Somatostatin displayed on filamentous phage as a receptor-specific agonist.
1. In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. 2. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. 3. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. 4. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand.
CESAME at Dept. Pathology, Maastricht University, The Netherlands.
M
M
Rousch
J T
JT
Lutgerink
J
J
Coote
A
A
de Bruïne
J W
JW
Arends
H R
HR
Hoogenboom
D000595:Amino Acid Sequence; D000818:Animals; D001435:Bacteriophages / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004279:DNA, Viral / Q000032:analysis; D019204:GTP-Binding Proteins / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D019151:Peptide Library; D017481:Receptors, Somatostatin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D013004:Somatostatin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D004279:DNA, Viral; D008024:Ligands; D019151:Peptide Library; D017481:Receptors, Somatostatin; D013004:Somatostatin; D019204:GTP-Binding Proteins