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Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors

Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are n...

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Autores principales: Bracamontes, John R., Akk, Gustav, Steinbach, Joe Henry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786271/
https://www.ncbi.nlm.nih.gov/pubmed/26963253
http://dx.doi.org/10.1371/journal.pone.0151071
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author Bracamontes, John R.
Akk, Gustav
Steinbach, Joe Henry
author_facet Bracamontes, John R.
Akk, Gustav
Steinbach, Joe Henry
author_sort Bracamontes, John R.
collection PubMed
description Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression.
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spelling pubmed-47862712016-03-23 Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors Bracamontes, John R. Akk, Gustav Steinbach, Joe Henry PLoS One Research Article Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression. Public Library of Science 2016-03-10 /pmc/articles/PMC4786271/ /pubmed/26963253 http://dx.doi.org/10.1371/journal.pone.0151071 Text en © 2016 Bracamontes et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Bracamontes, John R.
Akk, Gustav
Steinbach, Joe Henry
Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title_full Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title_fullStr Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title_full_unstemmed Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title_short Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors
title_sort introduced amino terminal epitopes can reduce surface expression of neuronal nicotinic receptors
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786271/
https://www.ncbi.nlm.nih.gov/pubmed/26963253
http://dx.doi.org/10.1371/journal.pone.0151071
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