The heterotrimeric G-protein alpha-subunit Galphaq regulates TCR-mediated immune responses through an Lck-dependent pathway.

Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Galphaq and Galphas, but not Galphai-2. Targeting of Galphas, Galphai-2 and Galphaq using siRNA demonstrated a specific role of Galphaq in TCR signaling.

Reduced Cbl phosphorylation and degradation of the zeta-chain of the T-cell receptor/CD3 complex in T cells with low Lck levels.

T cells with short interfering RNA-mediated Lck-knockdown (kd) display paradoxical hyper-responsiveness upon TCR ligation. We have previously reported a possible mechanism for T-cell activation in cells with low levels of Lck depending on Grb2-SOS1 recruitment to the zeta-chain of TCR/CD3 (Methi et al., Eur.

Hypophosphorylated TCR/CD3zeta signals through a Grb2-SOS1-Ras pathway in Lck knockdown cells.

Despite the loss of proximal TCR-dependent signaling events, downstream T cell responses are paradoxically augmented in T cells with siRNA-mediated Lck knockdown (Methi et al., J. Immunol.

Short-interfering RNA-mediated Lck knockdown results in augmented downstream T cell responses.

The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization.

Endogenous expression and protein kinase A-dependent phosphorylation of the guanine nucleotide exchange factor Ras-GRF1 in human embryonic kidney 293 cells.

We have previously reported the Ras-dependent activation of the mitogen-activated protein kinases p44 and p42, also termed extracellular signal-regulated kinases (ERK)1 and 2 (ERK1/2), mediated through Gs-coupled serotonin receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Whereas Gi- and Gq-coupled receptors have been shown to activate Ras through the guanine nucleotide exchange factor (GEF) called Ras-GRF1 (CDC25Mm) by binding of Ca2+/calmodulin to its N-terminal IQ domain, the mechanism of Ras activation through Gs-coupled receptors is not fully understood. We report the endogenous expression of Ras-GRF1 in HEK293 cells.

The cAMP-Epac-Rap1 pathway regulates cell spreading and cell adhesion to laminin-5 through the alpha3beta1 integrin but not the alpha6beta4 integrin.

Laminin-5 is an important constituent of the basal lamina. The receptors for laminin-5, the integrins alpha3beta1 and alpha6beta4, have been associated with epithelial wound migration and carcinoma invasion. The signal transduction mechanisms that regulate these integrins are not well understood.