14-3-3 zeta influences calcium-sensing receptor cell signalling through the Rho pathway
The calcium-sensing receptor (CaR) maintains calcium homeostasis, but also influences processes such as cell proliferation, differentiation and apoptosis through activation of diverse signalling pathways. The CaR has been shown to modulate Rho-dependent stimulation of serum-response element transcription requiring the protein filamin. In addition, filamin has been shown to protect the CaR against degradation through high affinity interaction at residues 962-981 of the CaR tail. To provide insight into mechanisms controlling CaR signalling, a yeast two-hybrid (Y2H) screen was performed using the CaR intracellular tail as bait. Several interacting proteins were identified including the 14-3-3 isoform zeta. 14-3-3 proteins are chaperones which readily form homodimers or heterodimers enabling them to influence a multitude of cellular processes.
Co-immunoprecipitation and confocal microscopy experiments have confirmed CaR-14-3-3 zeta in vivo interaction and co-localisation. Y2H deletion mapping delineated the interaction site for 14-3-3 zeta to residues 965-980 in the CaR tail, overlapping the filamin binding site. Therefore, to determine the possible role of 14-3-3 zeta in CaR-mediated Rho signalling involving filamin, HEK-293 cells stably expressing the CaR and endogenously expressing filamin were transfected with 14-3-3 zeta and a luciferase reporter gene which allowed for the measurement of luciferase activity through Rho-dependent stimulation of serum-response element transcription. Results demonstrated that 14-3-3 zeta inhibited Rho signalling. It is possible that 14-3-3 zeta interferes with the filamin interaction domain on the CaR by competitive binding leading to inhibition of Rho activity. These results suggest a role for 14-3-3 proteins as adapters in the regulation of CaR signalling involving filamin.
Arulpragasam, A., Ward, B. K., Magno, A. L., Ingley, E., Conigrave, A. D., & Ratajczak, T. (2008). 14-3-3 zeta influences calcium-sensing receptor cell signalling through the Rho pathway. Paper presented at the Australian Society for Medical Research Western Australia Scientific Symposium.