CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells

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

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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 α-subunits; Gα13 and Gα12 but not Gαq 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 α12/13- 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 α-subunit carboxy-terminal peptide from α13 and, to a lesser extent α12, also inhibited the effect of CCK, whereas the peptide from αq 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume285
Issue number5 54-5
StatePublished - Nov 2003
Externally publishedYes

Fingerprint

Cholecystokinin A Receptor
Cholecystokinin
Stress Fibers
GTP-Binding Proteins
Fibers
Actins
Assays
Bearings (structural)
Serum Response Element
Rho Guanine Nucleotide Exchange Factors
GTP-Binding Protein Regulators
Clostridium botulinum
rho-Associated Kinases
Clostridium
Peptides
Acinar Cells
Protein Subunits
Luciferases
Actin Cytoskeleton
Plasmids

Keywords

  • Actin
  • Cholecystokinin
  • Rho
  • Rho-kinase
  • Stress fibers

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells. / Le Page, Sophie L.; Bi, Yan; Williams, John A.

In: American Journal of Physiology - Cell Physiology, Vol. 285, No. 5 54-5, 11.2003.

Research output: Contribution to journalArticle

Le Page, Sophie L. ; Bi, Yan ; Williams, John A. / CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells. In: American Journal of Physiology - Cell Physiology. 2003 ; Vol. 285, No. 5 54-5.
@article{3a4af5b44bae4fc7941b73008db1aab8,
title = "CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells",
abstract = "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 α-subunits; Gα13 and Gα12 but not Gαq 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 α12/13- 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 α-subunit carboxy-terminal peptide from α13 and, to a lesser extent α12, also inhibited the effect of CCK, whereas the peptide from αq 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.",
keywords = "Actin, Cholecystokinin, Rho, Rho-kinase, Stress fibers",
author = "{Le Page}, {Sophie L.} and Yan Bi and Williams, {John A.}",
year = "2003",
month = "11",
language = "English (US)",
volume = "285",
journal = "American Journal of Physiology",
issn = "0363-6143",
publisher = "American Physiological Society",
number = "5 54-5",

}

TY - JOUR

T1 - CCK-A receptor activates RhoA through Gα12/13 in NIH3T3 cells

AU - Le Page, Sophie L.

AU - Bi, Yan

AU - Williams, John A.

PY - 2003/11

Y1 - 2003/11

N2 - 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 α-subunits; Gα13 and Gα12 but not Gαq 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 α12/13- 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 α-subunit carboxy-terminal peptide from α13 and, to a lesser extent α12, also inhibited the effect of CCK, whereas the peptide from αq 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.

AB - 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 α-subunits; Gα13 and Gα12 but not Gαq 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 α12/13- 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 α-subunit carboxy-terminal peptide from α13 and, to a lesser extent α12, also inhibited the effect of CCK, whereas the peptide from αq 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.

KW - Actin

KW - Cholecystokinin

KW - Rho

KW - Rho-kinase

KW - Stress fibers

UR - http://www.scopus.com/inward/record.url?scp=0142084710&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0142084710&partnerID=8YFLogxK

M3 - Article

C2 - 12853286

AN - SCOPUS:0142084710

VL - 285

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6143

IS - 5 54-5

ER -