TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins

Abstract

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 G_q/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 G_q/11α, Gβ, β-arrestin2 and phospholipase Cβ1, but not of G_iα1, 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 G_iα1 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.

Keywords:

• TRH receptor
• G_q/11 protein
• β-arrestin
• phospholipase Cβ1
• lateral mobility
• FRAP

Acknowledgments

The authors would like to thank Dr. Jan Krusek for his assistance with calcium imaging experiments.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Charles University Grant Agency (project No 402214) and co-financed by the European Regional Development Fund and the State Budget of the Czech Republic (CZ.1.05/4.1.00/16.0347).