G alpha 12 inhibits alpha2 beta1 integrin-mediated Madin-Darby canine kidney cell attachment and migration on collagen-I and blocks tubulogenesis.
Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.
Renal Division, Brigham and Women's Hospital, Harvard Institutes of Medicine, Boston, MA 02115, USA.
Tianqing
T
Kong
Daosong
D
Xu
Wanfeng
W
Yu
Ayumi
A
Takakura
Ilene
I
Boucher
Mei
M
Tran
Jordan A
JA
Kreidberg
Jagesh
J
Shah
Jing
J
Zhou
Bradley M
BM
Denker
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D038982:Integrin alpha2beta1 / Q000235:genetics / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism; D051419:Paxillin / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
D024042:Collagen Type I; D038982:Integrin alpha2beta1; D051419:Paxillin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020741:rho GTP-Binding Proteins