Proteomic Analysis Unveils Expressional Changes in Cytoskeleton- and Synaptic Plasticity-Associated Proteins in Rat Brain Six Months after Withdrawal from Morphine

Drug withdrawal is associated with abstinence symptoms including deficits in cognitive functions that may persist even after prolonged discontinuation of drug intake. Cognitive deficits are, at least partially, caused by alterations in synaptic plasticity but the precise molecular mechanisms have not yet been fully identified. In the present study, changes in proteomic and phosphoproteomic profiles of selected brain regions (cortex, hippocampus, striatum, and cerebellum) from rats abstaining for six months after cessation of chronic treatment with morphine were determined by label-free quantitative (LFQ) proteomic analysis. Interestingly, prolonged morphine withdrawal was found to be associated especially with alterations in protein phosphorylation and to a lesser extent in protein expression. Gene ontology (GO) term analysis revealed enrichment in biological processes related to synaptic plasticity, cytoskeleton organization, and GTPase activity. More specifically, significant changes were observed in proteins localized in synaptic vesicles (e.g., synapsin-1, SV2a, Rab3a), in the active zone of the presynaptic nerve terminal (e.g., Bassoon, Piccolo, Rims1), and in the postsynaptic density (e.g., cadherin 13, catenins, Arhgap35, Shank3, Arhgef7). Other differentially phosphorylated proteins were associated with microtubule dynamics (microtubule-associated proteins, Tppp, collapsin response mediator proteins) and the actin–spectrin network (e.g., spectrins, adducins, band 4.1-like protein 1). Taken together, a six-month morphine withdrawal was manifested by significant alterations in the phosphorylation of synaptic proteins. The altered phosphorylation patterns modulating the function of synaptic proteins may contribute to long-term neuroadaptations induced by drug use and withdrawal

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

<p><b>Objectives:</b> Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to G_q/11 proteins.</p> <p><b>Methods:</b> We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP).</p> <p><b>Results:</b> FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of G_q/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of G_iα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of G_iα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling.</p> <p><b>Conclusion:</b> These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.</p