Bill_et_al_(2014).pdf (923.79 kB)
High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor
journal contribution
posted on 2023-06-08, 19:46 authored by Anke Bill, Elizabeth M Rosethorne, Toby C Kent, Lindsay Fawcett, Lynn Burchell, Michiel T Van Diepen, Anthony Marelli, Sergey Batalov, Loren Miraglia, Anthony P Orth, Nicole A Renaud, Steven J Charlton, Martin Gosling, L Alex Gaither, Paul J Groot-KormelinkThe 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 structurefunction relationship of GPCRs. © 2014 Bill et al.
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Publication status
- Published
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- Published version
Journal
PLoS ONEISSN
1932-6203Publisher
Public Library of ScienceExternal DOI
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6Volume
9Article number
e97973Department affiliated with
- Chemistry Publications
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- Yes
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- Yes
Legacy Posted Date
2015-01-27First Open Access (FOA) Date
2015-01-27First Compliant Deposit (FCD) Date
2015-01-27Usage metrics
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