Protein Alterations Induced by Long-Term Agonist Treatment of HEK293 Cells Expressing Thyrotropin-Releasing Hormone Receptor and G(11)alpha Protein

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 G I a protein. Our previous experiments using silver-stained two-dimensional polyacrylanlide gel electrophoretograms did not reveal any significant changes in an overall composition of membrane rnicrodomain 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 21) 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 membrame-enriched fraction isolated from cells treated with TRH (I x 10(-5) M, 16h): 4 proteins disappeared, the level of 18 proteins decreased and the level of 39 proteins increased. Our concomitant In determinations a a clear down-regulation of Gq/1 1Q proteins in preparations from hornnone-treated cc.11s. 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. J. Cell. Biochem. 109: 255-264, 2010. (C) 2009 Wiley-Liss, Inc.