Direct observation of G-protein binding to the human delta-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 ( approximately 10 nm); the lowest when receptor was bound to an antagonist ( approximately 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 approximately 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 Galphao (Kd approximately 20 nm) and lowest affinity toward the Galphai2 ( approximately 590 nm) subtypes, whereas the agonist-bound state had highest affinity for the Galphao and Galphai2 subtypes (Kd approximately 9 nm and approximately 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.