Endothelin receptor in virus-like particles: ligand binding observed by fluorescence fluctuation spectroscopy.

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 (ET(A)R) 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 ET(A)Rs in the VLiPs. For the interaction between ET-1 and VLiP-integrated ET(A)Rs, K(D) 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 ET(A)Rs in membrane fragments derived from an ET(A)R-overexpressing mammalian cell line, which yielded a similar K(D) of 0.2 nM. In addition, we examined the binding competition of a set of reference compounds to VLiP-ET(A)Rs in the presence of ET-1 and obtained K(i) values similar to those reported in the literature. Our results demonstrate that integration into VLiPs does not change the binding properties of the ET(A)Rs. FIDA analysis of VLiP-integrated receptors shows great promise for highly miniaturized and fast compound testing in the pharmaceutical industry.