The rapid activation of N-Ras by alpha-thrombin in fibroblasts is mediated by the specific G-protein Galphai2-Gbeta1-Ggamma5 and occurs in lipid rafts

• Published in: Cellular signalling 2009-06, Vol.21 (6), p.1007-1014
• Main Authors: Lents, Nathan H, Irintcheva, Virginia, Goel, Reema, Wheeler, Leroy W, Baldassare, Joseph J
• Format: Article
• Language: English
• Subjects: Animals; CHO Cells; Cholesterol - metabolism; Cricetinae; Cricetulus; Fibroblasts - drug effects; Fibroblasts - metabolism; GTP-Binding Protein alpha Subunit, Gi2 - metabolism; GTP-Binding Protein alpha Subunits, G12-G13; GTP-Binding Protein alpha Subunits, Gq-G11; GTP-Binding Protein beta Subunits - metabolism; GTP-Binding Protein gamma Subunits - metabolism; Guanine Nucleotide-Releasing Factor 2 - metabolism; Humans; Membrane Microdomains - drug effects; Membrane Microdomains - metabolism; Protein Binding - drug effects; Protein Transport - drug effects; ras Proteins - metabolism; Signal Transduction - drug effects; Thrombin - pharmacology
• ISSN: 1873-3913
• DOI: 10.1016/j.cellsig.2009.02.016

alpha-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to alpha-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking alpha-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by alpha-thrombin. Further, although several Galpha subunits associate with PAR1 and are released following stimulation, only Galpha(i2) couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gbeta and Ggamma subunits, only a subset associates with Galpha(i2), and of those, only a single Gbetagamma dimer, Gbeta(1)gamma(5), participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by alpha-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking alpha-thrombin stimulation to the activation of Ras.