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Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection
G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPC...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10522717/ https://www.ncbi.nlm.nih.gov/pubmed/37752220 http://dx.doi.org/10.1038/s41598-023-43389-z |
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author | Watanabe, Ayami Nakajima, Ami Shiroishi, Mitsunori |
author_facet | Watanabe, Ayami Nakajima, Ami Shiroishi, Mitsunori |
author_sort | Watanabe, Ayami |
collection | PubMed |
description | G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and then an in vivo screening to obtain mutants that recovered the activity of the human histamine H(3) receptor (H(3)R), which loses its signaling function when expressed in yeast. Four mutations with recovered activity were identified after screening. Three of the mutations were identified near the DRY and NPxxY motifs of H(3)R, which are important for activation and are commonly found in class A GPCRs. The mutants responded exclusively to the yeast YB1 strain harboring G(i)-chimera proteins, showing retention of G protein specificity. Analysis of one of the mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The strategy used in this study may enable the recovery of the activity of other GPCRs that do not function in S. cerevisiae and may be useful in creating GPCRs mutants stabilized in their active conformations. |
format | Online Article Text |
id | pubmed-10522717 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-105227172023-09-28 Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection Watanabe, Ayami Nakajima, Ami Shiroishi, Mitsunori Sci Rep Article G protein-coupled receptors (GPCRs) are the largest protein family in humans and are important drug targets. Yeast, especially Saccharomyces cerevisiae, is a useful host for modifying the function and stability of GPCRs through protein engineering, which is advantageous for mammalian cells. When GPCRs are expressed in yeast, their function is often impaired. In this study, we performed random mutagenesis using error-prone PCR and then an in vivo screening to obtain mutants that recovered the activity of the human histamine H(3) receptor (H(3)R), which loses its signaling function when expressed in yeast. Four mutations with recovered activity were identified after screening. Three of the mutations were identified near the DRY and NPxxY motifs of H(3)R, which are important for activation and are commonly found in class A GPCRs. The mutants responded exclusively to the yeast YB1 strain harboring G(i)-chimera proteins, showing retention of G protein specificity. Analysis of one of the mutants with recovered activity, C415R, revealed that it maintained its ligand-binding characteristics. The strategy used in this study may enable the recovery of the activity of other GPCRs that do not function in S. cerevisiae and may be useful in creating GPCRs mutants stabilized in their active conformations. Nature Publishing Group UK 2023-09-26 /pmc/articles/PMC10522717/ /pubmed/37752220 http://dx.doi.org/10.1038/s41598-023-43389-z Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Watanabe, Ayami Nakajima, Ami Shiroishi, Mitsunori Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title | Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title_full | Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title_fullStr | Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title_full_unstemmed | Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title_short | Recovery of the histamine H(3) receptor activity lost in yeast cells through error-prone PCR and in vivo selection |
title_sort | recovery of the histamine h(3) receptor activity lost in yeast cells through error-prone pcr and in vivo selection |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10522717/ https://www.ncbi.nlm.nih.gov/pubmed/37752220 http://dx.doi.org/10.1038/s41598-023-43389-z |
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