title: High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.
abstract: 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.
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Anke
authors.initials: A
authors.lastname: Bill
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Rosethorne
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Toby C
authors.initials: TC
authors.lastname: Kent
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Fawcett
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Lynn
authors.initials: L
authors.lastname: Burchell
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Michiel T
authors.initials: MT
authors.lastname: van Diepen
authors.affiliation: Infectious Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Anthony
authors.initials: A
authors.lastname: Marelli
authors.affiliation: Infectious Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Sergey
authors.initials: S
authors.lastname: Batalov
authors.affiliation: Infectious Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Loren
authors.initials: L
authors.lastname: Miraglia
authors.affiliation: Infectious Diseases, Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Orth
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Renaud
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom.
authors.forename: Martin
authors.initials: M
authors.lastname: Gosling
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.
authors.forename: L Alex
authors.initials: LA
authors.lastname: Gaither
authors.affiliation: Respiratory Disease Area, Novartis Institutes for Biomedical Research, Horsham, United Kingdom; Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Basel, Switzerland.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Groot-Kormelink
mesh_terms: D000596:Amino Acids / Q000235:genetics*; D003198:Computer Simulation; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D019811:Hydroxylamine; D016296:Mutagenesis / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D059645:Mutation Rate; D016133:Polymerase Chain Reaction; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000235:genetics*
chemical_list: D000596:Amino Acids; C113936:PTGIR protein, human; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; D019811:Hydroxylamine