Protein alterations induced by long-term agonist treatment of HEK293 cells expressing thyrotropin-releasing hormone receptor and G11alpha protein
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
19911373
DOI
10.1002/jcb.22409
Knihovny.cz E-resources
- MeSH
- Electrophoresis, Gel, Two-Dimensional MeSH
- Apoptosis physiology MeSH
- Cell Line MeSH
- Gene Expression drug effects physiology MeSH
- Thyrotropin-Releasing Hormone metabolism pharmacology MeSH
- Nuclear Proteins metabolism MeSH
- Humans MeSH
- Membrane Proteins drug effects metabolism MeSH
- Protein Serine-Threonine Kinases metabolism MeSH
- Receptors, Thyrotropin-Releasing Hormone metabolism MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Thyrotropin-Releasing Hormone MeSH
- Nuclear Proteins MeSH
- Membrane Proteins MeSH
- Protein Serine-Threonine Kinases MeSH
- Receptors, Thyrotropin-Releasing Hormone MeSH
- STK19 protein, human MeSH Browser
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.
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Biochemical and physiological insights into TRH receptor-mediated signaling
