Serotonylation of the Rho Family GTPases: Investigation in Neuronal Cell Models

Serotonin is a well-known monoamine neurotransmitter that displays a wide range of biological activities, one of which is protein serotonylation. In this process serotonin is covalently linked to a protein by the calcium-dependent activity of the enzyme Transglutaminase-2. Many different proteins can be serotonylated, ranging from extracellular proteins such as fibronectin, to intracellular proteins such as the small GTPases. This investigation focused on the Rho family of small GTPases, in particular the proteins RhoA, Rac1 and Cdc42, which all have roles in modulating the polymerisation activity of the actin cytoskeleton. These proteins can be serotonylated, which leads to their constitutive activation. It is thought that these changes in activity play a role in altered dendritic spine morphologies seen in neuropsychiatric disorders. This thesis examined the serotonylation of these three GTPases in neuronal cell culture models using Liquid Chromatography Tandem Mass Spectrometry techniques. Additionally, the chemical synthesis of the serotonin analogue O-propargylserotonin was investigated for its application to detect serotonylation using Immunocytochemistry and Western Blotting methodologies. The results of the Liquid Chromatography Tandem Mass Spectrometry investigation indicated that there was no serotonylation of the GTPases RhoA, Rac1 and Cdc42 under the conditions applied, and that this may be due to the absence of specific receptor stimulation events. Investigation of O-propargylserotonin synthesis indicated that there were inconsistencies with the published methods, which required an alternate methodology to be used.