Phospholipase C, Ca²⁺, and calmodulin signaling are required for 5-HT_2A receptor-mediated transamidation of Rac1 by transglutaminase

Rationale: 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. Objectives: In this study, we explore the signaling pathway involved in 5-HT_2A receptor-mediated 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 Ca²⁺ concentration and TGase-modified Rac1 transamidation were monitored. The effect of manipulation of intracellular Ca²⁺ by a Ca²⁺ 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 Ca²⁺ concentration and TGase-modified Rac1 were significantly attenuated as compared to those pretreated with U73343, an inactive analog. The membrane-permeant Ca²⁺ chelator, BAPTA-AM strongly reduced TGase-catalyzed Rac1 transamidation upon DOI stimulation. Conversely, the Ca²⁺ ionophore ionomycin, at a concentration that induced an elevation of cytosolic Ca²⁺ 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 Ca²⁺ and CaM signaling are necessary for TGase-catalyzed Rac1 transamidation, and an increase in intracellular Ca²⁺ is sufficient to induce Rac1 transamidation.