The Chaperone Protein 14-3-3η Interacts with the Nicotinic Acetylcholine Receptor α4 Subunit

Abstract

By using the large cytoplasmic domain of the nicotinic acetylcholine receptor (AChR) ␣4 subunit as a bait in the yeast two-hybrid system, we isolated the first cytosolic protein, 14-3-3, known to interact directly with neuronal AChRs. 14-3-3 is a member of a family of proteins that function as regulatory or chaperone/ scaffolding/adaptor proteins. 14-3-3 interacted with the recombinant ␣4 subunit alone in tsA 201 cells following activation of cAMP-dependent protein kinase by forskolin. The interaction of 14-3-3 with recombinant ␣4 subunits was abolished when serine 441 of the ␣4 subunit was mutated to alanine (␣4 S441A ). The surface levels of recombinant wild-type ␣4␤2 AChRs were ϳ2-fold higher than those of mutant ␣4 S441A ␤2 AChRs. The interaction significantly increased the steady state levels of the ␣4 subunit and ␣4␤2 AChRs but not that of the mutant ␣4 S441A subunit or mutant ␣4 S441A ␤2 AChRs. The EC 50 values for activation by acetylcholine were not significantly different for ␣4␤2 AChRs and ␣4 S441A ␤2 AChRs coexpressed with 14-3-3 in oocytes following treatment with forskolin. 14-3-3 coimmunopurified with native ␣4 AChRs from brain. These results support a role for 14-3-3 in dynamically regulating the expression levels of ␣4␤2 AChRs through its interaction with the ␣4 subunit. Neuronal nicotinic acetylcholine receptors (AChR)1 are a family of ligand-gated, cation-selective, homo-or heteropentameric ion channels expressed in the peripheral and central nervous system (1, 2). A multitude of neuronal AChR subtypes assembled from different combinations of ␣2-␣9 and ␤2-␤4 subunits have been identified (3,4 (7), and show attenuated self-administration of nicotine (8) suggesting that ␣4␤2 AChRs have a role in mediating addiction to nicotine. The normal and pathophysiological functions mediated by ␣4␤2 AChRs are of significant importance to human health. Some inherited forms of epilepsy, such as the autosomal dominant nocturnal frontal lobe epilepsies, are caused by ␣4␤2 AChRs harboring at least two separate mutations within their ␣4 subunit (9 -12). Most recently, ␣4␤2 AChRs, among other ␤2 subunit-containing AChRs, have been implicated in neuronal survival during aging, as surmised from the neurodegeneration observed in ␤2-subunit knock-out mice (13).The ␣4 subunit, like the other AChR subunits, consists of an extracellular N-terminal domain, followed by three transmembrane domains (M1-M3), a large cytoplasmic domain, a fourth transmembrane domain (M4), and a short extracellular C terminus. The large cytoplasmic domain is highly divergent among the various subunits, and this sequence divergence presumably provides the diversity necessary for different AChR subtypes to interact directly with cytosolic proteins of different function. To identify such proteins associated with ␣4␤2 AChRs, we used the large cytoplasmic domain of the ␣4 subunit as a bait to screen a mouse brain cDNA yeast two-hybrid library. Here we describe the isolation of a known protein termed 14-3-3. The 14-3-3 proteins family consists of sev...

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…14-3-3 is present in postsynaptic densities isolated from rat brain (Martin et al, 1994;Couve et al, 2001;Rajan et al, 2002). Interaction with 14-3-3 promotes the surface expression of selected neurotransmitter receptors and ion channels (Jeanclos et al, 2001;Rajan et al, 2002;Exley et al, 2006). In particular, 14-3-3 binds directly to another neuronal nAChR subunit, the long cytoplasmic loop of α4, a major subtype in the CNS, and this interaction increases surface expression of α4*nAChRs in heterologous cell expression studies (Jeanclos et al, 2001;Exley et al, 2006).…”

“…Interaction with 14-3-3 promotes the surface expression of selected neurotransmitter receptors and ion channels (Jeanclos et al, 2001;Rajan et al, 2002;Exley et al, 2006). In particular, 14-3-3 binds directly to another neuronal nAChR subunit, the long cytoplasmic loop of α4, a major subtype in the CNS, and this interaction increases surface expression of α4*nAChRs in heterologous cell expression studies (Jeanclos et al, 2001;Exley et al, 2006). 14-3-3 binding to the α4 loop overrides retention signals in the endoplasmic reticulum and facilitates forward transport of the assembled receptor (O'Kelly et al, 2002).…”

Nicotinic α7 receptors: Synaptic options and downstream signaling in neurons

“…Few molecules have been identified as candidates for organizing postsynaptic nicotinic receptors on neurons (Huh and Fuhrer, 2002). Yeast two-hybrid screens have shown the chaperone protein 14-3-3 capable of associating with nAChR ␣4 subunits, and immunoprecipitation experiments confirm that the chaperone is associated with ␣4␤2-nAChRs in brain (Jeanclos et al, 2001). The association leads to an increase in the number of ␣4␤2-nAChRs found on the surface of cotransfected cells.…”

Regulatory mechanisms that govern nicotinic synapse formation in neurons

“…Similarly, a recent yeast two-hybrid genetic screen has identified a protein that directly binds nAChR subunits and influences surface expression. 14-3-3, a protein chaperone, binds to the long intracellular loop of the ␣4 subunit (Jeanclos et al, 2001). Heterologous coexpression of 14-3-3 with ␣4 and ␤2 nAChR subunits significantly increases surface expression and stabilizes the nAChRs.…”