Leukotriene D4 activates distinct G-proteins in intestinal epithelial cells to regulate stress fibre formation and to generate intracellular Ca2+ mobilisation and ERK1/2 activation

Abstract

We have shown that the pro-inflammatory mediator LTD4, via its G-protein-coupled receptor CysLT1, signals through both pertussis-toxin-sensitive and -insensitive G-proteins to induce various cellular responses. To further characterise the initial step of the different signalling pathways emanating from the CysLT1 receptor, we transfected intestinal epithelial cells, Int 407, with different mini vectors that each express a specific inhibitory peptide directed against a unique alpha subunit of a specific heterotrimeric G-protein. Our results revealed that LTD4-induced stress fibre formation is inhibited approximately 80% by a vector expressing an inhibitory peptide against the pertussis-toxin-insensitive Galpha12-protein in intestinal epithelial Int 407 cells. Control experiments revealed that the LPA-induced stress fibre formation, mediated via the Galpha12-protein in other cell types, was blocked by the same peptide in intestinal Int 407 cells. Furthermore, the CysLT1-receptor-mediated calcium signal and activation of the proliferative ERK1/2 kinase are blocked in cells transfected with a vector expressing an inhibitory peptide against the Galphai3-protein, whereas in cells transfected with an empty ECFP-vector or vectors expressing inhibitory peptides against the Galphai1-2-, Galpha12-, GalphaR-proteins these signals are not significantly affected. Consequently, the CysLT1 receptor has the capacity to activate at least two distinctly different heterotrimeric G-proteins that transduce activation of unique downstream cellular events.