Endothelin Receptor in Virus-Like Particles:  Ligand Binding Observed by Fluorescence Fluctuation Spectroscopy†

Lenka Zemanová,‡ Andreas Schenk,‡ Nicholas Hunt,§ G. Ulrich Nienhaus,‡‖ and Ralf Heilker*
Department of Biophysics, University of Ulm, Albert Einstein Allee 11, D-89081 Ulm, Evotec OAI, Schnackenburgallee 114, D-22525 Hamburg, Germany, Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, and Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str.65, D-88397 Biberach, Germany
Biochemistry, 2004, 43 (28), pp 9021–9028
DOI: 10.1021/bi035901+
Publication Date (Web): June 19, 2004
Copyright © 2004 American Chemical Society
Mammalian Hormones

Abstract

Abstract Image

The functional analysis of transmembrane receptor proteins is frequently hampered by the difficulty to produce sufficiently homogeneous receptor preparations that preserve the physiological biomembrane integration of the receptor protein. To improve the receptor protein density in the lipid bilayer and to maintain the physiological lipid−protein environment, a novel method has been established that enables the selective integration of transmembrane receptors into a virus-like particle (VLiP). Here we have studied the binding of tetramethylrhodamine-labeled endothelin-1 (TMR-ET-1) to VLiP-integrated endothelin A receptor (ETAR) by fluorescence fluctuation spectroscopy. The concentration of TMR-ET-1 was determined by fluorescence correlation spectroscopy (FCS). These measurements also confirmed that the free ligand is monomeric in solution in our experiments. Fluorescence intensity distribution analysis (FIDA) was used to quantify the fraction of ligands bound to ETARs in the VLiPs. For the interaction between ET-1 and VLiP-integrated ETARs, KD values of 0.5 nM and 0.3 nM were determined from ligand and receptor titration experiments, respectively. For comparison, a FIDA analysis was also carried out with ETARs in membrane fragments derived from an ETAR-overexpressing mammalian cell line, which yielded a similar KD of 0.2 nM. In addition, we examined the binding competition of a set of reference compounds to VLiP-ETARs in the presence of ET-1 and obtained Ki values similar to those reported in the literature. Our results demonstrate that integration into VLiPs does not change the binding properties of the ETARs. FIDA analysis of VLiP-integrated receptors shows great promise for highly miniaturized and fast compound testing in the pharmaceutical industry.

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History

  • Published In Issue July 20, 2004
  • Received October 24, 2003
    Revised Manuscript Received May 18, 2004

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