Determination of the two-dimensional interaction rate constants of a cytokine receptor complex.
Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFNalpha2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.
Institute of Biochemistry, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Martynas
M
Gavutis
Eva
E
Jaks
Peter
P
Lamken
Jacob
J
Piehler
D003198:Computer Simulation; D016207:Cytokines / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D025941:Protein Interaction Mapping; D018121:Receptors, Cytokine / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D016207:Cytokines; D008024:Ligands; D008051:Lipid Bilayers; D018121:Receptors, Cytokine; D011994:Recombinant Proteins