Efficiency of acetylcholine receptor subunit assembly and its regulation by cAMP.

The primary target for nicotine in the brain is the neuronal nicotinic acetylcholine receptor (nAChR). It has been well documented that nAChRs respond to chronic nicotine exposure by up-regulation of receptor numbers, which may underlie some aspects of nicotine addiction. In order to investigate the mechanism of nicotine-induced nAChR up-regulation, we have developed a cell culture system to assess membrane trafficking and nicotine-induced up-regulation of surface-expressed ␣ 4 ␤ 2 nAChRs. Previous reports have implicated stabilization of the nAChRs at the plasma membrane as the potential mechanism of up-regulation. We have found that whereas nicotine exposure results in up-regulation of surface receptors in our system, it does not alter surface receptor internalization from the plasma membrane, postendocytic trafficking, or lysosomal degradation. Instead, we find that transport of nAChRs through the secretory pathway to the plasma membrane is required for nicotine-induced up-regulation of surface receptors. Therefore, nicotine appears to regulate surface receptor levels at a step prior to initial insertion in the plasma membrane rather than by altering their endocytic trafficking or degradation rates as had been previously suggested. Neuronal nicotinic acetylcholine receptors (nAChRs)1 in mammalian brain compose a family of proteins encoded by 11 genes (␣ 2-7 , ␣ 9 -10 , and ␤ 2-4 ) that assemble into pentameric ligandgated ion channels (1-4). Although all combinations of subunits that form functional nicotinic receptors can bind nicotine, ␣ 4 -and ␤ 2 -containing receptors form the high affinity nicotine binding site (5-9) and are therefore thought to be the primary nAChR subtype affected by the relatively low nicotine concentrations (100 -500 nM) found in the blood of smokers (10).Chronic exposure of ␣4␤2 receptors to nicotine, as occurs in smokers, results initially in receptor desensitization, followed by subsequent up-regulation of high affinity nicotine binding sites (11). Upon nicotine withdrawal, the increased number of nAChRs recover from desensitization, resulting in excess activity in the nAChR system. It has been proposed that this cycle of nicotine-induced receptor up-regulation may contribute to the negative symptoms associated with nicotine withdrawal, resulting in continued tobacco consumption and, eventually, nicotine dependence (12).The mechanisms and cellular machinery required for nicotine-induced up-regulation remain unknown. However, a large body of research supports a consensus on several relevant points. First, up-regulation is observed in the brains of human smokers (13) as well as in chronically nicotine-treated animal models (8,14) and in cultured cells heterologously expressing nAChRs (15-18), suggesting that up-regulation requires basic, conserved cellular processes. It has been convincingly shown that increased nicotine binding reflects an increase in receptor number rather than receptor affinity (15, 19 -21), implying that up-regulation involves receptor protein dynamics rather th...

Section: Discussionsupporting

confidence: 81%

Exocytic Trafficking Is Required for Nicotine-induced Up-regulation of α4β2 Nicotinic Acetylcholine Receptors

Darsow

Booker

Piña‐Crespo

et al. 2005

“…This indicates that intracellular ␣␤ 128 ␥␦ complexes are similar in size and composition to intracellular ␣␤␥␦ complexes with the exception of the ␣ 2 ␤␥␦ complexes in the 9 S peak. The broad profile observed for the intracellular AChR complexes relative to the cell surface ␣ 2 ␤␥␦ 9 S peak has been seen in other studies both for the Torpedo subunits at reduced temperature (4,8,40) and the mouse subunits at 37°C (3,5,6,8). D, the effects of the ␣ 128/142 and ␤ 128 subunits on subunit assembly are consistent with the model shown.…”

Section: Elimination Of Cystine Loop Disulfide Bond On the ␣ Subunit supporting

confidence: 77%

The Role of the Cystine Loop in Acetylcholine Receptor Assembly

Green

Wanamaker

1997

Nicotinic acetylcholine receptors (AChRs) are composed of ␣, ␤, ␥, and ␦ subunits, assembled into ␣ 2 ␤␥␦ pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into ␣␤␥ trimers, but the subsequent steps in assembly are blocked. When ␣ subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when ␤ subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of ␣␤␥␦ tetramers and before the addition of the second ␣ subunit. After formation of the cystine loop, the ␣ subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of ␣␤␥ trimers and before the addition of the ␦ subunit. The data indicate that the ␣ subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.The molecular events involved in subunit folding and assembly of large oligomeric proteins remain largely uncharacterized. The subunit folding and oligomerization events that take place during the assembly of ion channels are particularly complex, since the finished product requires the correct oligomeric arrangement and subunit stoichiometry for proper function (1). In terms of their structure, the best characterized ion channels are the muscle-type nicotinic acetylcholine receptors (AChRs), 1 which are the neurotransmitter receptors responsible for rapid signaling between motor neurons and skeletal muscle. Muscle-type AChRs are composed of four distinct, homologous subunits, ␣, ␤, ␥, and ␦, which assemble into pentamers with the subunit stoichiometry of ␣ 2 ␤␥␦.Although AChR assembly is a slow process that takes ϳ2 h to complete (2), assembly intermediates have been difficult to isolate. A number of laboratories turned to expression of less than the full complement of subunits in different heterologous expression systems to isolate assembly intermediates (3-6).Based on their findings, the "heterodimer" model was proposed, where the ␣ subunit must first fold or "mature," as assayed by the formation of the ␣-bungarotoxin (BuTx) binding site and antigenic epitopes, before assembling with other subunits. The mature ␣ subunit assembles with ␥ or ␦ subunits in parallel to form ␣␥ and ␣␦ heterodimers, and the heterodimers associate together and with ␤ subunits to form ␣ 2 ␤␥␦ pentamers.We have developed techniques that have allowed isolation of assembly intermediates in cells stably expressing all four AChR subunits (7, 8) and have obtained results at odds with the heterodimer model. Instead of heterodimers, two partially assembled complexes, ␣␤␥ trimers and ␣␤␥␦ tetramers, were isol...

“…Interestingly, it has been demonstrated that both the ␥ and ␦ subunits are phosphorylated in vivo, and the ␦ subunits are more highly phosphorylated in the unassembled than in the assembled state indicating that phosphorylation precedes assembly and that phosphorylation/dephosphorylation mechanisms control the AChR subunit (36). Furthermore, using Torpedo AChR subunits expressed in mouse fibroblasts, it has been demonstrated previously that cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of the unassembled ␥ subunit assembly (37). But the underlying mechanism by which this phosphorylation increases the efficiency of subunit assembly and increased surface AChR expression has not been elucidated.…”

Section: Discussionmentioning

confidence: 99%

“…In muscle-type AChRs, pulse-chase experiments and immunofluorescent microscopy indicate that AChR subunit assembly is complete in the ER following which AChR oligomers move rapidly through the Golgi membrane onto the plasma membrane (37). Interestingly, it has been demonstrated that both the ␥ and ␦ subunits are phosphorylated in vivo, and the ␦ subunits are more highly phosphorylated in the unassembled than in the assembled state indicating that phosphorylation precedes assembly and that phosphorylation/dephosphorylation mechanisms control the AChR subunit (36).…”

Section: Discussionmentioning

confidence: 99%

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

Jeanclos

Lin

Treuil

et al. 2001

147

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...