OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of Giα1, β-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and β-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.
- Klíčová slova
- FRAP, Gq/11 protein, TRH receptor, lateral mobility, phospholipase Cβ1, β-arrestin,
- MeSH
- beta arrestiny chemie genetika MeSH
- buněčná membrána účinky léků MeSH
- FRAP MeSH
- HEK293 buňky MeSH
- hormon uvolňující thyreotropin chemie metabolismus MeSH
- kinasa 2 receptorů spřažených s G-proteiny chemie MeSH
- lidé MeSH
- ligandy MeSH
- midazolam farmakologie MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go chemie genetika MeSH
- receptory thyroliberinu agonisté antagonisté a inhibitory chemie genetika MeSH
- signální transdukce účinky léků genetika MeSH
- vazba proteinů účinky léků MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- beta arrestiny MeSH
- hormon uvolňující thyreotropin MeSH
- kinasa 2 receptorů spřažených s G-proteiny MeSH
- ligandy MeSH
- midazolam MeSH
- proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
- receptory thyroliberinu MeSH
