- Publikační typ
- abstrakt z konference MeSH
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.
- MeSH
- 2D gelová elektroforéza MeSH
- apoptóza fyziologie MeSH
- buněčné linie MeSH
- exprese genu fyziologie účinky léků MeSH
- hormon uvolňující thyreotropin metabolismus MeSH
- lidé MeSH
- membránové proteiny metabolismus účinky léků MeSH
- protein-serin-threoninkinasy metabolismus MeSH
- receptory thyroliberinu metabolismus MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Agonist-induced subcellular redistribution of G-protein coupled receptors (GPCR) and of trimeric guanine-nucleotide binding regulatory proteins (G-proteins) represent mechanisms of desensitization of hormone response, which have been studied in our laboratory since 1989. This review brings a short summary of these results and also presents information about related literature data covering at least small part of research carried out in this area. We have also mentioned sodium plus potassium dependent adenosine triphosphatase (Na, K-ATPase) and 3H-ouabain binding as useful reference standard of plasma membrane purity in the brain.
- MeSH
- buněčná membrána enzymologie metabolismus MeSH
- down regulace MeSH
- heterotrimerní G-proteiny chemie metabolismus MeSH
- hormony metabolismus MeSH
- křečci praví MeSH
- krysa rodu rattus MeSH
- mozek metabolismus MeSH
- multimerizace proteinu MeSH
- proteiny vázající GTP chemie metabolismus MeSH
- receptory spřažené s G-proteiny metabolismus MeSH
- signální transdukce fyziologie MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- subcelulární frakce metabolismus MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
We monitored the radioligand-binding characteristics of thyrotropin-releasing hormone (TRH) receptors, functional activity of G(q/11)alpha proteins, and functional status of the whole signaling cascade in HEK293 expressing high levels of TRH receptors and G(11)alpha. Our analyses indicated that disruption of plasma membrane microdomains by cholesterol depletion did not markedly influence the binding parameters of TRH receptors, but it altered efficacy of signal transduction. The functional coupling between TRH receptor and G(q/11)alpha was assessed by agonist-stimulated [(35)S]GTPgammaS binding, and results of these measurements pointed out to significantly lower potency of TRH to mediate G protein activation in the plasma membrane fraction isolated from cholesterol-depleted cells; there was a shift in sensitivity by one order of magnitude to the higher concentrations. A markedly lower sensitivity to stimulation with TRH was also observed in our experiments dealing with determination of hormone-induced Ca(2+) response. These data suggest that the intact structure of plasma membranes is an important optimum signal transduction initiated by TRH receptors and mediated by G(q/11)alpha proteins.
- MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- buněčné linie MeSH
- cholesterol fyziologie MeSH
- financování organizované MeSH
- guanosin 5'-O-(3-thiotrifosfát) metabolismus MeSH
- hormon uvolňující thyreotropin metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- membránové mikrodomény metabolismus MeSH
- proteiny vázající GTP - alfa-podjednotky Gq-G11 metabolismus MeSH
- receptory thyroliberinu metabolismus MeSH
- signální transdukce MeSH
- vápník metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- MeSH
- beta-1-adrenergní receptory fyziologie MeSH
- finanční podpora výzkumu jako téma MeSH
- heterotrimerní G-proteiny agonisté chemie metabolismus MeSH
- kaveoly chemie MeSH
- signální transdukce MeSH
- sodíko-draslíková ATPasa fyziologie chemie MeSH
- srdeční glykosidy farmakologie MeSH
- zpětná vazba fyziologická fyziologie MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- baklofen farmakologie MeSH
- finanční podpora výzkumu jako téma MeSH
- GABA agonisté farmakologie MeSH
- GTP-fosfohydrolasy metabolismus MeSH
- lékové interakce MeSH
- mozková kůra růst a vývoj účinky léků MeSH
- proteiny vázající GTP fyziologie MeSH
- stárnutí fyziologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
- MeSH
- katecholaminy fyziologie MeSH
- leukocyty mononukleární fyziologie MeSH
- lidé MeSH
- termogeneze fyziologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- kongresy MeSH
- techniky in vitro MeSH
