The ζ isoform of 14-3-3 proteins interacts with the third intracellular loop of different α2-adrenergic receptor subtypes

Abstract

The α2-adrenergic receptors (α2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the α(2A)AR (Val217-Ala377), α(2B)AR (Lys210- Trp354), and α(2C)AR (Arg248-Va1363) were used as ligands in gel overlay assays. A protein doublet of ~30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (α(2B) ≥ α(2C) >> α(2A)) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the ζ isoform of 14-3-3 proteins. [35S]Met-14-3-3ζ binds to all three native α2AR subtypes, assessed using a solid phase binding assay (α(2A)≥/α(2B)> α(2C)), and this binding depends on the presence of the 3i loops. Attenuation of the α2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the α2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.