Transglutaminase-Catalyzed Transamidation: A Novel Mechanism for Rac1 Activation by 5-Hydroxytryptamine_2AReceptor Stimulation

Abstract

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 receptorsignaling cascade in neurons.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…Examples in the literature include muscarinic receptor-mediated increases in TG2 activity in SH-SY5Y cells (Zhang et al, 1998) and 5-HT2A receptor-mediated transamidation (TG-catalyzed) of the small G-protein Rac1 in the rat cortical cell line A1A1v (Dai et al, 2008). The study by Zhang et al (1998) measured in situ TG2 activity (polyamine incorporation) triggered by the non-selective muscarinic agonist carbachol, whereas, Dai et al (2008) reported TG2 catalysed incorporation of 5-hydroxytryptamine into Rac1. 5-HT2A receptor-mediated incorporation of 5-HT into the small GTPases RhoA and Rab4 has also been observed in platelets (Walther et al, 2003).…”

“…However, the regulation of intracellular TG2 following stimulation of GPCRs is not well understood. Examples include muscarinic receptor-mediated increases in TG2 activity in SH-SY5Y cells (Zhang et al, 1998), 5-HT2A receptor-mediated transamidation of Rac1 in the rat A1A1v cortical cell line (Dai et al, 2008) and A1 adenosine receptor-mediated increases in TG2 transamidase activity in rat H9c2 cardiomyoblasts (Vyas et al, 2016).…”

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival

“…Examples in the literature include muscarinic receptor-mediated increases in TG2 activity in SH-SY5Y cells [38] and 5-HT2A receptor mediated transamidation (TG-catalyzed) of the small G-protein Rac1 in the rat A1A1v cells [39].…”

“…Zhang et al, [38] measured in situ TG2 activity (polyamine incorporation) triggered by the muscarinic agonist carbachol, whereas Dai et al, [39] reported TG2 catalysed incorporation of 5-hydroxytryptamine into Rac1. 5-HT2A receptor-mediated incorporation of 5-HT into the small GTPases RhoA and Rab4 was also observed in platelets [40].…”

Brief serotonin exposure initiates arteriolar inward remodeling processes in vivo that involve transglutaminase activation and actin cytoskeleton reorganization

“…These results are in accordance with previous findings implicating 5-HT in the activation of transglutaminases. It has been demonstrated that 5-HT activation of 5-HT 2A receptors increases transglutaminase activity in the rat cortical cell line A1A1v (11). In rat aortic smooth muscle cells, a 10-min exposure to 5-HT induces activation of RhoA, via 5-HT 1B/1D receptors in a transglutaminase-dependent manner.…”