Direct observation of G-protein binding to the human δ-opioid receptor using plasmon-waveguide resonance spectroscopy

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791 - 2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain delta-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist (similar to10 nM); the lowest when receptor was bound to an antagonist (similar to 500 nM); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of similar to 60 nM. Furthermore, GTP binding to the receptor . G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the Galpha(o) (K-d similar to 20 nM) and lowest affinity toward the Galpha(i2) (similar to 590 nM) subtypes, whereas the agonist-bound state had highest affinity for the Galpha(o) and Galpha(i2) subtypes (K-d similar to 9 nM and similar to 7 nM, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.