Beschreibung:
Caveolae are small invaginations of the plasma membrane, which can expand upon stimulation and be internalized as endocytic vesicles. In addition to their role in clathrin‐independent endocytosis, caveolae play a role in many different signalling pathways.The major caveolar membrane protein, caveolin‐1 (CAV1) forms a dense coat of approx. 150 monomers assembled at the surface of the caveolae. CAV1 is believed to possess a “scaffolding domain” capable of interacting directly with numerous signalling proteins; its high oligomeric state amplifies its potency as a signalling platform.Here we have conducted a comprehensive in vitro screen of potential interacting partners to verify this “caveolin signalling” hypothesis. We have developped a binding assay using co‐expression of interactors and CAV1 in a cell‐free system which mimics the folding and insertion of caveolin in native membranes. Electron tomography shows that CAV1 generates membrane domains of uniform size and curvature and that we can re‐create small decorated vesicles that closely resemble endocytic caveolae.We measured binding of 40 potential interactors on these “reconstituted” caveolae using single‐molecule fluorescence and screened for interaction at the monomer level (non‐inserted CAV1) using a binding assay (AlphaScreen). We reveal exquisite selectivity of the assembled caveolae compared to monomeric CAV1. CAV1 phosphorylation, as found in cellular stress, showed amplification of binding to a subset of interactors. We further characterized binding sites by testing different mutations and truncations that alter the structure and oligomerisation state of CAV1. The results were confirmed in cells by proximity ligation assay, validating protein‐protein interactions and the effect of protein posttranslational modifications.Taken together, our findings create a novel picture of the role of caveolae in cell signalling and stress‐response.