title: Monitoring the dynamics of ligand-receptor complexes on model membranes.
abstract: Ligand-induced cross-linking of cell surface receptors is a basic paradigm of signal activation by many transmembrane receptors. After ligand binding, the receptor complexes formed on the membrane are dynamically maintained by two-dimensional protein-protein interactions on the membrane. The biophysical principles governing the dynamics of such interactions have not been understood, mainly because the measurement of lateral interactions on membranes so far has not been experimentally addressed. Here, we describe a generic approach for measuring two-dimensional dissociation rate constants in vitro using a novel high-affinity chelator lipid for reconstituting a ternary cytokine-receptor complex on solid-supported membranes. While monitoring the interaction between the ligand and one of the receptor subunits on the membrane by fluorescence resonance energy transfer, the equilibrium on the surface was perturbed by rapidly tethering a large excess of the unlabeled receptor subunit. Displacement of labeled by unlabeled protein in the ternary complex was detected as a recovery of the donor quenching. Since the dissociation of the ligand-receptor complex in plane of the membrane was the rate-limiting step under these conditions, the two-dimensional rate constant of this process was determined. Strikingly, the two-dimensional dissociation was much slower than ligand dissociation into solution, suggesting that membrane tethering significantly affects the dissociation process. This result highlights the importance of studying ligand-receptor complexes tethered to membranes for understanding the principles governing signal activation by ligand-induced receptor assembling.
authors.affiliation: Institute of Biochemistry, Goethe University Frankfurt/Main, Marie-Curie-Strasse 9, 60439 Frankfurt/Main, Germany.
authors.forename: Suman
authors.initials: S
authors.lastname: Lata
authors.forename: Martynas
authors.initials: M
authors.lastname: Gavutis
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
mesh_terms: D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D016207:Cytokines / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D016898:Interferon-alpha / Q000737:chemistry; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008567:Membranes, Artificial; D008958:Models, Molecular; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D053633:Receptor, Interferon alpha-beta; D018121:Receptors, Cytokine / Q000737:chemistry* / Q000378:metabolism; D017471:Receptors, Interferon / Q000737:chemistry
chemical_list: D002614:Chelating Agents; D016207:Cytokines; D016898:Interferon-alpha; D008563:Membrane Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D018121:Receptors, Cytokine; D017471:Receptors, Interferon; D053633:Receptor, Interferon alpha-beta; D009571:Nitrilotriacetic Acid