Large scale preparation and characterization of membrane-bound and detergent-solubilized muscarinic acetylcholine receptor from pig atria

The muscarinic acetylcholine receptor (mAcChR) has been prepared from pig atrial membranes by new large scale procedures which result in 30-40 fold enrichment of the receptor in the membrane-bound state and a further three fold enrichment during solubilization. The membrane-bound receptor was prepared by differential and sucrose density gradient centrifugation in 25 mM imidazole, 1 mM EDTA, pH 7.4. A double extraction procedure using a mixed digitonin/cholate detergent was used to solubilize the receptor at a 60-70% yield. The membrane and solubilized preparations had specific activities of 3.5-5 and 8-12 pmol [3H]L-quinuclidinyl benzilate (QNB) binding sites per mg of protein, respectively. The presence of imidazole, which behaved as a weak muscarinic ligand, stabilized the receptor during solubilization and storage. Both the membrane-bound and detergent-solubilized mAcChR bound antagonists at a single class of sites and agonists at two subclasses of QNB sites. The proportion of high affinity agonist sites in the solubilized receptor was about 1/3 that in the membrane receptor. [3H]Propylbenzilylcholine mustard covalently labeled a single prominent atropine-sensitive component with an apparent molecular weight of 70-74,000 on SDS-polyacrylamide gels for both the membrane and solubilized receptor.     

Reconstitution of the purified porcine atrial muscarinic acetylcholine receptor with purified porcine atrial inhibitory guanine nucleotide binding protein

Purified porcine atrial muscarinic receptor (mAcChR) was reconstituted with purified porcine atrial inhibitory guanine nucleotide binding protein (Gi) in a lipid mixture consisting of phosphatidylcholine, phosphatidylserine, and cholesterol (1:1:0.1 w/w). 5'-Guanylyl imidodiphosphate (0.1 mM) had no effect on the binding of the muscarinic antagonist L-quinuclidinyl benzilate but converted high-affinity carbachol binding sites (Kd equal to 1 microM) in the reconstituted preparation to the low-affinity state (Kd equal to about 100 microM). Steady-state kinetic measurements of GTPase activity showed that the turnover number was increased from 0.19 min-1 in the presence of the muscarinic antagonist L-hyoscyamine to 2.11 min-1 for the agonist carbachol. The affinity of Gi for GDP was reduced by about 50-fold upon interaction with the carbachol-mAcChR complex, and the observed rate constant for GDP dissociation was increased by 38-fold from 0.12 to 4.5 min-1. Thus, the increase in steady-state GTPase activity observed for muscarinic agonists is largely, if not exclusively, due to the increase in GDP dissociation from Gi--probably the rate-limiting step in the steady-state mechanism. Carbachol-stimulated GTPase was sensitive to ADP-ribosylation of the reconstituted Gi by pertussis toxin, but the high-affinity agonist binding was uncoupled only when the reconstituted preparation was treated with pertussis toxin in the presence of GTP and the agonist acetylcholine. These results suggest that association with the mAcChR protects Gi from ADP-ribosylation by pertussis toxin.     

The heterogeneity and nucleotide modulation of cholinergic muscarinic receptors are restored by poly (ethylene) glycol-6000 precipitation of solubilized atrial receptors

The membrane bound muscarinic receptor exhibits heterogeneity of binding sites for agonists that are modulated by guanine nucleotides. These properties are generally lost upon receptor solubilization by several detergents, but may be recovered after rapid detergent removal by poly (ethylene) glycol-6000 precipitation of the digitonin plus cholate solubilized atrial muscarinic receptor. The results are discussed in terms of muscarinic receptor interactions in the intact membrane.     

Phosphorylation of the porcine atrial muscarinic acetylcholine receptor by cyclic AMP dependent protein kinase

cAMP-dependent protein kinase, protein kinase C, cGMP-dependent protein kinase, smooth muscle myosin light-chain kinase, and phosphorylase kinase were examined with respect to their ability to phosphorylate porcine atrial muscarinic receptors (mAcChRs). Experiments were performed both in detergent solution and in a reconstituted system containing the mAcChR alone or in the presence of the purified porcine atrial inhibitor guanine nucleotide binding protein (Gi). Only cAMP-dependent protein kinase was capable of phosphorylating the receptor under any of the experimental conditions examined. Phosphorylation of the mAcChR in the detergent-solubilized state resulted in a loss of ligand binding sites that was reversible upon treatment with calcineurin in the presence of calcium and calmodulin. Upon reconstitution, the apparent stoichiometry of phosphorylation was increased by about 15-fold. Carbachol-stimulated covalent incorporation of phosphate was found only in the reconstituted system in the presence of Gi, suggesting that the large agonist-stimulated increase in phosphorylation observed in vivo [Kwatra, M. M., & Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432] may in part result from a unique receptor conformation that occurs upon association with this protein. Ligand binding studies indicated that phosphorylation of the mAcChR in the detergent-solubilized or reconstituted state did not affect its interaction with carbachol or L-quinuclidinyl benzilate in vitro. Carbachol-induced stimulation of the GTPase activity of Gi in the reconstituted system was also unaffected by phosphorylation.     

Muscarinic receptor-detergent complexes with different biochemical properties: selective solubilization, lectin affinity chromatography and ligand binding studies

Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.     

Fluidity of detergent micelles plays an important role in muscarinic receptor solubilization

In order to find a suitable reagent for extracting the muscarinic receptor from rat brain membranes 14 different detergents were tested. Only the plant glycoside digitonin efficiently solubilized the receptor protein in its native form. At the same time microviscosity of detergent micelles was determined by measuring the fluorescence polarization of a hydrophobic fluorescent probe diphenylhexatriene incorporated into the micelles. In the case of digitonin the polarization value was close to the corresponding value obtained for rat brain membrane fragments, while for the other detergents studied it remained considerably lower. The results obtained indicate that the fluidity of detergent micelles may play an important role in retaining the active conformation of the solubilized muscarinic receptor.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.     

Characterization of Neurotensin Binding Sites in Intact and Solubilized Bovine Brain Membranes

Analysis of the equilibrium binding of [3H]-neurotensin(1-13) at 25 degrees C to its receptor sites in bovine cortex membranes indicated a single population of sites with an apparent equilibrium dissociation constant (KD) of 3.3 nM and a density (Bmax) of 350 fmol/mg protein (Hill coefficient nH = 0.97). Kinetic dissociation studies revealed the presence of a second class of sites comprising less than 10% of the total. KD values of 0.3 and 2.0 nM were obtained for the higher and lower affinity classes of sites, respectively, from association-dissociation kinetic studies. The binding of [3H]neurotensin was decreased by cations (monovalent and divalent) and by a nonhydrolysable guanine nucleotide analogue. Competition studies gave a potency ranking of [Gln4]neurotensin greater than neurotensin(8-13) greater than neurotensin(1-13). Smaller neurotensin analogues and neurotensin-like peptides were unable to compete with [3H]neurotensin. Stable binding activity for [3H]neurotensin in detergent solution (Kd = 5.5 nM, Bmax = 250 fmol/mg protein, nH = 1.0) was obtained in 2% digitonin/1 mM Mg2+ extracts of membranes which had been preincubated (25 degrees C, 1 h) with 1 mM Mg2+ prior to solubilization. Association-dissociation kinetic studies then revealed the presence of two classes of sites (KD1 = 0.5 nM, KD2 = 3.6 nM) in a similar proportion to that found in the membranes. The solubilized [3H]-neurotensin activity retained its sensitivity to cations and guanine nucleotide.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Solubilisation and Molecular Characterisation of Muscarinic Acetylcholine Receptors

Abstract

Stable, soluble preparations of rat brain muscarinic receptors can be prepared by extracting membranes with digitonin, or with combinations of sodium cholate and sodium chloride. The stability of the cholate/NaCl extract is enhanced by the addition of egg phosphatidylcholine, which, at the same time, suppresses the considerable dispersity apparent in the hydrodynamic behaviour of the solubilised receptor. The Stokes radius of the brain muscarinic receptor in cholate/NaCl/lecithin extracts is 6.7 nm, with very similar values in other detergents, including digitonin and sodium dodecyl sulphate. Its sedimentation coefficient is 3.78s, and its molecular weight approximately 110,000 after correction for detergent binding. The isoelectric point of the digitonin - solubilised receptor is approximately 4.5.     

Pharmacological and biochemical characterization of complexes of muscarinic acetylcholine receptor and guanine nucleotide-binding protein

Complexes of agonist-bound muscarinic acetylcholine receptor (mAChR) and guanine nucleotide-binding protein (G protein) were solubilized and isolated from rat heart. Heart membranes were incubated with mAChR agonists or antagonists, solubilized using digitonin and cholate, and subjected to chromatography over wheat germ agglutinin-Affi-Gel. Eluted fractions were precipitated using a cardiac-selective anti-mAChR antibody (Luetje, C. W., Brumwell, C., Norman, M. G., Peterson, G. L., Schimerlik, M. I., and Nathanson, N. M. (1987) Biochemistry 26, 6892-6898). Using samples obtained from membranes initially incubated with carbachol (10 nM, 100 nM, or 1 mM), G alpha immunoreactivity was detected on Western blots probed using antibodies with specificity for G alpha subunits. The G alpha immunoreactivity was not detected when atropine alone (10 nM or 1 microM) or when excess atropine (1 microM) plus carbachol (100 nM) was used during the membrane preincubation. G beta immunoreactivity, when detectable on Western blots, was present in substoichiometric amounts relative to that of G alpha. The G alpha immunoreactivity was not present if GTP was included during incubation of membranes with agonist and following membrane solubilization. Further results indicate that although agonist binding to receptors is rapidly reversed by GTP or GDP (t1/2 less than 10 min), the mAChR-G protein complex is reversed more slowly or not at all. It was also shown that at high agonist concentrations, the cardiac mAChR interacts with both Go and Gi-like proteins. Together, these results demonstrate the utility of an immunoaffinity approach to the purification and biochemical study of receptor-G protein interactions.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Solubilization of the D-1 dopamine receptor from rat striatum

The D-1 dopamine receptor was extracted from rat striatal membranes with 0.7% sodium cholate and 1 M NaCl. Pretreatment of the membranes with a D-1 specific agonist, inclusion of crude phospholipids in the solubilization buffer, and subsequent removal of the detergent led to a maximal extraction of 48% of the receptor binding sites. The D-1 antagonist, [125I]SCH 23982, bound to single class of sites with a Kd of 1.8 nM and a Bmax of 1.65 pmol/mg protein. The solubilized receptors retained the ability to discriminate between active and inactive enantiomers of agonists and antagonists selective for the D-1 receptor.     

Ultrastructure of reconstituted membranes containing the muscarinic receptor

In this paper the demonstration is made that membrane vesicles (liposomes) containing the muscarinic receptor can be formed by polyethylene glycol (PEG) precipitation of detergent extracts of bovine atrial membranes. The incorporation of the muscarinic receptor in these vesicles may be related to the restoration of the heterogeneity and nucleotide modulation of muscarinic agonist binding by PEG precipitation of atrial detergent extracts, previously reported. Vesicles are also formed when detergent solubilized asolectin lipids, alone or in combination with membrane detergent extracts, are precipitated by PEG. The structure of the vesicles seems depend on their lipid and protein composition and the procedure employed for the removal of the dispersing medium. These results indicate that PEG precipitation could be used for the reconstitution of the muscarinic receptor into the liposomes of exogenous lipids.Special Issue dedicated to Prof. Eduardo De Robertis.     

Glycoprotein Properties of the Solubilized Atrial Muscarinic Acetylcholine Receptor

Abstract: The muscarinic acetylcholine receptor from porcine atria exhibits sialoglycoprotein characteristics based on its sensitivity to neuraminidase digestion and its ability to interact specifically with lectin affinity resins when solubilized with a digitonin/cholate mixed detergent system. Differential lectin binding properties of the neuraminidase-treated and untreated receptor suggest that high-affinity binding to immobilized wheat germ agglutinin is accomplished through the presence of both terminal sialic acid and internal N -acetylglucosamine or its β(1→4)-linked oligomers.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.