Lateral ligand-receptor interactions on membranes probed by simultaneous fluorescence-interference detection

¹ Goethe-Universität Frankfurt am Main

^* To whom correspondence should be addressed. E-mail: j.piehler{at}em.uni-frankfurt.de.

Submitted on November 6, 2004
Revised on December 23, 2004
Accepted on 15 February 2005

	Abstract

We describe an experimental approach for studying ligand-receptor interactions in plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFN2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e. association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFN2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.

Key Words: Total internal reflection fluorescence spectroscopy, protein-protein interaction, reduction of dimensionality, reflectance interferometry, solid-supported membrane, type I interferon receptor

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