CCK-A receptor activates RhoA through G{alpha}12/13 in NIH3T3 cells

Sophie L. Le Page,* Yan Bi,* and John A. Williams

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G13- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active {alpha}-subunits; G{alpha}13 and G{alpha}12 but not G{alpha}q induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G12/13 inhibited active {alpha}12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit Gq, had no effect. Cotransfection with plasmids coding for the G protein {alpha}-subunit carboxy-terminal peptide from {alpha}13 and, to a lesser extent {alpha}12, also inhibited the effect of CCK, whereas the peptide from {alpha}q did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G13, leading to rearrangement of the actin cytoskeleton.

actin; cholecystokinin; Rho; Rho-kinase; stress fibers


CHOLECYSTOKININ (CCK) is a gut hormone with major actions on the pancreas, gallbladder, and stomach. Its mechanism of action has been studied extensively in rodent pancreatic acinar cells, where CCK stimulates digestive enzyme secretion, protein synthesis, and mitogenesis (50, 51). Secretion of digestive enzymes is triggered by a rise in intracellular Ca2+ and activation of PKC, both of which result from the CCK receptor activating a phosphoinositide-specific phospholipase C through the heterotrimeric G proteins of the Gq family (52, 53). CCK receptors also activate a number of other intracellular signal transduction pathways, including the three MAP kinase pathways leading to ERKs, JNKs, and p38 and the phosphatidylinositol 3-kinase (PI3-K) pathway leading to p70S6 kinase (50). How the CCK receptor couples to the initial steps in these pathways, however, is not yet known.

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 G{alpha}12 and G{alpha}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{alpha}12 and G{alpha}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{alpha}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 {alpha}-subunits, inhibitor minigene constructs coding for the carboxy terminus of the {alpha}-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{alpha}12/13 and, most specifically, G{alpha}13.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and plasmids. Sulfated cholecystokinin octapeptide (CCK) was purchased from Research Plus (Bayonne, NJ), LPA from Sigma (St. Louis, MO), and the Rho-kinase inhibitor Y-27632 from BioMol (Plymouth Meeting, PA). Beetle luciferin was purchased from Promega (Madison, WI). Other chemicals were purchased from Sigma. Triple hemaglutinin antigen (HA)-tagged RhoV14, RhoN19, and RacN17, which are cloned into the PKH3 plasmid (25), were obtained from Ian Macara (University of Virginia, Charlottesville, VA). The constitutively active glutamine (Q)209leucine (L) mutant of the {alpha}-subunit of the heterotrimeric G protein Gq in pcDNA1 and pCEV KZ-C3, coding for C. botulinum C3-transferase, were provided by Silvio Gutkind (National Institutes of Health, Bethesda, MD). Expression plasmids encoding the constitutively active, GTPase-deficient mutations of G{alpha}12 (Q229L) and G{alpha}13 (Q226L) were obtained from Diane Barber (University of California, San Francisco, CA) and were described previously (48). Constructs for the carboxy-terminal undecapeptide of the G protein-coupled receptor {alpha}-subunits, Gq, G12, and G13 in pcDNA3.1(-) were obtained from Cue BIOtech (Chicago, IL) and were described previously (10, 11). A 528-bp p115 RhoGEF RGS domain was obtained by PCR from mouse pancreas RNA and inserted into pEGFP-N1 by using Xho1 and BamH1 sites. Expression clones for RGS2 and RGS4 in pcDNA3.1 were obtained from R. McKenzie (Parke Davis, Ann Arbor, MI). The rhotekinglutathione S-transferase (GST) plasmid was a gift from Martin Schwartz and Xiang-Dong Ren (Scripps Research Institute, La Jolla, CA). The serum response element (SRE)-luciferase reporter plasmid was obtained from T. Kozasa (University of Illinois, Chicago, IL) and was described previously (49).

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.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CCK induces stress fiber formation. To determine whether CCK stimulates the formation of actin stress fibers, we used a cell line stably transfected with rat CCK-A receptors. Serum-starved cells showed minimal F-actin staining with few fibers extending through the entire cell body (Fig. 1A). Cells treated with 1 nM CCK for 5 min demonstrated a moderate number of stress fibers when compared with serum-starved cells (Fig. 1B). Stimulation for 30 min with CCK led to a large number of prominent stress fibers (Fig. 1C), and ~30% of the cells rounded up. The concentration of 1 nM CCK was chosen because 100 pM CCK induced a lower number of stress fibers and fiber formation occurred in <100% of cells, whereas 10 nM CCK had a larger effect and caused a greater proportion of cells to round up. As a positive control, cells were treated with LPA, a lipid component of serum that was previously shown to be an activator of RhoA and to induce stress fiber formation (33). LPA-stimulated cells showed strong induction of stress fibers and appeared similar to the cells treated with CCK (Fig. 1D). Quantitative analysis of rhodamine phalloidin fluorescence inside cells showed a 2.55 ± 0.17-fold increase (n = 4) after 30-min treatment with 1 nM CCK.



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Fig. 1. Treatment of NIH3T3-CCKAR cells with cholecystokinin (CCK) or lysophosphatidic acid (LPA) induces actin stress fibers. Serum-starved cells were treated with 1 nM CCK or 1 µM LPA and then fixed and stained for filamentous actin with rhodaminephalloidin. A: serum-free cells. B: 5-min CCK. C: 30-min CCK. D: 30-min LPA.

 

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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Fig. 2. RhoN19 inhibits whereas RhoV14 mimics the action of CCK to induce stress fibers. A: cells were simultaneously transfected with RhoN19 and pEGFP to identify the transfected cells and then serum starved and treated with 1 nM CCK for 30 min. The transfected cell shows blockage of stress fiber formation. B: cells cotransfected with RhoV14 and pEGFP. The transfected cell show very strong stress fiber induction.

 


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Fig. 3. C3 exoenzyme blocks stress fiber formation induced by CCK. A: cells cotransfected with C3 exoenzyme and pEGFP. B: cells cotransfected with C3 exoenzyme and pEGFP and treated with 1 nM CCK for 30 min. In transfected cells indicated by green nuclei, stress fibers are essentially abolished.

 

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-{alpha}) 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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Fig. 4. Rho-kinase inhibitor Y-27632 blocks induction of stress fibers induced by CCK or LPA. A: stimulation with 1 µM LPA for 30 min. B: Y-27632 (10 µM) preincubation for 2 h followed by 30-min stimulation with LPA. C: stimulation with 1 nM CCK for 30 min. D: Y-27632 preincubation for 2 h followed by 30-min stimulation with CCK.

 

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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Fig. 5. Activation of RhoA by LPA and CCK. Subconfluent NIH3T3-CCKAR cells were serum starved and then treated with either 1 µM LPA (A) or 1 nM CCK (B) for the indicated times. Cells were then rapidly lysed, active Rho was pulled down with glutathione S-transferase (GST)-rhotekin, and RhoA was visualized by Western blotting. C: densitometric measurements (means ± SE) from 3 experiments each. *P < 0.05 compared with LPA control; #P < 0.05 compared with CCK control.

 


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Fig. 6. CCK dose-dependently activates a RhoA-dependent reporter, which can be blocked by C3 exoenzyme or dominant-negative RhoN19. A: NIH3T3-CCKAR cells were transfected with a 0.1 µg/well serum response element (SRE)-luc reporter gene; 24 h after transfection, cells were serum starved for 12 h and then stimulated with the indicated concentrations of CCK and LPA or 10% serum for 5 h. Luciferase assay was carried out as described in METHODS. B: cells were transfected with 0.1 µg/well SRE-luc reporter and either control pcDNA3 or plasmids coding for C3 exoenzyme or RhoN19 at 1.5 µg/well. CCK stimulation was at 10 nM. All results shown are means and SE of at least 4 experiments.

 

Active G{alpha}12 or G{alpha}13 but not G{alpha}q, mimics CCK in inducing stress fibers. Although it is well known that the CCK-A receptor couples to G{alpha}q to activate PLC and thereby mobilize Ca2+ and activate PKC, it is unknown whether G{alpha}q also mediates cytoskeletal remodeling or whether the CCK-A receptor is able to activate G{alpha}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{alpha}12(Q229L), G{alpha}13(Q226L), and G{alpha}q(Q209L) for 24 h, serum starved for 6 h, and stained with rhodaminephalloidin. G{alpha}13QL-transfected cells showed intense stress fibers, and 90% of cells rounded up (Fig. 7D). G{alpha}12QL-transfected cells showed moderate stress fibers (Fig. 7C), whereas G{alpha}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{alpha}12QL and G{alpha}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{alpha}12QL and G{alpha}13QL still induced stress fibers, whereas as much as 2 µg of G{alpha}qQL plasmid had no effect. The combination of 0.125 µg of plasmid coding for each of G{alpha}12QL and G{alpha}13QL induced a greater amount of stress fibers and cell rounding than did 0.125 µg of either alone.



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Fig. 7. Active G{alpha}12 and G{alpha}13, but not G{alpha}q, mimic the effect of CCK to induce stress fibers. Cells were cotransfected with pEGFP and activated G protein {alpha}-subunits for {alpha}q[glutamine (Q)209leucine (L)], {alpha}12(Q229L), or {alpha}13(Q226L). A: pEGFP alone. B: pEGFP and {alpha}q(Q209L). C: pEGFP and {alpha}12(Q229L). D: pEGFP and {alpha}13(Q226L).

 

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 G{alpha}12/13 to Rho through its RGS domain and at the same time to act as a GAP for G{alpha}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{alpha}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{alpha}12QL- and G{alpha}13QL-induced stress fibers. p115 RhoGEF RGS domain totally abolished G{alpha}13QL-induced stress fibers and moderately inhibited the effect of G{alpha}12QL, whereas RGS2 and RGS4 had no effect (data not shown).



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Fig. 8. p115 RhoGEF regulator of G protein signaling (RGS) domain, but not RGS2 or RGS4, blocks CCK-induced stress fiber formation. NIH3T3-CCKAR cells were cotransfected with the indicated RGS construct for 48 h before serum starvation and 30-min stimulation with 1 nM CCK. Green fluorescent protein (GFP) was used to indicate the transfected cells. A: RGS2. B: RGS4. C: p115 RhoGEF RGS (p115 RGS). D: quantitation of rhodamine phalloidin in control unstimulated cells and after stimulation with CCK in cells transfected with GFP alone or GFP combined with specified RGS constructs. All values are the means and SE for 3 experiments. *P < 0.05 compared with GFP alone.

 

Carboxy-terminal minigenes inhibit CCK-induced stress fibers. G protein {alpha}-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{alpha}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{alpha}12 partially blocked (Fig. 9, C and E) and that of G{alpha}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{alpha}13 had no effect to block stress fibers induced by RhoV14 (data not shown).



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Fig. 9. The carboxy-terminal minigene for G{alpha}13 blocks the stress fiber formation induced by CCK. NIH3T3-CCKAR cells were cotransfected with carboxy-terminal minigenes in combination with pEGFP for 48 h before 30-min stimulation with 1 nM CCK. GFP was used to indicate the transfected cells. A: pEGFP. B: carboxy-terminal minigene of G{alpha}q. C: carboxy-terminal minigene of G{alpha}12. D: carboxy-terminal minigene of G{alpha}13. E: quantitation of rhodaminephalloidin in unstimulated cells (Ctrl) and after stimulation with CCK in cells transfected with GFP alone or GFP combined with specified minigene constructs. All values are the means and SE for 3 experiments. *P < 0.05 compared with GFP alone.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CCK has a number of effects on pancreatic acinar and gallbladder and intestinal smooth muscle cells that involve contractile proteins, especially actin. RhoA is known to regulate actin, especially in model cells, but fully differentiated cells such as pancreatic acini are more difficult to study. Therefore, to determine whether CCK-A receptors activate Rho and to determine involvement of a specific heterotrimeric G protein, we used NIH3T3 cells as a model. These cells are readily transfectable and demonstrate an easily observed manifestation of Rho activation, namely, the formation of actin stress fibers. Using this system, we showed that CCK treatment could induce actin stress fibers similar to the known inducer LPA. That this action involved Rho was shown by the fact that it could be mimicked with constitutively active RhoA and blocked by dominant-negative RhoA and C. botulinum C3 transferase (Figs. 2 and 3). The specificity of the dominant-negative Rho was shown by the lack of effect of dominant-negative Rac1. Although we did not carry out further studies, CCK most likely also activates Rac and Cdc42, because it induced a modest amount of membrane ruffles and microspikes, hallmarks of activation of these two small G proteins. Finally, CCK also activated Rho, as shown with a GST-rhotekin pull-down assay and a SRE-luciferase reporter assay. These actions were shown previously for other calcium-mobilizing G protein-coupled receptors including bombesin, angiotensin II, gastrin, and thrombin on a variety of cultured cell models (40).

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) (GTP{gamma}S) 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 {alpha}-subunits {alpha}12 and {alpha}13 are distinct from {alpha}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{alpha}12/13 and not with G{alpha}q (Fig. 7). More definitively, two separate approaches targeted at G{alpha}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{alpha}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{alpha}12 and G{alpha}13 but not G{alpha}q minigenes (Fig. 9). Thus, although G{alpha}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 {alpha}-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{alpha}12 and G{alpha}13 in vitro but shows much higher stimulatory effect on G{alpha}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.


    DISCLOSURES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41122 and DK-52860 (to J. A. Williams), Michigan Gastrointestinal Peptide Center Grant P30 DK-34933, and Michigan Diabetes Research and Training Center Grant P60 DK-20572.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Williams, Dept. of Molecular & Integrative Physiology, Univ. of Michigan, 7744 Medical Science II, Ann Arbor, MI 48109-0622 (E-mail: jawillms{at}umich.edu).

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. Back


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