Use of a modified bacteriophage to probe the interactions between peptides and ion channel receptors in mammalian cells.
Besides natural peptide ligands, screening of random peptide libraries has yielded novel bioactive peptides for cell surface receptors. A method is described that uses a modified bacteriophage as a detection reagent to monitor the expression of receptor channels in mammalian cells and to probe the molecular interaction between phage-tethered peptides (phi T-peptides) and specific receptor targets. By taking advantage of a specific multivalent interaction between phi T-peptides and the receptor target, assays have been developed that use phi T-peptides specific for the N-methyl-D-aspartate glutamate receptor, an important ligand-gated ion channel in the nervous system, to monitor the receptor expression in cultured mammalian cells. Combining these phi T-peptide binding assays with fluorescence-activated cell sorting, 10(4) random glutamate receptor mutants were screened and candidate interaction residues were identified. This dual heterologous expression system offers a powerful approach to the molecular studies of protein-protein interactions.
Li
M
M
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. min-li@qmail.bs.jhu.edu
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D005434:Flow Cytometry; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017470:Receptors, Glutamate / Q000737:chemistry* / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites
D017931:DNA Primers; D019151:Peptide Library; D010455:Peptides; D017470:Receptors, Glutamate; D016194:Receptors, N-Methyl-D-Aspartate; D011994:Recombinant Proteins