Characterization of Solubilized Opioid Receptors: Reconstitution and Uncoupling of Guanine Nucleotide‐Sensitive Agonist Binding

Abstract

Opioid receptors were solubilized from bovine striatal membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS). High concentrations of NaCl (0.5-1.0 M) were necessary to ensure optimal yields, which ranged from 40 to 50% of membrane-bound receptors. This requirement was found to be specific for sodium, with only lithium able to substitute partially, as previously reported for solubilization with digitonin. Opioid antagonists, but not agonists, were able to bind to soluble receptors with high affinity. High-affinity binding of mu, delta, and kappa agonists was reconstituted following polyethylene glycol precipitation and resuspension of CHAPS extract. Evidence is presented suggesting that this is the result of inclusion of receptors in liposomes. Competition and saturation studies indicate that the three opioid receptor types retain their selectivity and that they exist in the reconstituted CHAPS extract in a ratio (50:15:35) identical to that in the membranes. In reconstituted CHAPS extract, as in membranes, mu-agonist binding was found to be coupled to a guanine nucleotide binding protein (G protein), as demonstrated by the sensitivity of [3H][D-Ala2,N-methyl-Phe4,Gly5-ol]-enkephalin ([3H]DAGO) binding to guanosine 5'-O-(thiotriphosphate) (GTP gamma S). In the reconstituted CHAPS extract, complete and irreversible uncoupling by GTP gamma S was observed, whereas membrane-bound receptors were uncoupled only partially. Treatment with GTP gamma S, at concentrations that uncoupled the mu receptors almost completely, resulted in a fourfold decrease in the Bmax of [3H]DAGO binding with a relatively small change in the KD. Competition experiments showed that the Ki of DAGO against [3H]bremazocine was increased 200-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and pharmacological characterization of the human μ‐opioid receptor in the methylotrophic yeast Pichia pastoris

“…Our data obtained by expressing a wild-type and a truncated (61 amino-terminal residues) HUMOR in COS-M6 cells (Emorine L.J., unpublished) showed that this was indeed true for antagonists but that K~ values were increased from 7 to 60 nM for DAGO and from 10 to 80 nM for morphine in wild-type and truncated receptors, respectively. Additionally, in mammalian cells, Gpp(NH)p a non-hydrolysable analogue of GTP, is able to shift receptors from a high-affinity to a low-affinity state for agonists by uncoupling the receptors and G-proteins [24,28]. We showed here that 100 gM Gpp(NH)p had no such effects on agonist binding (Table 1), certainly reflecting the lack of functional interactions of the HUMOR with host G-proteins.…”

Solubilization of Serotonin1AReceptors Heterologously Expressed in Chinese Hamster Ovary Cells

“…PEG precipitation of the CHAPS-solubilized membrane was performed to remove NaCl from the solubilized extract since the agonist binding of the 5-HT 1A receptor is inhibited by NaCl (Harikumar and Chattopadhyay, 1998a). This procedure constitutes a method in which the detergent along with salt is rapidly removed (Gal et al, 1983, Kremenetzky andAtlas, 1984;Ofri et al, 1992) thus overcoming the problem of long dialysis periods (Aguilar and Ochoa, 1986;Medrano et al, 1989). The solubilization efficiency is moderate when 0.25 M NaCl is used.…”

“…CHAPS has very low absorbance at 280 nm (unlike Triton X-100) and does not have circular dichroic activity in the far UV region, making it ideal for optical studies of proteins. These factors have led to extensive use of CHAPS in solubilization of membrane proteins and receptors (Banerjee et al, 1995;Ofri et al, 1992). We have previously used CHAPS at low concentration to solubilize 5-HT 1A receptors from bovine hippocampus in a functional form (Chattopadhyay et al, 2002;Chattopadhyay and Harikumar, 1996).…”

Dependence of critical micelle concentration of a zwitterionic detergent on ionic strength: implications in receptor solubilization

“…In addition, CHAPS has very low absorbance at 280 nm (unlike Triton X-100) and does not have circular dichroic activity in the far UV region, making it ideal for optical studies of proteins. These factors have led to extensive use of CHAPS in solubilization of membrane proteins and receptors [17,[26][27][28][29][30][31].…”

“…This procedure is believed to remove the salt [19,20]. PEG precipitation was carried out by diluting the extract with equal volume of 40% PEG-8000 in buffer C [17]. Following a vigorous vortexing and incubation for 10 min on ice, the samples were centrifuged at 15,000×g for 10 rain at 4°C.…”