Mapping of the ICOS binding surface of murine B7h using an unbiased, cellular library of B7h mutants created by cyclical packaging rescue.
Functional studies of immunologically relevant molecules often involve time-consuming generation and cloning of gene mutations prior to introduction into mammalian cells. We describe here an alternative mutagenesis approach that relies solely on transfer of helper-free retroviral supernatants to rapidly create in virtually any cell line of interest a large cellular library that retrovirally expresses a defined number of independent point mutations in a gene of interest. Using this rapid non-cloning approach, we generated a 3T3 cellular library retrovirally expressing 2 x 10(5) mutants of the murine costimulatory B7h gene. Screening of this unbiased cellular library identified six residues of murine B7h that are critical for binding to the ICOS receptor. These residues are located on the same strands of human B7h that were identified by targeted mutagenesis [Chattopadhyay, K., Bhatia, S., Fiser, A., Almo, S.C., Nathenson, S.G. (2006). Structural basis of inducible costimulator ligand costimulatory function: determination of the cell surface oligomeric state and functional mapping of the receptor-binding site of the protein. J. Immunol. 177, 3920], indicating that the ICOS receptor-binding interface is similar in mouse and human B7h. Based on this proof-of-principle study, CPR-based mutagenesis is applicable to studies of gene function in a variety of mammalian cells.
Bakkour
Sonia
S
Cancer Research Laboratory, 441 LSA, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200 United States.
Sha
William C
WC
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D018604:Epitope Mapping / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D060945:Inducible T-Cell Co-Stimulator Ligand; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods; D025941:Protein Interaction Mapping; D011506:Proteins / Q000235:genetics / Q000276:immunology*; D012190:Retroviridae / Q000235:genetics; D012680:Sensitivity and Specificity; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C498514:ICOSLG protein, human; C400230:Icosl protein, mouse; D060945:Inducible T-Cell Co-Stimulator Ligand; D019151:Peptide Library; D011506:Proteins