Protein alterations induced by long‐term agonist treatment of HEK293 cells expressing thyrotropin‐releasing hormone receptor and G₁₁α protein

Abstract

This study aimed to determine whether sustained stimulation with thyrotropin-releasing hormone (TRH), a peptide with important physiological functions, can possibly affect expression of plasma membrane proteins in HEK293 cells expressing high levels of TRH receptor and G(11)alpha protein. Our previous experiments using silver-stained two-dimensional polyacrylamide gel electrophoretograms did not reveal any significant changes in an overall composition of membrane microdomain proteins after long-term treatment with TRH of these cells (Matousek et al. 2005 Cell Biochem Biophys 42: 21-40). Here we used a purified plasma membrane fraction prepared by Percoll gradient centrifugation and proteins resolved by 2D electrophoresis were stained with SYPRO Ruby gel stain. The high enrichment in plasma membrane proteins of this preparation was confirmed by a multifold increase in the number of TRH receptors and agonist stimulated G-protein activity, compared to postnuclear supernatant. By a combination of these approaches we were able to determine a number of clearly discernible protein changes in the plasma membrane-enriched fraction isolated from cells treated with TRH (1 x 10(-5) M, 16 h): 4 proteins disappeared, the level of 18 proteins decreased and the level of 39 proteins increased. Our concomitant immunochemical determinations also indicated a clear down-regulation of G(q/11)alpha proteins in preparations from hormone-treated cells. In parallel, we observed decrease in caspase 3 and alterations in some other apoptotic marker proteins, which were in line with the presumed antiapoptotic effect of TRH.

Prolonged morphine administration alters protein expression in the rat myocardium

“…Protein spots showing altered abundance levels were excised from the gel, in-gel digested with trypsin, and analyzed by MALDI-TOF MS/MS as described previously [16]. Peptide mass-fingerprinting and subsequent database searching revealed the identity of altered proteins as summarized in Table 1.…”

“…Strips were equilibrated for 15 min in 4 ml of EQ buffer (50 mM Tris, pH 6.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% DTT, followed by a second 15-min equilibration in EQ buffer containing 4% iodacetamide and separated in the second dimension on 10% SDS-polyacrylamide gels using the Hoefer apparatus. Gels were stained with the fluorescent dye SYPRO Ruby or colloidal Coomassie G-250 for MALDI-TOF MS/MS and spot density was quantitatively analyzed by PDQuest software (Bio-Rad, Hercules, CA, USA) as described previously [16]. Proteins with altered expression were identified using a 4800 Plus MALDI TOF/TOF Analyzer [16].…”

“…Gels were stained with the fluorescent dye SYPRO Ruby or colloidal Coomassie G-250 for MALDI-TOF MS/MS and spot density was quantitatively analyzed by PDQuest software (Bio-Rad, Hercules, CA, USA) as described previously [16]. Proteins with altered expression were identified using a 4800 Plus MALDI TOF/TOF Analyzer [16]. The obtained MS spectra were searched against the rat IPI database by Mascot v. 2.1 software (Matrix Science Ltd., London, UK).…”

Identification and subcellular localization of molecular complexes of G<sub>q/11</sub>α protein in HEK293 cells

“…However, we have previously found that the addition of 10 mM TRH to the HEK293 cell line stably expressing TRH-R1 and G 11 m protein does not lead to apoptosis, although the cells stop growing [38]. On the contrary, a prolonged incubation of these cells with TRH increased the Bcl-2/Bax ratio and elevated the level of some proteins with anti-apoptotic and protective effects.…”

The role of Thyrotropin Releasing Hormone in aging and neurodegenerative diseases

“…TRH has also been demonstrated to exert trophic actions in lower motor neurons of the spinal cord (Van den Bergh et al, 1991), and pro-TRH gene expression has been shown to play a role in preventing neuronal apoptosis in the CNS. Furthermore, long-term stimulation of clonal human fibroblast cells (HEK293) cells with TRH can engender multiple antiapoptotic effects, such as a decrease in caspase 3 and increased expression of mitochondrial proteins thought to play a role in antiapoptotic activity (Drastichova et al, 2010). Many of the CNS effects of TRH are coordinated through the coherent modulation of neurotransmitters such as glutamate, dopamine, norepinephrine, serotonin, acetylcholine, and GABA (Malthe-Soressen et al, 1978; Yarbrough, 1979; Deng et al, 2006; Jantas et al, 2009).…”