Title

The role of 14-3-3 proteins in calcium-sensing receptor-mediated Rho signalling

Abstract

The calcium-sensing receptor (CaR) is a class C, G protein-coupled receptor which is pivotal in maintaining calcium homeostasis. The receptor also influences a number of other cellular processes including cell proliferation, differentiation and apoptosis. To achieve this, the CaR activates a number of different cell signalling pathways including Rho-dependent serum-response element (SRE) activation, which is enhanced by the partner binding protein, filamin.

To provide further insight into mechanisms controlling CaR signalling, a yeast two-hybrid screen was performed using the CaR intracellular tail as bait. A number of interacting proteins were identified including 14-3-3 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins which are most often phosphorylated. The proteins influence a multitude of cellular processes, including cell signalling and have been shown to be involved in various aspects of the Rho signalling pathway.

Yeast two-hybrid mapping studies delineated the interaction site for both 14-3-3 isoforms to residues 865-922 on the CaR tail, and co-immunoprecipitation studies confirmed CaR/14-3-3 theta and 14-3-3 zeta in vivo interaction in mammalian cells. To investigate the possible role of 14-3-3 theta and 14-3-3 zeta in CaR-mediated SRE activation, HEK-293 cells stably expressing the CaR (HEK-293/CaR) were transfected with either 14-3-3 theta or 14-3-3 zeta and an SRE luciferase reporter, which allowed for the measurement of SRE activation. Results demonstrated that over-expression of both 14-3-3 theta and 14-3-3 zeta inhibited CaR-mediated SRE activation in these cells. This effect was not seen in CaR-transfected M2 cells unable to express filamin nor in CaR-transfected M2 cells stably expressing filamin. We propose a mechanism whereby 14-3-3 theta and/or 14-3-3 zeta either competitively bind with or sequester filamin leading to the inhibition of CaR-mediated SRE activation in HEK-293/CaR cells.

Keywords

Poster presentation, Abstract only

Comments

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