title: DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells.
abstract: The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.
authors.affiliation: Department of Biosciences and Informatics, Keio University, Yokohama, Japan. doi@bio.keio.ac.jp
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
authors.forename: Natsuko
authors.initials: N
authors.lastname: Yamakawa
authors.forename: Hideaki
authors.initials: H
authors.lastname: Matsumoto
authors.forename: Yasutsugu
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nagano
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Matsumura
authors.forename: Kenichi
authors.initials: K
authors.lastname: Horisawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000737:chemistry / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D008024:Ligands; D019151:Peptide Library; D010455:Peptides; D044140:Receptor, Angiotensin, Type 1; D011994:Recombinant Proteins; D000804:Angiotensin II; D004247:DNA; D002118:Calcium