CCK activates RhoA and Rac1 differentially through G?13and G?qin mouse
Maria E. Sabbatini,1Yan Bi,1* Baoan Ji,1Stephen A. Ernst,2and John A. Williams1
Departments of1Molecular and Integrative Physiology and2Cell and Developmental Biology, The University of Michigan,
Ann Arbor, Michigan
Submitted 7 October 2009; accepted in final form 23 November 2009
Sabbatini ME, Bi Y, Ji B, Ernst SA, Williams JA. CCK activates
acini. Am J Physiol Cell Physiol 298: C592–C601, 2010. First published
November 25, 2009; doi:10.1152/ajpcell.00448.2009.—Cholecystokinin
(CCK) has been shown to activate RhoA and Rac1, as well as
reorganize the actin cytoskeleton and, thereby, modify acinar mor-
phology and amylase secretion in mouse pancreatic acini. The aim of
the present study was to determine which heterotrimeric G proteins
activate RhoA and Rac1 upon CCK stimulation. G?13, but not G?12,
was identified in mouse pancreatic acini by RT-PCR and Western
blotting. Using specific assays for RhoA and Rac1 activation, we
showed that only active G?13 activated RhoA. By contrast, active
G?13 and G?q, but not G?s, slightly increased GTP-bound Rac1
levels. A greater increase in Rac1 activation was observed when
active G?13and active G?qwere coexpressed. G?iwas not required
for CCK-induced RhoA or Rac1 activation. The regulator of G protein
signaling (RGS) domain of p115-Rho guanine nucleotide exchange
factor (p115-RGS), a specific inhibitor of G?12/13-mediated signaling,
abolished CCK-stimulated RhoA activation. By contrast, both RGS-2,
an inhibitor of G?q, and p115-RGS abolished CCK-induced Rac1
activation, which was PLC pathway-independent. Active G?q and
G?13, but not G?s, induced morphological changes and actin redis-
tribution similar to 1 nM CCK. CCK-induced actin cytoskeletal
reorganization was inhibited by RGS-2, but not by p115-RGS,
whereas CCK-induced amylase secretion was blocked by both inhib-
itors. Together, these findings indicate that, in mouse pancreatic acini,
G?13links CCK stimulation to the activation of RhoA, whereas both
G?13 and G?q link CCK stimulation to the activation of Rac1.
CCK-induced actin cytoskeletal reorganization is mainly mediated by
G?q. By contrast, G?13 and G?q signaling are required for CCK-
induced amylase secretion.
actin cytoskeleton; amylase secretion; bleb formation
CHOLECYSTOKININ (CCK) activates multiple signaling pathways
in pancreatic acinar cells through a specific G protein-coupled
receptor, CCK1, which is coupled to heterotrimeric G proteins
(38, 55). Activated G protein-coupled receptors interact with
and produce a conformational change in G protein ?-subunits
(G?), which promotes exchange of GDP for GTP. GTP-bound
G? dissociates from G?? and activates downstream effectors.
Activation of G?q by CCK results in stimulation of PLC?,
leading to formation of inositol-1,4,5-trisphosphate, which in-
creases intracellular Ca2?levels, and diacylglycerol, which
activates PKC (49). Activation of G?s stimulates adenylate
cyclase (14), which in pancreatic acinar cells is associated with
an increase in cAMP-dependent protein kinase activity (26).
CCK activates several forms of G?i in rat pancreatic acinar
cells (38), which in various cells inhibits adenylate cyclase. In
pancreatic acini, pertussis toxin blocks G?i and, thereby, in-
creases cAMP formation (46) but has no effect on Ca2?
mobilization (28, 46). G12/13 is another well-characterized
heterotrimeric G protein family that can initiate intracellular
signaling (51). A previous study showed that, in NIH 3T3 cells
expressing the CCK1 receptor, CCK activates G?12/13 and,
thereby, the small GTP-binding protein RhoA, inducing actin
cytoskeletal reorganization (23). In intestinal smooth muscle,
CCK stimulates phospholipase D activity via RhoA through
Rho proteins, members of the Ras superfamily of small
GTP-binding proteins, participate in several cellular processes,
including cytoskeletal rearrangement, cell cycle progression,
gene transcription, and cytokinesis (5, 17). The Rho family
includes at least three well-studied subfamilies: Rho, Rac, and
Cdc42. Like all small GTP-binding proteins, members of the
Rho family cycle between two forms: an active GTP-bound
form and an inactive GDP-bound form. This cycle is regulated
by three groups of regulatory proteins: guanine nucleotide
exchange factors (GEFs), which induce the binding of GTP to
the small GTP-binding protein; GTPase-activating proteins,
which induce inactivation by hydrolysis of GTP; and guanine
nucleotide-dissociation inhibitors, which, in the cytosol, bind
the GDP-bound form of the small GTP-binding proteins,
thereby preventing exchange to the GTP-bound form (17). One
group of RhoGEFs, including p115-RhoGEF, contains a reg-
ulator of G protein signaling (RGS) domain, which interacts
with the ?-subunit of the heterotrimeric G protein G12/13and,
when expressed as an isolated domain, inhibits G12/13signaling
(17). A number of RacGEFs are known, and some are activated
by second messengers downstream of G?q(5).
In pancreatic acinar cells, RhoA and Rac1 have been impli-
cated in the regulation of CCK-induced amylase secretion
through an actin cytoskeleton-dependent cellular process (3, 4).
CCK not only increases the amount of GTP-bound RhoA and
GTP-bound Rac1, but it also induces translocation of both
from the cytosol to the membrane (4). Constitutively active
RhoA and Rac1 expression induces morphological changes,
actin cytoskeletal reorganization, and amylase secretion (3),
whereas dominant-negative RhoA and Rac1 expression re-
duces CCK-induced acinar morphological changes, actin reor-
ganization, and amylase secretion (3, 4).
Because little is known of the upstream regulators of RhoA
and Rac1 in mouse pancreatic acini, the aim of the present
study was to determine which heterotrimeric G proteins link
CCK stimulation to activation of RhoA and Rac1. Four fami-
lies of heterotrimeric G proteins were studied, Gq, Gi, Gs, and
G12/13, which have been implicated in the activation of RhoA
and Rac1 in several cell types (9, 12, 13, 23, 27, 32, 44, 45).
* Y. Bi should be considered as co-first author.
Address for reprint requests and other correspondence: M. E. Sabbatini,
7703 Medical Science Bldg. II, 1150 W. Medical Center Dr., Ann Arbor, MI
48109-5266 (e-mail: email@example.com).
Am J Physiol Cell Physiol 298: C592–C601, 2010.
First published November 25, 2009; doi:10.1152/ajpcell.00448.2009.
0363-6143/10 $8.00 Copyright © 2010 the American Physiological Societyhttp://www.ajpcell.orgC592
We used pull-down assays to study RhoA and Rac1 activation
and isolated pancreatic acini infected with adenoviruses encod-
ing the constitutively active ?-subunits, G?q, G?13, and G?s,
as well as RGS domains of the p115-RhoGEF and RGS-2,
which inhibit G?12/13and G?q, respectively. Pretreatment with
pertussis toxin was used to study the participation of G?i. The
present findings show that G?13 is the only member of the
G12/13 family expressed in mouse pancreatic acini. We also
demonstrate that G?13 activation mediates the response to
CCK by RhoA, whereas G?qand G?13activation mediates the
response to CCK by Rac1 in a PLC pathway-independent
manner. Finally, we found that although G?13is able to induce
actin reorganization and bleb formation via the RhoA/Rho
kinase pathway, G?q is implicated in CCK-induced actin
cytoskeletal reorganization and acinar morphological changes,
whereas G?qand G?13are required for CCK-induced amylase
MATERIALS AND METHODS
Materials. Collagenase was purchased from Crescent Chemical
(Islandia, NY); BSA and soybean trypsin inhibitor from Sigma Chem-
ical (St. Louis, MO), and DMEM and Alexa 594-conjugated phalloi-
din from Invitrogen (Carlsbad, CA). The following stimuli and inhib-
itors were used: sulfated CCK octapeptide (Research Plus, Bayonne,
NJ); vasoactive intestinal polypeptide (VIP; American Peptide,
Sunnyvale, CA); A-23187 and GF-109203X (Calbiochem, La Jolla,
CA); and PMA, Y-27632, pertussis toxin, and U-73122 (Sigma
Chemical). All other chemicals were of reagent grade.
Antibodies against the following proteins were used: rabbit poly-
clonal antibodies to Rap1, RhoA, G?q, G?12, and G?13(Santa Cruz
Biotechnology, Santa Cruz, CA); rabbit polyclonal antibody to G?12
(Abcam, Cambridge, MA); mouse monoclonal antibody to hemagglu-
tinin (HA)- and Myc-tagged proteins (Cell Signaling Technology,
Beverly, MA); and mouse monoclonal antibody to Rac1 (Pierce
Biotechnology, Rockford, IL).
Fed male ICR mice (22–27 g body wt) were used in the experi-
ments. All the experimental protocols were approved by The Univer-
sity of Michigan University Committee on the Use and Care of
Construction of recombinant adenoviruses. Plasmids of constitu-
tively active G protein ?-subunits, G?q(Q209L) and G?13(Q226L),
denoted active G?q and G?13, respectively, were obtained from J.
Silvio Gutkind (National Institutes of Health, Bethesda, MD) and
Diana Barber (University of California, San Francisco, CA). The
plasmid of G?s(Q227L), denoted G?s, was purchased from American
Type Culture Collection (Manassas, VA). Recombinant adenoviruses
were prepared using the pAdTrack System (Addgene, Cambridge,
MA) according to the method of He et al. (16), as described previ-
ously (3), and amplified and purified using the ViraBind adenovirus
purification kit (Cell Biolabs, San Diego, CA). Adenoviruses express-
ing the RGS domain of p115-RhoGEF, Myc-tagged p115-RGS, the
mutant of the RGS domain of p115-RhoGEF, Myc-tagged p115-
RGS(E29K), and HA-tagged RGS-2 were obtained from Patrick J.
Casey (Duke University, Durham, NC). Adenovirus expressing ?-ga-
lactosidase (?-Gal) was used as a control.
Preparation, short-term culture, and viral infection of pancreatic
acini. Mouse pancreatic acini from ICR mice were prepared by
enzymatic digestion with collagenase followed by mechanical shear-
ing, as previously described (4). Acini were cultured in suspension
without shaking at low density in 10-cm petri dishes in DMEM
enriched with 0.1% BSA, 0.01% soybean trypsin inhibitor, and
antibiotics and incubated overnight at 37°C with 5% CO2. For the
viral infection experiments, 106plaque-forming units/ml of constitu-
tively active G?13, G?q, and G?s or 107plaque-forming units/ml of
p115-RGS, p115-RGS(E29K), and RGS-2 were added to the culture
medium at the beginning of the overnight incubation, unless otherwise
indicated. Under such conditions, ?95% of acinar cells express
adenoviral-driven proteins (4). In another experiment, acutely disso-
ciated acini were incubated with or without pertussis toxin (2 ?g/ml)
for 2 h at 37°C, as previously described (36).
Detection of G?12and G?13expression in mouse pancreatic acini.
Expression of G?12and G?13in mouse pancreatic acini was assessed
Fig. 1. Expression of active G?13activates RhoA, whereas expression of active G?qand G?13additively activates Rac1. ?-Galactosidase [?-Gal (vector control)],
G?13, G?q, and G?s were expressed in isolated mouse pancreatic acini by means of recombinant adenoviruses with overnight incubation. In another group of
acini, active G?13 and G?q were coexpressed. ?-Gal-expressing pancreatic acini were treated with or without 1 nM cholecystokinin (CCK) for 10 min. Acini
were lysed and assayed for activation of RhoA (A) and Rac1 (B and C) using rhotekin Rho-binding domain (RBD) and p21-activated kinase binding domain
(PAK1-PBD), respectively, as activation-specific probes. Expression of active G?13 increased GTP-RhoA and GTP-Rac1 levels, whereas expression of active
G?qincreased GTP-Rac1 levels only. Coexpression of active G?13and G?qinduces an additive effect on Rac1 activation (C). Top: representative immunoblots
for GTP-RhoA, GTP-Rac1, total RhoA, total Rac1, G?13, and G?q. Bottom: quantitative analysis of RhoA and Rac1 activation. Values are means ? SE (n ?
4–5 experiments). *P ? 0.05; **P ? 0.01 vs. control. CTL, control.
G?13 AND G?q ARE UPSTREAM REGULATORS OF RhoA AND Rac1
AJP-Cell Physiol • VOL 298 • MARCH 2010 • www.ajpcell.org
21. Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG,
Bollag G, Sternweis PC. p115 RhoGEF, a GTPase activating protein for
G?12 and G?13. Science 280: 2109–2111, 1998.
22. Kusama K, Nozu F, Awai T, Tanaka S, Honma I, Tsunoda Y,
Mitamura K. Deactivation of ROCK-II by Y-27632 enhances basolateral
pancreatic enzyme secretion and acute pancreatitis induced by CCK
analogues. Biochem Biophys Res Commun 305: 339–344, 2003.
23. Le Page S, Bi Y, Williams JA. CCK-A receptor activates RhoA through
G?12/13 in NIH 3T3 cells. Am J Physiol Cell Physiol 285: C1197–C1206,
24. Lecuona E, Ridge K, Pesce L, Batlle D, Sznajder JI. The GTP-binding
protein RhoA mediates Na,K-ATPase exocytosis in alveolar epithelial
cells. Mol Biol Cell 14: 3888–3897, 2003.
25. Li C, Chen X, Williams JA. Regulation of CCK-induced amylase release
by PKC-? in rat pancreatic acinar cells. Am J Physiol Gastrointest Liver
Physiol 287: G764–G771, 2004.
26. Marino CR, Leach SD, Schaefer JF, Miller LJ, Gorelick FS. Charac-
terization of cAMP-dependent protein kinase activation by CCK in rat
pancreas. FEBS Lett 316: 48–52, 1993.
27. Masci AM, Galgani M, Cassano S, De Simone S, Gallo A, De Rosa V,
Zappacosta S, Racioppi L. HIV-1 gp120 induces anergy in naive T
lymphocytes through CD4-independent protein kinase-A-mediated signal-
ing. J Leukoc Biol 74: 1117–1124, 2003.
28. Matozaki T, Sakamoto C, Nagao M, Nishizaki H, Baba S. G protein in
stimulation of PI hydrolysis by CCK in isolated rat pancreatic acinar cells.
Am J Physiol Endocrinol Metab 255: E652–E659, 1988.
29. Meshki J, Douglas SD, Lai JP, Schwartz L, Kilpatrick LE, Tuluc F.
Neurokinin 1 receptor mediates membrane blebbing in HEK293 cells
through a Rho/Rho-associated coiled-coil kinase-dependent mechanism. J
Biol Chem 284: 9280–9289, 2009.
30. Murthy KS, Zhou H, Grider JR, Makhlouf GM. Sequential activation
of heterotrimeric and monomeric G proteins mediates PLD activity in
smooth muscle. Am J Physiol Gastrointest Liver Physiol 280: G381–
31. Murthy KS, Zhou H, Huang J, Pentyala SN. Activation of PLC-?1 by
Gi/o-coupled receptor agonists. Am J Physiol Cell Physiol 287: C1679–
32. O’Connor KL, Mercurio AM. Protein kinase A regulates Rac and is
required for the growth factor-stimulated migration of carcinoma cells. J
Biol Chem 276: 47895–47900, 2001.
33. Ren XD, Kiosses WB, Schwartz MA. Regulation of the small GTP-
binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 18:
34. Riobo NA, Manning DR. Receptors coupled to heterotrimeric G proteins
of the G12 family. Trends Pharmacol Sci 26: 146–154, 2005.
35. Sabbatini ME, Chen X, Ernst SA, Williams JA. Rap1 activation plays
a regulatory role in pancreatic amylase secretion. J Biol Chem 283:
36. Sabbatini ME, Vatta MS, Davio CA, Bianciotti LG. Atrial natriuretic
factor negatively modulates secretin intracellular signaling in the exocrine
pancreas. Am J Physiol Gastrointest Liver Physiol 292: G349–G357,
37. Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen
RA, Michiels F, Collard JG. Matrix-dependent Tiam1/Rac signaling in
epithelial cells promotes either cell-cell adhesion or cell migration and is
regulated by phosphatidylinositol 3-kinase. J Cell Biol 143: 1385–1398,
38. Schnefel S, Profrock A, Hinsch KD, Schulz I. Cholecystokinin activates
Gi1, Gi2, Gi3and several Gsproteins in rat pancreatic acinar cells. Biochem
J 269: 483–488, 1990.
39. Shafer SH, Williams CL. Elevated Rac1 activity changes the M3 mus-
carinic acetylcholine receptor-mediated inhibition of proliferation to in-
duction of cell death. Mol Pharmacol 65: 1080–1091, 2004.
40. Shi CS, Sinnarajah S, Cho H, Kozasa T, Kehrl JH. G13?-mediated
PYK2 activation. PYK2 is a mediator of G13?-induced serum response
element-dependent transcription. J Biol Chem 275: 24470–24476, 2000.
41. Spicher K, Kalkbrenner F, Zobel A, Harhammer R, Nurnberg B,
Soling A, Schultz G. G12 and G13?-subunits are immunochemically
detectable in most membranes of various mammalian cells and tissues.
Biochem Biophys Res Commun 198: 906–914, 1994.
42. Szaszi K, Kurashima K, Kapus A, Paulsen A, Kaibuchi K, Grinstein
S, Orlowski J. RhoA and Rho kinase regulate the epithelial Na?/H?
exchanger NHE3. Role of myosin light chain phosphorylation. J Biol
Chem 275: 28599–28606, 2000.
43. Torgerson RR, McNiven MA. The actin-myosin cytoskeleton mediates
reversible agonist-induced membrane blebbing. J Cell Sci 111: 2911–
44. Vogt A, Lutz S, Rumenapp U, Han L, Jakobs KH, Schmidt M,
Wieland T. Regulator of G-protein signaling 3 redirects prototypical
Gi-coupled receptors from Rac1 to RhoA activation. Cell Signal 19:
45. Vogt S, Grosse R, Schultz G, Offermanns S. Receptor-dependent RhoA
activation in G12/G13-deficient cells: genetic evidence for an involvement
of Gq/G11. J Biol Chem 278: 28743–28749, 2003.
46. Willems PH, Tilly RH, de Pont JJ. Pertussis toxin stimulates cholecys-
tokinin-induced cyclic AMP formation but is without effect on secreta-
gogue-induced calcium mobilization in exocrine pancreas. Biochim Bio-
phys Acta 928: 179–185, 1987.
47. Williams JA, Chen X, Sabbatini ME. Small G proteins as key regulators
of pancreatic digestive enzyme secretion. Am J Physiol Endocrinol Metab
296: E405–E414, 2009.
48. Williams JA, Korc M, Dormer RL. Action of secretagogues on a new
preparation of functionally intact, isolated pancreatic acini. Am J Physiol
235: 517–524, 1978.
49. Williams JA, Yule DI. Stimulus secretion coupling in pancreatic acinar
cells. In: Physiology of the Gastrointestinal Tract (4th ed.), edited by
Johnson LR. Burlington, MA: Academic, 2006, p. 1337–1369.
50. Wojciak-Stothard B, Tsang LY, Haworth SG. Rac and Rho play
opposing roles in the regulation of hypoxia/reoxygenation-induced per-
meability changes in pulmonary artery endothelial cells. Am J Physiol
Lung Cell Mol Physiol 288: L749–L760, 2005.
51. Worzfeld T, Wettschureck N, Offermanns S. G12/G13-mediated signal-
ing in mammalian physiology and disease. Trends Pharmacol Sci 29:
52. Xu X, Zeng W, Popov S, Berman DM, Davignon I, Yu K, Yowe D,
Offermanns S, Muallem S, Wilkie TM. RGS proteins determine signal-
ing specificity of Gq-coupled receptors. J Biol Chem 274: 3549–3556,
53. Yamazaki J, Katoh H, Yamaguchi Y, Negishi M. Two G12 family G
proteins, G?12and G?13, show different subcellular localization. Biochem
Biophys Res Commun 332: 782–786, 2005.
54. Yang SA, Carpenter CL, Abrams CS. Rho and Rho-kinase mediate
thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in
platelets. J Biol Chem 279: 42331–42336, 2004.
55. Yule DI, Baker CW, Williams JA. Calcium signaling in rat pancreatic
acinar cells: a role for G?q, G?11, and G?14. Am J Physiol Gastrointest
Liver Physiol 276: G271–G279, 1999.
56. Yule DI, Williams JA. U73122 inhibits Ca2?oscillations in response to
cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar
cells. J Biol Chem 267: 13830–13835, 1992.
G?13 AND G?q ARE UPSTREAM REGULATORS OF RhoA AND Rac1
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