title: IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.
abstract: Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.
authors.affiliation: Department of Immunology, Mayo Clinic College of Medicine, Rochester, MN, USA.
authors.forename: Stephen E P
authors.initials: SE
authors.lastname: Smith
authors.forename: Anya T
authors.initials: AT
authors.lastname: Bida
authors.forename: Tessa R
authors.initials: TR
authors.lastname: Davis
authors.forename: Hugues
authors.initials: H
authors.lastname: Sicotte
authors.forename: Steven E
authors.initials: SE
authors.lastname: Patterson
authors.forename: Diana
authors.initials: D
authors.lastname: Gil
authors.forename: Adam G
authors.initials: AG
authors.lastname: Schrum
mesh_terms: D015153:Blotting, Western; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism
chemical_list: D011948:Receptors, Antigen, T-Cell; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human