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Analysis of Early Events in Acetylcholine Receptor Assembly

Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into α2β γ δ pentamers, we analyzed the...

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Published in:The Journal of cell biology 1991-06, Vol.113 (6), p.1371-1384
Main Authors: Paulson, Henry L., Ross, Anthony F., Green, William N., Claudio, Toni
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creator Paulson, Henry L.
Ross, Anthony F.
Green, William N.
Claudio, Toni
description Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into α2β γ δ pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains. We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of α, β, γ, and δ subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of α, contain some disulfide-linked subunits. The coprecipitation of α subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associate with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. This fraction increases at temperatures more permissive for assembly, further indicating the importance of the monomer as the precursor to assembly of α2β γ δ pentamers.
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Psychology</topic><topic>Gels</topic><topic>Immunoblotting</topic><topic>L cells</topic><topic>Marine</topic><topic>Molecular and cellular biology</topic><topic>Molecular Weight</topic><topic>Monomers</topic><topic>Muscles - cytology</topic><topic>Muscles - metabolism</topic><topic>Precipitin Tests</topic><topic>proteins</topic><topic>Receptors, Cholinergic - biosynthesis</topic><topic>Torpedo</topic><topic>Torpedo californica</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Paulson, Henry L.</creatorcontrib><creatorcontrib>Ross, Anthony F.</creatorcontrib><creatorcontrib>Green, William N.</creatorcontrib><creatorcontrib>Claudio, Toni</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Technology Research Database</collection><collection>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Engineering Research Database</collection><collection>Aquatic Science &amp; Fisheries Abstracts (ASFA) 1: Biological Sciences &amp; Living Resources</collection><collection>Aquatic Science &amp; Fisheries Abstracts (ASFA) Professional</collection><collection>Biochemistry Abstracts 1</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Paulson, Henry L.</au><au>Ross, Anthony F.</au><au>Green, William N.</au><au>Claudio, Toni</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Analysis of Early Events in Acetylcholine Receptor Assembly</atitle><jtitle>The Journal of cell biology</jtitle><addtitle>J Cell Biol</addtitle><date>1991-06-01</date><risdate>1991</risdate><volume>113</volume><issue>6</issue><spage>1371</spage><epage>1384</epage><pages>1371-1384</pages><issn>0021-9525</issn><eissn>1540-8140</eissn><coden>JCLBA3</coden><abstract>Mammalian cell lines expressing nicotinic acetylcholine receptor (AChR) subunit cDNAs from Torpedo californica were used to study early events in AChR assembly. To test the hypothesis that individual subunits form homooligomeric intermediates before assembling into α2β γ δ pentamers, we analyzed the sedimentation on sucrose density gradients of each subunit expressed separately in cell lines. We have shown previously that the acute temperature sensitivity of Torpedo AChR subunit assembly is due, in part, to misfolding of the polypeptide chains. We use this phenomenon to further analyze putative assembly-competent intermediates. In nonionic detergent at an assembly-permissive temperature, the majority of α, β, γ, and δ subunits sediment neither as 3-4S monomers nor as 9S complexes, but rather as 6S species whether synthesized in fibroblasts, myoblasts, or differentiated myosyncytia. Several results indicate that the 6S species are complexes comprised predominantly of incorrectly folded subunit polypeptides. The complexes represent homoaggregates which form rapidly within the cell, are stable to mild SDS treatment and, in the case of α, contain some disulfide-linked subunits. The coprecipitation of α subunit with BiP or GRP78, a resident protein of the ER, further indicates that at least some of these internally sequestered subunits also associate with an endogenous protein implicated in protein folding. The majority of subunits expressed in these cell lines appear to be aggregates of subunits which are not assembly intermediates and are not assembly-competent. The portion which migrates as monomer, in contrast, appears to be the fraction which is assembly competent. 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identifier ISSN: 0021-9525
ispartof The Journal of cell biology, 1991-06, Vol.113 (6), p.1371-1384
issn 0021-9525
1540-8140
language eng
recordid cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_2289024
source Alma/SFX Local Collection
subjects 3T3 cells
acetylcholine
Animals
Biological and medical sciences
biological membranes
biosynthesis
Cell aggregates
Cell Differentiation
Cell Line
Cell lines
cell membranes
Cell structures and functions
Cells
Centrifugation, Density Gradient
Complementary DNA
Dimers
Disulfides - metabolism
Electrophoresis, Polyacrylamide Gel
Fibroblasts
Fibroblasts - cytology
Fibroblasts - metabolism
Fundamental and applied biological sciences. Psychology
Gels
Immunoblotting
L cells
Marine
Molecular and cellular biology
Molecular Weight
Monomers
Muscles - cytology
Muscles - metabolism
Precipitin Tests
proteins
Receptors, Cholinergic - biosynthesis
Torpedo
Torpedo californica
title Analysis of Early Events in Acetylcholine Receptor Assembly
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