Fluorescently labelled versions of the κ opioid receptor for the development of biophysical assays

Abstract

G-protein coupled receptors, GPCRs, are located in the plasma membrane of eukaryotic cells. They participate in the highly complex cell communication and regulation pathways by transmitting the signals from extracellular signalling molecules to the interior of the cell and thereby inducing a cellular response. The κ opioid receptor, KOR, is a GPCR, which takes part in the modulation of e.g. pain, stress, mood and addiction.

In this project, two different fluorescent versions of the κ opioid receptor were developed by site-specific cysteine labelling of the receptor with different fluorescent probes. One of the labelled KOR variants is brightly fluorescent and highly photostable while the fluorescence of the KOR labelled with the other fluorescent probe is sensitive to changes in the molecular environment of the probe. Thus, the two labelled κ opioid receptors can be applied to investigate the receptor in different ways. In this project it was investigated whether conformational changes induced by addition of ligand to the KOR labelled with the sensitively fluorescent probe, could be detected as a change in fluorescence due to differences in the surroundings of the fluorophores in the receptor in different conformations. When increasing concentrations of ligand were added to a sample of the labelled KOR, the fluorescence was seen to change until a certain concentration of ligand was reached, after which it remained constant. The detected change in fluorescence may be caused by ligand-induced structural rearrangements, thus the data suggests that the system can be improved to clearly communicate conformational changes in the receptor. A perspective of this assay is the development of a biosensor for drug screening assays.

Time and date

June 26, 2009 at 13:15. HCØ, Aud. 5.