SOMATOSTATIN DISPLAYED ON FILAMENTOUS PHAGE AS A RECEPTOR-SPECIFIC AGONIST

I In search of methods to identify bio-active ligands specific for G p rotein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional scr eening method. 2 First, methods for panning peptide phage on cells wer e established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into pha ge(mid) vectors and fusion to either pIII or pVIII viral coat proteins . Peptide displaying phage bound to a polyclonal anti-somatostatin ser um, and, more importantly, to several somatostatin receptor subtypes ( Sst) expressed on transfected CHO-K1 cells, in a pattern which was dep endent on the used display method. Binding was competed with somatosta tin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selecti ons of phage libraries. 3 Binding of somatostatin displaying phage to sst(2) on a reporter cell line, in which binding of natural ligand red uces secretion of alkaline phosphatase (via a cyclic AMP responsive el ement sensitive promoter), proved that the phage particles act as rece ptor-specific agonists. Less than 100 phage particles per cell were re quired for this activity, which is approximately 1000 fold less than s oluble somatostatin, suggesting that phage binding interferes with nor mal receptor desensitization and/or recycling. 4 The combination of bi opanning of phage libraries on cells with functional screening of phag e particles for receptor triggering activity, may be used to select no vel, bio-active ligands from phage libraries of random peptides, antib ody fragments, or libraries based on the natural receptor ligand.