The thrombin receptor, PAR-1, causes transformation by activation of Rho-mediated signaling pathways.
We utilized a cDNA expression library derived from the B6SutA(1) mouse myeloid progenitor cell line to search for novel oncogenes that promote growth transformation of NIH3T3 cells. A 2.2 kb transforming cDNA was recovered that encodes the wild type thrombin-stimulated G protein-coupled receptor PAR-1. In addition to its potent focus forming activity, constitutive overexpression of PAR-1 in NIH3T3 cells promoted the loss of anchorage- and serum-dependent growth. Although inhibitors of thrombin failed to block PAR-1 transforming activity, a PAR-1 mutant that cannot be cleaved by thrombin was nontransforming. Since the foci of transformed cells induced by PAR-1 bear a striking resemblance to those induced by activated RhoA, we determined if PAR-1 transformation was due to the aberrant activation of a specific Rho family member. Like RhoA, PAR-1 cooperated with activated Raf-1 and caused synergistic enhancement of transforming activity, induced stress fibers when microinjected into porcine aortic endothelial cells, stimulated the activity of the serum response factor and NF-kappaB transcription factors, and PAR-1 transformation was blocked by co-expression of dominant negative RhoA. Finally, PAR-1 transforming activity was blocked by pertussis toxin and by co-expression of the RGS domain of Lsc, implicating Galpha(i) and Galpha(12)/Galpha(13) subunits, respectively, as mediators of PAR-1 transformation. Taken together, these observations suggest that PAR-1 growth transformation is mediated, in part, by activation of RhoA.
Martin
C B
CB
Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina NC 27599-7295, USA.
Mahon
G M
GM
Klinger
M B
MB
Kay
R J
RJ
Symons
M
M
Der
C J
CJ
Whitehead
I P
IP
D016475:3T3 Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D000199:Actins / Q000378:metabolism; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000502:physiology; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D051379:Mice; D022423:Myeloid Cells / Q000502:physiology; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D000199:Actins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D020962:Heterotrimeric GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein