Title

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

Abstract

Introduction: The calcium-sensing receptor (CaR) is a receptor of biomedical importance being pivotal in maintaining calcium homeostasis, and also influencing processes such as cell proliferation, differentiation and apoptosis through activation of signalling pathways such as Rho and ERK1/2. 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 isoforms theta and zeta. 14-3-3 proteins are chaperones which bind to numerous partner proteins, preferring targets containing phosphorylated motifs. They influence a multitude of cellular processes, including cell signalling.

The CaR has been shown to be involved in Rho signalling leading to subsequent serum response element (SRE)-mediated gene transcription, and activation of Rho signalling by the CaR is enhanced by the structural protein, filamin. The region of the CaR involved in Rho signalling is localised to residues 906-980. Furthermore, filamin has been demonstrated to interact with the CaR tail at residues 962-981. Preliminary Y2H studies in our laboratory have established that both 14-3-3 theta and zeta interact with the CaR tail. These findings implicate a possible role for 14-3-3 proteins in CaR-mediated Rho signalling involving filamin.

Objectives:

(1) To confirm CaR and 14-3-3 protein interaction in the mammalian system

(2) To delineate the 14-3-3 binding region on the CaR

(3) To determine whether 14-3-3 proteins modulate CaR-mediated Rho signalling

Methods:

(1) Co-immunoprecipitation studies to confirm the interaction between 14-3-3 proteins and the CaR

(2) A yeast two-hybrid beta-galactosidase interaction assay to delineate 14-3-3 binding regions on the CaR

(3) A luciferase reporter assay (as a measure of Rho-dependent stimulation of SRE-mediated gene transcription) to determine whether 14-3-3 proteins modulate CaR-mediated Rho signalling in HEK-293 cells stably expressing the CaR (HEK-293/CaR), and in melanoma cells not expressing filamin (M2 cells) and stably expressing filamin (A7 cells).

Results: Co-immunoprecipitation studies confirmed CaR-14-3-3 in vivo interaction for both 14-3-3 theta and zeta. Y2H mapping studies delineated the interaction site for 14-3-3 zeta to residues 965-980 on the CaR tail, which overlaps the filamin interaction domain. By contrast, the 14-3-3 theta interaction site is confined to an upstream domain (residues 865-923).

To determine the possible role of 14-3-3 theta or zeta in CaR-mediated Rho signalling, HEK-293/CaR cells were transfected with 14-3-3 theta or zeta and a SRE luciferase reporter which allowed for the measurement of luciferase activity. Results demonstrated that over-expression of both 14-3-3 theta or zeta inhibited CaR-mediated Rho signalling in HEK-293/CaR cells. This effect was not seen in cells unable to express filamin (M2 cells). Further examination of Rho signalling in M2 cells stably expressing filamin (A7 cells) demonstrated that re-introducing filamin did not allow modulation of CaR-mediated Rho signalling by over-expression of 14-3-3 protein as seen in HEK-293/CaR.

Conclusions: We have demonstrated that both 14-3-3 theta and zeta interact with the CaR in vivo and bind differentially to the CaR tail. The inhibition of CaR-mediated Rho signalling by over-expression of 14-3-3 proteins suggests a regulatory role for the proteins in Rho signalling through competitive binding with filamin on the CaR tail. The inability to restore this inhibition in A7 cells may have been due to low level expression of filamin in these cells compared to HEK-293 cells.

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