Direct Observation of G-protein Binding to the Human δ-Opioid Receptor Using Plasmon-Waveguide Resonance Spectroscopy

Abstract

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 ␦-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 (ϳ10 nM); the lowest when receptor was bound to an antagonist (ϳ500 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 ϳ60 nM. Furthermore, GTP binding to the receptor⅐G-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 G␣ o (K d ϳ 20 nM) and lowest affinity toward the G␣ i2 (ϳ590 nM) subtypes, whereas the agonist-bound state had highest affinity for the G␣ o and G␣ i2 subtypes (K d ϳ 9 nM and ϳ 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.Opioid receptors belong to the superfamily of GPCRs.1 Their predicted topology is that of a polytopic integral membrane protein with seven membrane-spanning helical segments, an extracellular N terminus and an intracellular C terminus. These receptors and their endogenous ligands, opioid peptides such as endorphins and enkephalins, form a neuromodulatory system that is involved in stress-induced analgesia, that affects locomotive activity and regulates neuroendocrine physiology and autonomic functions such as respiration, blood pressure, and gastrointestinal motility. The opioid system has also been shown to play a role in learning and memory and possibly in the modulation of the immune system (1) and is an important factor in pain modulation and drug abuse. Studies to determine the G-protein subtypes that mediate the intracellular signaling of the opioid receptor systems have shown that functional coupling occurs to the G i -G o family of G-proteins (2-4). The human brain ␦-opioid receptor has been cloned (5), stably transfected in Chinese hamster ovary (CHO) cells, and characterized (6).Due to their integral membrane nature and their low cellular concentrations, there is little information on the structures and functional mechanisms of the opioid receptors. Furthermore, classic pharmacological methods only give indirect information about the interaction of GPCRs with G-protein...

Unique agonist-bound cannabinoid CB1 receptor conformations indicate agonist specificity in signaling

“…As seen in Fig 10A, addition of GTPγS to WIN 55,212-2 bound human cannabinoid CB 1 -myc-His 6 receptor led to a decrease in the resonance angle, indicating a decrease in the proteolipid mass, presumably due to the dissociation of the agonist/ human cannabinoid CB 1 -myc-His 6 receptor /G iα1 ternary complex and release of the alpha subunit from the bilayer. This is similar to results obtained with the Δ-opioid receptor (Alves et al 2003). The PWR shift was GTPγS dose-dependent and reached saturation at 10 nM GTPγS concentration.…”

“…Previous investigations have demonstrated (Alves et al, 2003;Alves et al, 2004a,b) that ligand-bound G-protein coupled receptors incorporate into the lipid bilayer randomly, with either their extracellular (ligand binding) or their intracellular (G protein binding) surfaces facing the aqueous compartment of the PWR cell. Thus, upon addition of G proteins into the aqueous compartment we can directly measure the affinity of ligand-bound receptor conformations to individual G protein types.…”

“…Since it is known that GPCR receptor incorporation proceeds bidirectionally (Alves et al, 2003(Alves et al, , 2004a, in this way a fraction of ligand-bound receptors will have their G-protein binding site accessible to the external aqueous medium. Subsequently, small aliquots of recombinant rat G iα1 (Calbiochem, San Diego,CA) and a βγ subunit mixture purified from bovine brain (Calbiochem, San Diego,CA) (1:1 ratio) were added to the equilibrated proteolipid system and PWR spectral changes were determined.…”

“…Addition of a GTP molecule results in the dissociation of the high affinity ternary complex and the activation of the G protein. Therefore, measuring the affinity of a non-hydrolyzable GTPanalogue (GTPγS) to the agonist/receptor/G protein ternary complex can provide information about the potency of the given agonist-bound receptor conformation to activate G protein types (Alves et al, 2003;Alves et al, 2004b;Subramaniam et al, 2005). As seen in Fig 10A, addition of GTPγS to WIN 55,212-2 bound human cannabinoid CB 1 -myc-His 6 receptor led to a decrease in the resonance angle, indicating a decrease in the proteolipid mass, presumably due to the dissociation of the agonist/ human cannabinoid CB 1 -myc-His 6 receptor /G iα1 ternary complex and release of the alpha subunit from the bilayer.…”

“…PWR has been successfully used previously to evaluate ligand-mediated changes in the conformation of G-protein coupled receptors, to determine the affinities of G protein subtypes to ligand-bound receptors and to assess the efficacies of ligands to activate individual G proteins (Salamon et al, 2000;Alves et al, 2003;Alves et al, 2004a;Alves et al, 2004b).…”