14-3-3 protein isoforms interact differentially with the calcium-sensing receptor intracellular tail

Abstract

The main role of the calcium-sensing receptor (CaR) is to maintain calcium homeostasis but the receptor also participates in other functions such as cell proliferation and apoptosis mediated by various intracellular signalling pathways. To provide insight into mechanisms that control CaR signalling, yeast two-hybrid (Y2H) studies in our laboratory, using the CaR tail as bait, identified a number of interacting proteins including the 14-3-3 isoforms zeta and theta. 14-3-3 proteins are ubiquitously expressed and highly conserved, and are emerging as a group of multifunctional adapter proteins with a recognised role as chaperones. 14-3-3 proteins bind to numerous partner proteins having a preference for phosphorylated targets. Y2H deletion mapping studies have delineated the interaction region for 14-3-3 zeta to residues 923-1078 in the CaR tail. By contrast, the 14-3-3 theta interaction site is confined to residues 865-923 in the CaR tail. This region of 14-3-3 theta interaction contains a consensus 14-3-3 binding motif that includes a phosphorylated serine, residue 895. In vivo interaction between the CaR and 14-3-3 theta has been demonstrated by co-immunoprecipitation in mammalian cells. In addition, a direct in vitro interaction has been confirmed between 14-3-3 theta and the CaR tail using pulldown assays. Site-directed mutagenesis and subsequent co-immunoprecipitation experiments have shown that the phosphoserine residue (S895) in the identified consensus motif in the CaR tail is not primarily responsible for mediating CaR and 14-3-3 theta interaction. Differential binding of the 14-3-3 isoforms to the CaR tail points to differences in the way these isoforms might influence CaR-mediated signalling.

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