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NEUROPHARMACOLOGY

Transglutaminase-Catalyzed Transamidation: A Novel Mechanism for Rac1 Activation by 5-Hydroxytryptamine_2A Receptor Stimulation

Neuroscience Program, Loyola University Medical Center, Maywood, Illinois (Y.D.); Department of Pharmacology and Experimental Therapeutics, School of Medicine, Loyola University Chicago, Maywood, Illinois (N.L.D., T.B.P.); and Department of Pharmacology and Toxicology, School of Pharmacy, University of Kansas, Lawrence, Kansas (N.A.M.)

Transglutaminase (TGase)-induced activation of small G proteins via 5-hydroxytryptamine (HT)_2A receptor signaling leads to platelet aggregation (Cell 115:851–862, 2003). We hypothesize that stimulation of 5-HT_2A receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that, in rat cortical cell line A1A1v, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT_2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine, but not the 5-HT_1A receptor agonist 5-hydroxy-2-dipropylamino tetralin, increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT_2A receptors mediate the transamidation reaction because expression of 5-HT_2C receptors was not detectable and the selective 5-HT_2A receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 min of serotonin treatment, but returns it to control levels after 30 min. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or small interfering RNA reduces TGase modification of Rac1, and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT_2A receptor stimulation as demonstrated by coimmunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT_2A receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT_2A receptor-signaling cascade in neurons.