Conclusions
These results indicate that 5-HT(2A) receptor-coupled PLC activation and subsequent Ca(2+) and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca(2+) is sufficient to induce Rac1 transamidation.
Methods
A1A1v cells were pretreated with pharmacological inhibitors of phospholipase C (PLC) or calmodulin (CaM), and then stimulated by the 5-HT(2A) receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI). Intracellular Ca(2+) concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca(2+) by a Ca(2+) ionophore or a chelating agent on Rac1 transamidation was also evaluated.
Results
In cells pretreated with a PLC inhibitor U73122, DOI-stimulated increases in the intracellular Ca(2+) concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca(2+) chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca(2+) ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca(2+) to a level comparable to cells treated with DOI, produced an increase in TGase-modified Rac1 without 5-HT(2A) receptor activation. Moreover, the CaM inhibitor W-7, significantly decreased Rac1 transamidation in a dose-dependent manner in DOI-treated cells. Conclusions: These results indicate that 5-HT(2A) receptor-coupled PLC activation and subsequent Ca(2+) and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca(2+) is sufficient to induce Rac1 transamidation.