The goal of this proposal is to identify and characterize novel proteins and peptide motifs that mediate cell adhesion and migration using a novel functional proteomic approach. We propose to develop further a cell-screening strategy that utilizes a GFP-tagged cDNA library transfected into adherent/spread cells as well as actively migrating cells. Candidate clones are identified by their (1) localization in focal adhesions, (2) perturbation or alteration of adhesive structures and/or (3) perturbation of cell migration. Secondary screens that study more carefully the adhesive and migratory properties of the cells expressing the cDNAs, along with sequence homologies, are used to determine which clones will be studied in depth. We have already identified several novel or uncharacterized focal adhesion genes by this method, and I am particularly interested in characterizing one of them since it is novel, localizes in focal adhesions, and its over expression appears to affect cell migration. This screening process has also been used to identify several truncated peptides, i.e., 20-200 amino acid random peptides, fused to GFP, that also localize to focal adhesions or show strong phenotypes when ectopically expressed. These peptides, in essence, are an intracellular analogue of a phage display library that can be used to identify protein targeting motifs for focal adhesions and provide specific reagents for perturbing function. We have already identified a 100 amino acid peptide that has homology to the disintegrin family of proteins and appears to inhibit cell spreading. My initial studies demonstrate the feasibility and utility of these approaches to identify clones that contribute to adhesion and migration. These screens can be modified into a high throughput format for screening cDNA, peptide, and chemical libraries.
