Phospholipase C, Ca2, and calmodulin signaling are required for 5-HT2A receptor-mediated transamidation of Rac1 by transglutaminase.
Serotonin and especially serotonin 2A (5-HT(2A)) receptor signaling are important in the etiology and treatment of schizophrenia and affective disorders. We previously reported a novel 5-HT(2A) receptor effector, increased transglutaminase (TGase)-catalyzed transamidation, and activation of the small G protein Rac1 in A1A1v cells, a rat embryonic cortical cell line. In this study, we explore the signaling pathway involved in 5-HT(2A) receptor-mediated Rac1 transamidation. 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. 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. 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.