The ζ Isoform of 14-3-3 Proteins Interacts with the Third Intracellular Loop of Different α2-Adrenergic Receptor Subtypes

Abstract

The ␣ 2 -adrenergic receptors (␣ 2 ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. 35 S]Met-14-3-3 binds to all three native ␣ 2 AR subtypes, assessed using a solid phase binding assay (␣ 2A >␣ 2B > ␣ 2C ), and this binding depends on the presence of the 3i loops. Attenuation of the ␣ 2 AR-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 ␣ 2 AR 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.The three ␣ 2 -adrenergic receptor (␣ 2 AR) 1 subtypes, encoded by distinct genes (1), all couple via the G i /G o family of GTPbinding proteins to inhibition of adenylyl cyclase, suppression of voltage-sensitive calcium channels, and activation of receptor-operated potassium channels (2). These receptors also couple to activation of Ras (3, 4), the mitogen-activated protein kinase cascade (3, 5-7), and to activation of phospholipase D (8, 9).Despite the qualitatively similar signaling properties of the three ␣ 2 AR subtypes, differences in trafficking of these receptors have been reported. For example, subtype-selective differences in agonist-elicited ␣ 2 AR redistribution occur (10 -15). In addition, selective itineraries for the ␣ 2 AR subtypes are observed in polarized Madin-Darby canine kidney (MDCKII) renal epithelial cells. Thus, the ␣ 2A AR subtype is targeted directly to the basolateral surface (16), whereas the ␣ 2B AR subtype is delivered randomly to both the apical and basolateral surfaces but is rapidly lost from the apical (t1 ⁄2 ϭ 5-15 min) and selectively retained on the basolateral (t1 ⁄2 ϭ 10 -12 h) surface (17). These findings suggest that there is a molecular mechanism responsible for the selective retention of the ␣ 2B AR on the basolateral domain of MDCK cells that may be shared by all three ␣ 2 AR subtypes, as they manifest comparable halflives on that surface (17).Receptor retention on the lateral subdomain of MDCKII cells likely involves the third intracellular loop of the ␣ 2A AR, since deletion of this loop, creating the mutant ␣ 2A ⌬3iAR, results in accelerated basolateral turnover (t1 ⁄2 Х 4.5 h) when compared with that for the wild-type receptor or with ␣ 2A AR structures that have been mutated in the N terminus or the C-terminal tail (all possessing a t1 ⁄2 of 10 -12 h) (18). The accelerated turnover of the ␣ 2A ⌬3iAR when compared with the wild-type ␣ 2A AR structure suggests that the third intracellular loop interacts with proteins that either tether ␣ 2A AR to a particular surface domain or, alternatively, mask the ␣ 2A AR from interacting with endocytosis machiner...

14-3-3ζ Protein Regulates Anterograde Transport of the Human κ-Opioid Receptor (hKOPR)

“…Our finding is consistent with the observation that interaction of 14-3-3 with all three types of ␣ 2 -adrenergic receptors does not require receptor phosphorylation (44). In addition, the parathyroid hormone receptor interacts with 14-3-3 in the C-tail, and the binding is reduced by phosphorylation of the C-tail by protein kinase A (45).…”

“…Expression of 14-3-3 attenuated the Ca 2ϩ -sensing receptor-mediated Rho signaling but had no effect on ERK1/2 signaling, whereas expression of 14-3-3 significantly reduced plasma membrane expression of the receptor (49). 14-3-3 interacts with the third intracellular loops of all three types of ␣ 2 -adrenergic receptors (44). The parathyroid hormone receptor interacts with 14-3-3 in the C-tail, which is reduced by treatment of the C-tail with protein kinase A (45).…”

A comparison of agonist‐specific coupling of cloned human α2‐adrenoceptor subtypes

“…See Kukkonen et al, 1998;Peltonen et al, 1998;Pihlavisto et al, 1998;Audubert et al, 1999;Olli-LaÈ hdesmaÈ ki et al, 1999;Prezeau et al, 1999;Takesono et al, 1999). The ability of the three a 2 -adrenoceptor subtypes to regulate the cyclic AMP second messenger pathway, has been extensively studied in numerous cell lines (Duzic & Lanier, 1992;Eason et al, 1992;Jansson et al, 1994b;NaÈ sman et al, 1997;Pohjanoksa et al, 1997).…”

Regulation of α2AR trafficking and signaling by interacting proteins

“…Interactions of 14-3-3ζ are proposed to occur with the α 2 AR in its inactive state. This interpretation derives from the finding that α 2 AR-14-3-3ζ interactions can be competed for by phosphorylated Raf peptides, but not by corresponding non-phosphorylated peptides [42]. Thus, the α 2 AR appears to interact with 14-3-3ζ at a site shared by phosphorylated Raf, as initially demonstrated by Muslin [47;48].…”

“…Two scaffolding proteins, 14-3-3ζ [42] and spinophilin [43], were identified to interact with the 3i loops of all three α 2 AR subtypes by gel overlay analysis and GST pull-down assay.…”