title: Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11alpha 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.
authors.affiliation: Faculty of Science, Department of Physiology, Charles University, Vinicna 7, Prague, Czech Republic.
authors.forename: Zdenka
authors.initials: Z
authors.lastname: Drastichova
authors.forename: Lenka
authors.initials: L
authors.lastname: Bourova
authors.forename: Lucie
authors.initials: L
authors.lastname: Hejnova
authors.forename: Petr
authors.initials: P
authors.lastname: Jedelsky
authors.forename: Petr
authors.initials: P
authors.lastname: Svoboda
authors.forename: Jiri
authors.initials: J
authors.lastname: Novotny
mesh_terms: D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D008565:Membrane Proteins; D009687:Nuclear Proteins; D018025:Receptors, Thyrotropin-Releasing Hormone; D013973:Thyrotropin-Releasing Hormone; D017346:Protein Serine-Threonine Kinases; C087996:STK19 protein, human