and to disrupt signaling but not by RGS4 and RGS2 proteins (Fig. 8). A similar p115 RhoGEF RGS domain had no effect on M3 muscarinic receptor-activated phospholipase C stimulation in HEK293 cells, which was blocked, however, by RGS4, thus demonstrating the specificity of this approach (35). Independent confirmation in our study was provided by inhibition of stress fibers by inhibitory carboxy-terminal G
12 and G
13 but not G
q minigenes (Fig. 9). Thus, although G
q might still contribute in a secondary manner to Rho activation, this does not appear to be the major mechanism.
G12 and G13 belong to a new family of G proteins that appear to be ubiquitously expressed and exhibit 67% of amino acid identity with each other and only 35-44% identity with other -subunits of other classes. They often induce similar responses in various cell types including transformation, activation of the Na+/H+ antiporter, actin stress fiber formation, and focal adhesion assembly (14). However, differences in the signal transduction mechanisms involved in their activation are also emerging. G12 and G13 both can physically associate with RGS-domain containing RhoGEFs, notably p115 RhoGEF, leukemia-associated RhoGEF (LARG), and PDZ-RhoGEF, to activate Rho and modify the actin cytoskeleton (7). Only G13 association with p115 RhoGef stimulates its guanine nucleotide exchange activity toward Rho (16). The RGS domain of p115 RhoGEF displays GAP activity on both G
12 and G
13 in vitro but shows much higher stimulatory effect on G
13 (24). Another difference is that G12 signaling is dependent on the presence of heat shock protein (HSP)90 whereas G13 signaling does not require HSP90 (47). A third difference is that the activation of G12 but not G13 requires tyrosine phosphorylation of LARG RhoGEF (41). Our data support the hypothesis that CCK remodels the cytoskeleton through G13 more than through G12. Active G13 induced more prominent stress fibers than G12 did (Fig. 7). In addition, p115 RhoGEF RGS domain construct, which is a more specific inhibitor for G13 than G12, abolished CCK-induced stress fibers (Fig. 8) and the carboxy-terminal minigene for G13 inhibited CCK-induced stress fibers stronger than the carboxy-terminal minigene for G12 (Fig. 9).
Pancreatic acinar cells possess an actin cytoskeleton that can also be altered by CCK stimulation with dissolution of the apical terminal web staining and enhancement of basal lateral actin staining. These changes are quite distinct from the induction of stress fibers reported here, and the regulatory mechanisms are likely to be more complex. Existing data suggest Rho is likely involved in the actin remodeling in pancreatic acinar cells. Botulinum C3 transferase incubated with isolated acini has been reported to inhibit CCK-induced tyrosine phosphorylation of focal adhesion kinase (FAK) associated with the actin cytoskeleton (9, 34), and a recent report also showed similar results in permeabilized acini (22). Our preliminary study using an adenoviral delivery strategy showed that expression of constitutively active Rho in mouse acini led to changes in the actin cytoskeleton that could be blocked by expression of C. botulinum C3 transferase (Bi Y and Williams JA, unpublished data). However, little is known of how Rho is activated or the nature of its downstream effectors in acinar cells. Our studies in NIH3T3 cells clearly suggest experiments for acini and other differentiated cells, but these will require more complex strategies for protein expression.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* S. L. Le Page and Y. Bi contributed equally to this work.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Amano M, Fukata Y, and Kaibuchi K. Regulation and functions of Rho-associated kinase. Exp Cell Res 261: 44-51, 2000.[ISI][Medline]
3. Bishop AL and Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241-255, 2002.
4. Buhl AM, Johnson NL, Dhanaskekaran N, and Johnson GL. G12 and G
13 stimulate Rho-dependent stress fiber formation and focal adhesion assembly. J Biol Chem 270: 24631-24634, 1995.
5. Fukuhara S, Chikumi H, and Gutkind JS. Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G12 family to Rho. FEBS Lett 485: 183-188, 2000.[ISI][Medline]
7. Fukuhara S, Chikumi H, and Gutkind JS. RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene 20: 1661-1668, 2001.[ISI][Medline]
8. Fukuhara S, Murga C, Zohar M, Igishi T, and Gutkind JS. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem 274: 5868-5879, 1999.
9. Garcia LJ, Rosado JA, González A, and Jensen RT. Cholecystokinin-stimulated tyrosine phosphorylation of p125FAK and paxillin is mediated by phospholipase C-dependent and -independent mechanisms and requires the integrity of the actin cytoskeleton and participation of p21rho. Biochem J 327: 461-472, 1997.[ISI][Medline]
10. Gilchrist A, Bunemann M, Li A, Hosey M, and Hamm HE. A dominant-negative strategy for studying roles of G proteins in vivo. J Biol Chem 274: 6610-6616, 1999.
11. Gilchrist A, Vanhauwe JF, Li A, Thomas TO, Voyno-Yasenetskaya T, and Hamm HE. G minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation. J Biol Chem 276: 25672-25679, 2001.
12. Gohla A, Harhammer R, and Schultz G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem 273: 4653-4659, 1998.
13. Gohla A, Offermanns S, Wilkie TM, and Schultz G. Differential involvement of G12 and G
13 in receptor-mediated stress fiber formation. J Biol Chem 274: 17901-17907, 1999.
14. Gutkind JS, Coso OA, and Xu N. G12- and G
13-subunits of heterotrimeric G-proteins. In: G Proteins, Receptors and Disease, edited by Spiegel AM. Totowa, NJ: Humana, 1998, p. 101-117.
15. Hall A. Rho GTPases and the actin cytoskeleton. Science 279: 509-514, 1998.
16. Hart M, Trang S, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, and Bollag G. Direct stimulation of the guanine nucleotide exchange activity of p115RhoGEF by G13. Science 280: 2112-2114, 1998.
17. Hepler JR, Berman DM, Gilman AG, and Kozasa T. RGS4 and GAIP are GTPase-activating proteins for Gq alpha and block activation of phospholipase C beta by gamma-thio-GTP-Gq alpha. Proc Natl Acad Sci USA 94: 428-432, 1997.
18. Heximer SP, Watson N, Linder ME, Blumer KJ, and Helper JR. RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc Natl Acad Sci USA 94: 14389-14393, 1997.
19. Hill CS, Wynne J, and Treisman R. The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF. Cell 81: 1159-1170, 1995.[ISI][Medline]
20. Jungermann J, Lerch MM, Weidenbach LH, Lutz MP, Kruger B, and Adler G. Disassembly of rat pancreatic acinar cell cytoskeleton during supramaximal secretagogue stimulation. Am J Physiol Gastrointest Liver Physiol 268: G328-G338, 1995.
21. Katoh H, Aoki J, Yamaguchi Y, Kitano Y, Ichikawa A, and Negishi M. Constitutively active G12, G
13, and G
q induced Rho-dependent neurite retraction through different signaling pathways. J Biol Chem 273: 28700-28707, 1998.
22. Kiehne K, Herzig KH, and Fölsch UR. CCK-stimulated changes in pancreatic acinar morphology are mediated by Rho. Digestion 65: 47-55, 2002.[ISI][Medline]
23. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245-248, 1996.[Abstract]
24. Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bolag G, and Sternweis PC. p115 RhoGEF, a GTPase activating protein for G12 and G
13. Science 280: 2109-2111, 1998.
25. Mattingly RR, Sorisky A, Brann MR, and Macara IG. Muscarinic receptors transform NIH3T3 cells through a Ras-dependent signaling pathway inhibited by the Ras-GTPase-activating protein SH3 domain. Mol Cell Biol 14: 7943-7952, 1994.[Abstract]
26. Muallem S, Kwiatkowska K, Xu X, and Yin HL. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J Cell Biol 128: 589-598, 1995.[Abstract]
27. Murthy KS, Zhou H, Grider JR, and Makhlouf GM. Sequential activation of heterotrimeric and monomeric G proteins mediates PLD activity in smooth muscle. Am J Physiol Gastrointest Liver Physiol 280: G381-G388, 2001.
28. Nobes CD and Hall A. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81: 53-62, 1995.[ISI][Medline]
29. O'Konski MS and Pandol SJ. Effects of caerulein on the apical cytoskeleton of the pancreatic acinar cell. J Clin Invest 86: 1649-1657, 1990.[ISI][Medline]
30. Piotrowicz RS, Hickey E, and Levin EG. Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J 12: 1481-1490, 1998.
31. Ren XD, Kiosses WB, and Schwartz MA. Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18: 578-585, 1999.
32. Ridley AJ. Rho family proteins: coordinating cell responses. Trends Cell Biol 11: 471-477, 2001.[ISI][Medline]
33. Ridley AJ and Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389-399, 1992.[ISI][Medline]
34. Rosado JA, Salido GM, Jensen RT, and Garcia LJ. Are tyrosine phosphorylation of p125FAK and paxillin or the small GTP binding protein, Rho, needed for CCK-stimulated pancreatic amylase secretion? Biochim Biophys Acta 14: 412-426, 1998.
35. Rümenapp U, Asmus M, Schablowski H, Woznicki M, Han L, Jakobs KH, Fahimi-Vahid M, Michalet C, Wieland T, and Schmidt M. The M3 muscarinic acetylcholine receptor expressed in HEK-293 cells signals to phospholipase D via G12 but not Gq-type G proteins. J Biol Chem 276: 2474-2479, 2001.
36. Sah VP, Seascholtz TM, Sagi SA, and Heller-Brown J. The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40: 459-489, 2000.[ISI][Medline]
37. Schäfer C, Clapp P, Welsh MJ, Benndorf R, and Williams JA. Hsp27 expression regulates CCK-induced changes of the actin cytoskeleton in CHO-CCKA cells. Am J Physiol Cell Physiol 277: C1032-C1043, 1999.
38. Schäfer C, Ross SE, Bragado MJ, Groblewski GE, Ernst SA, and Williams JA. A role for the p38 mitogen-activated protein kinase/Hsp 27 pathway in cholecystokinin-induced changes in the actin cytoskeleton in rat pancreatic acini. J Biol Chem 273: 24173-24180, 1998.
39. Schnefel S, Profrock A, Hinsch KD, and Schultz I. Cholecystokinin activates Gi1-, Gi2-, Gi3- and several Gs-proteins in rat pancreatic acinar cells. Biochem J 269: 483-488, 1990.[ISI][Medline]
40. Seasholtz TM, Majumdar M, and Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 55: 949-956, 1999.
41. Suzukini N, Nakamura S, Mano H, and Kozasa T. G alpha 12 activates Rho GTPase through tyrosine-phosphorylated leukemia-associated RhoGEF. Proc Natl Acad Sci USA 100: 733-738, 2003.
42. Tarasova NI, Stauber NH, Choi JK, Hudson EA, Czerwinski G, Miller JL, Pavlakiss GN, Michejda CJ, and Wank SA. Visualisation of G-protein receptor trafficking with the aid of the green fluorescent protein. J Biol Chem 272: 14817-14824, 1997.
43. Tashiro M, Schäfer C, Yao H, Ernst SA, and Williams JA. Arginine induced acute pancreatitis alters the actin cytoskeleton and increases heat shock protein expression in rat pancreatic acinar cells. Gut 49: 241-250, 2001.
44. Totsukawa G, Yamakita Y, Yamashiro S, Hartshorne DJ, Sasaki Y, and Matsumura F. Distinct roles of ROCK (Rhokinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J Cell Biol 150: 797-806, 2000.
45. Treisman R, Albert AS, and Sahai E. Regulation of SRF activity by Rho family GTPases. Cold Spring Harb Symp Quant Biol 63: 643-651, 1998.[ISI][Medline]
46. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990-994, 1997.[ISI][Medline]
47. Vaiskuhaite R, Kozasa T, and Voyno-Yasenetskaya TA. Interaction between the G subunit of heterotrimeric G12 protein and HSP90 is required for G
12 signaling. J Biol Chem 276: 46088-46093, 2001.
48. Voyno-Yasenetskaya T, Conklin BR, Gilbert RL, Hooley R, Bourne HR, and Barber DL. G13 stimulates Na-H exchange. J Biol Chem 269: 4721-4724, 1994.
49. Wells CD, Gutowski S, Bollag G, and Sternweis PC. Identification of potential mechanisms for regulation of p115RhoGEF through analysis of endogenous and mutant forms of the exchange factor. J Biol Chem 276: 28897-28905, 2001.
50. Williams JA. Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 63: 77-97, 2001.[ISI][Medline]
51. Williams JA and Blevins GT Jr. Cholecystokinin and regulation of pancreatic acinar cells function. Physiol Rev 73: 701-721, 1993.
52. Yule DI, Baker CW, and Williams JA. Calcium signaling in rat pancreatic acinar cells: a role for Gq, G
11, and G
14. Am J Physiol Gastrointest Liver Physiol 276: G271-G279, 1999.
53. Yule DI and Williams JA. Stimulus-secretion coupling in the pancreatic acinus. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by Johnson LR. New York: Raven, 1994, chapt. 39, p. 1447-1472.