Structural Determinants of α4β2 Nicotinic Acetylcholine Receptor Trafficking

Ren, Xiao‐Qin; Cheng, Shi-Bin; Treuil, Magdalen W.; Mukherjee, Jayanta; Rao, Jayaraman; Braunewell, K. H.; Lindstrom, Jon; Anand, René

doi:10.1523/jneurosci.1079-05.2005

Cited by 49 publications

(72 citation statements)

References 47 publications

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“…The M3-M4 loop of nAChRs mediates important receptor properties such as export from the endoplasmic reticulum (ER) (25, 26), targeting to axonal or dendritic compartments (27), and interactions with scaffold and signaling molecules (8,28). To identify proteins that associate with the ␤2-M3-M4 loop, we generated a fusion protein encoding the M3-M4 loop (amino acid residues 326-454 of mouse ␤2) in association with GST [GST-␤2 (M3-M4)], and we used a pulldown strategy to isolate the interactions of GST-␤2 (M3-M4) from mouse brain [supporting information (SI) Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The movement of receptors within cells requires the activity of molecules such as dynamin, a GTPase that mediates vesicle budding, and clathrin, a major constituent of the vesicle coat (8,25). Several ligand-gated ion channels, such as GABA A , are found to traffic in a dynamin-dependent fashion (43).…”

Section: Interestingly Alterations In the Expression Of Nsf And Dynamentioning

confidence: 99%

“…Studies show that Ϸ90% of the high-affinity nicotine receptor sites within the brain consist of the ␣4␤2-nAChR (5, 6). Topological analysis of nAChR subunits indicates the presence of a large, highly divergent intracellular loop (M3-M4 loop) that is implicated in trafficking and targeting properties of nAChRs (7,8). Recent analysis of a prokaryotic homolog of the nAChR family reveals that the M3-M4 loop is unique to the evolution of the nAChR in eukaryotes (9).…”

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Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Kabbani

Woll

Levenson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The ␤2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrixassisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the ␤2 subunit and components of immunoprecipitated ␤2-nAChR complexes from mouse brain. Using the ␤2-knockout mouse to exclude nonspecific binding to the ␤2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the ␤2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the ␤2-nAChR. Interactions of the ␤2 subunit with NIPs such as G protein ␣, G protein-regulated inducer of neurite outgrowth 1, and G proteinactivated K ؉ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the ␤2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.mass spectrometry ͉ receptor complex N icotine is a common drug of addiction and a leading cause of preventable deaths in developed countries (1, 2). The target of nicotine is a class of nicotinic acetylcholine receptors (nAChRs) existing as pentameric channels made up of various receptor subunits and distributed throughout the brain (3). To date, 11 neuronal subunits have been identified: ␣2-␣9 and ␤2-␤4. The specific combination of these subunits confers the pharmacological and physiological properties of the nAChR (4). Studies show that Ϸ90% of the high-affinity nicotine receptor sites within the brain consist of the ␣4␤2-nAChR (5, 6). Topological analysis of nAChR subunits indicates the presence of a large, highly divergent intracellular loop (M3-M4 loop) that is implicated in trafficking and targeting properties of nAChRs (7,8). Recent analysis of a prokaryotic homolog of the nAChR family reveals that the M3-M4 loop is unique to the evolution of the nAChR in eukaryotes (9).The generation of subunit-specific knockout (KO) mice has proven valuable in defining the role of individual nAChR subunits in brain physiology and animal behavior (10). Experiments in mice lacking the ␤2 subunit (␤2 Ϫ/Ϫ ) demonstrate an important role for this receptor subunit in neuronal development and plasticity (10), protection from excitotoxicity (11), and the mechanisms underlying cognitive and social behaviors (12). ␤2 Ϫ/Ϫ mice also exhibit a loss of nicotine self-administration and nicotine-elicited firing and dopamine release from dopaminergic neurons of the ventral tegmental area (13,14). Because reexpression of the ␤2 subunit in ␤2 Ϫ/Ϫ mice was found sufficient to restore the electrophysiological and beh...

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Section: Resultsmentioning

confidence: 99%

Section: Interestingly Alterations In the Expression Of Nsf And Dynamentioning

confidence: 99%

mentioning

confidence: 99%

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Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Kabbani

Woll

Levenson

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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“…ER export signals have also been demonstrated to enhance the recruitment of receptors into ER exit sites (24). In addition, degradation signals present in the cytoplasmic M3-M4 subunit loop (25,26) must be masked for ER exit licensing. The cytoplasmic M3-M4 loop is also involved in interactions with adaptor proteins, such as the small cytoplasmic chaperone 14-3-3 (27) or the UBXD4 protein (28).…”

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confidence: 99%

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

Richard

Boulin

Robert

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

110

146

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The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6), a previously uncharacterized gene in C. elegans. emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABA A receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

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“…Site-directed mutagenesis, in combination with heterologous expression, has however been used successfully to examine phenomena such as, subunit glycosylation [225][226][227], the role of disulfide-linked cysteines [80,228], cell surface receptor trafficking [214,229,230], interactions with agonists and antagonists [231][232][233], modulation by zinc [234,235] and by other allosteric modulators [199,200], calcium permeability [236] and channel gating [202,[237][238][239][240][241]. Analysis of mutated nAChRs has also provided insights into how subunit domains may move during receptor activation [241,242].…”

Section: Expression Of Nachrs Altered By Site-directed Mutagenesismentioning

confidence: 99%

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

Millar

2009

Biochemical Pharmacology

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Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels, a family that also includes receptors for γ-aminobutyric acid, glycine and 5-hydroxytryptamine. In humans, nAChRs have been implicated in several neurological and psychiatric disorders and are major targets for pharmaceutical drug discovery. In addition, nAChRs are important targets for neuroactive pesticides in insects and in other invertebrates. Historically, nAChRs have been one of the most intensively studied families of neurotransmitter receptors. They were the first neurotransmitter receptors to be biochemically purified and the first to be characterized by molecular cloning and heterologous expression. Although much has been learnt from studies of native nAChRs, the expression of recombinant nAChRs has provided dramatic advances in the characterization of these important receptors. This review will provide a brief history of the characterization of nAChRs by heterologous expression. It will focus, in particular, upon studies of recombinant nAChRs, work that has been conducted by many hundreds of scientists during a period of almost 30 years since the molecular cloning of nAChR subunits in the early 1980's.

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Structural Determinants of α4β2 Nicotinic Acetylcholine Receptor Trafficking

Cited by 49 publications

References 47 publications

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

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