High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocard...

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Main Authors: Bill, Anke, Rosethorne, Elizabeth M., Kent, Toby C., Fawcett, Lindsay, Burchell, Lynn, van Diepen, Michiel T., Marelli, Anthony, Batalov, Sergey, Miraglia, Loren, Orth, Anthony P., Renaud, Nicole A., Charlton, Steven J., Gosling, Martin, Gaither, L. Alex, Groot-Kormelink, Paul J.
Format: Online
Language:English
Published: Public Library of Science 2014
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041722/
id pubmed-4041722
recordtype oai_dc
spelling pubmed-40417222014-06-09 High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function Bill, Anke Rosethorne, Elizabeth M. Kent, Toby C. Fawcett, Lindsay Burchell, Lynn van Diepen, Michiel T. Marelli, Anthony Batalov, Sergey Miraglia, Loren Orth, Anthony P. Renaud, Nicole A. Charlton, Steven J. Gosling, Martin Gaither, L. Alex Groot-Kormelink, Paul J. Research Article The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. Public Library of Science 2014-06-02 /pmc/articles/PMC4041722/ /pubmed/24886841 http://dx.doi.org/10.1371/journal.pone.0097973 Text en © 2014 Bill 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author Bill, Anke
Rosethorne, Elizabeth M.
Kent, Toby C.
Fawcett, Lindsay
Burchell, Lynn
van Diepen, Michiel T.
Marelli, Anthony
Batalov, Sergey
Miraglia, Loren
Orth, Anthony P.
Renaud, Nicole A.
Charlton, Steven J.
Gosling, Martin
Gaither, L. Alex
Groot-Kormelink, Paul J.
spellingShingle Bill, Anke
Rosethorne, Elizabeth M.
Kent, Toby C.
Fawcett, Lindsay
Burchell, Lynn
van Diepen, Michiel T.
Marelli, Anthony
Batalov, Sergey
Miraglia, Loren
Orth, Anthony P.
Renaud, Nicole A.
Charlton, Steven J.
Gosling, Martin
Gaither, L. Alex
Groot-Kormelink, Paul J.
High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
author_facet Bill, Anke
Rosethorne, Elizabeth M.
Kent, Toby C.
Fawcett, Lindsay
Burchell, Lynn
van Diepen, Michiel T.
Marelli, Anthony
Batalov, Sergey
Miraglia, Loren
Orth, Anthony P.
Renaud, Nicole A.
Charlton, Steven J.
Gosling, Martin
Gaither, L. Alex
Groot-Kormelink, Paul J.
author_sort Bill, Anke
title High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
title_short High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
title_full High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
title_fullStr High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
title_full_unstemmed High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function
title_sort high throughput mutagenesis for identification of residues regulating human prostacyclin (hip) receptor expression and function
description The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.
publisher Public Library of Science
publishDate 2014
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041722/
_version_ 1612095432333197312

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