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1.
FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations. 相似文献
2.
The present study was performed on retinas of chick embryos receiving at day 8 of incubation an intracerebral injection of 0.02 microgram of corticosterone. We had previously shown with the use of [3H]quinuclidinylbenzilate [( 3H]QNB) that such treatment induced the appearance of two muscarinic binding sites in the treated retinas, whereas only one was detectable in the controls. In the present study we investigated muscarinic cholinergic receptor subclasses with agonist and antagonist binding. Agonist binding was studied by varying the concentrations of carbachol and acetylcholine (10(-9) M-10(-5) M) in the presence of a constant concentration (0.2 nM) of [3H]QNB. Two subpopulations of receptors were revealed, a high- and a low-affinity receptor, in both treated and control retinas. However, in the hormone-treated retinas, the two subpopulations significantly differed from the controls in their affinity and in their relative percentage among the total receptor population. Moreover, using pirenzepine, an antagonist known to have the capacity to distinguish between muscarinic cholinergic subclasses, two receptor subpopulations were found to be present in the hormone-treated retinas but a single one in the controls. It is suggested that hormone treatment can either induce the appearance of a new subclass of muscarinic cholinergic receptors or favor the maturation of a population of retinal cells having these receptors. Pirenzepine binding in retinas from intact embryos of 7, 9, and 11 days of incubation revealed one receptor subpopulation. Thus, these findings are more consistent with the hypothesis that corticosterone effects the target cells, either inducing changes in muscarinic receptor and/or modifying the receptor environment. 相似文献
3.
Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor 总被引:1,自引:0,他引:1
Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone. 相似文献
4.
Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Gαo, Gαi1-3, and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, α-subunits, and caveolins in various combinations. Homooligomers of αi2 were detected on Western blots, and heterooligomers of αi2 and αo were identified by coimmunoprecipitation; thus, a complex may contain at least two α-subunits. Other combinations of α-subunit were not detected. The ratio of total α-subunit to receptor was near 1, as measured by [35S]GTPγS and the antagonist [3H]quinuclidinylbenzilate, and the binding of [35S]GTPγS was manifestly biphasic. The ratio of αo to αi1,2 also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of α-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of α-subunit and the receptor. 相似文献
5.
The glycoprotein nature of solubilized muscarinic acetylcholine receptors from bovine cerebral cortex 总被引:4,自引:0,他引:4
O Shirakawa T Kuno C Tanaka 《Biochemical and biophysical research communications》1983,115(3):814-819
Muscarinic acetylcholine receptors were solubilized from bovine cerebral cortex with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The so-obtained receptors could be precipitated by Wheat Germ (73%), Concanavalin A (55%), Lens Culinaris (36%) and Ricinus Communis (26%), but not by Peanut, Dolichus Biflorus and Ulex Europaeus. On Wheat Germ- and Concanavalin A-affinity chromatography, the solubilized muscarinic receptors were retained on both columns and subsequently eluted with N-acetylglucosamine and alpha-methyl-D-mannoside, respectively. A high concentration (100 micrograms/ml) of Wheat Germ or Concanavalin A did not interfere with Z-[3H]quinuclidinyl benzilate binding, thereby suggesting that the lectin binding sites are not directly involved in the receptor binding function. These solubilized muscarinic receptors are postulated to contain carbohydrate residues, N-acetyl-glucosamine, mannose and galactose, as glycoprotein. 相似文献
6.
Oligomeric potential of the M2 muscarinic cholinergic receptor 总被引:1,自引:0,他引:1
G protein-coupled receptors are known to exist as oligomers. Although such aggregates often are referred to as dimers, there is little direct evidence regarding their oligomeric size. In the present investigation, c-Myc-, FLAG-, and influenza hemagglutinin (HA)-tagged forms of the M2 muscarinic receptor have been coexpressed in Sf9 cells to probe for aggregates larger than a dimer. Immunochromatography, immunoprecipitation, and immunoblotting were carried out with various combinations of antibodies directed against the different epitopes to demonstrate that all three tagged forms of the receptor can be immunopurified within a single complex. Extracts of the M2 muscarinic receptor from Sf9 cells therefore contain aggregates that are at least trimeric, and the levels detected point to the existence of larger complexes. The data also suggest that the oligomers coexist with a sizeable population of monomers. 相似文献
7.
At least four allosteric sites have been found to mediate the dose-dependent effects of gallamine on the binding of [(3)H]quinuclidinylbenzilate (QNB) and N-[(3)H]methylscopolamine (NMS) to M(2) muscarinic receptors in membranes and solubilized preparations from porcine atria, CHO cells, and Sf9 cells. The rate of dissociation of [(3)H]QNB was affected in a bell-shaped manner with at least one Hill coefficient (n(H)) greater than 1, indicating that at least three allosteric sites are involved. The level of binding of [(3)H]QNB was decreased in a biphasic manner, revealing at least two allosteric sites; binding of [(3)H]NMS was affected in a triphasic, serpentine manner, revealing at least three sites, and values of n(H) >1 pointed to at least four sites. Several lines of evidence indicate that all effects of gallamine were allosteric in nature and could be observed at equilibrium. The rates of equilibration and dissociation suggest that the receptor was predominately oligomeric, and the heterogeneity revealed by gallamine can be attributed to differences in its affinity for the constituent protomers of a tetramer. Those differences appear to arise from inter- and intramolecular cooperativity between gallamine and the radioligand. 相似文献
8.
Using peptide affinity purification, we identified an interaction between somatostatin receptors SSTR4 and SSTR1 and PDZ domains 1 and 2 of the postsynaptic proteins postsynaptic density protein of 95kDa (PSD-95) and PSD-93. The existence of the SSTR4/PSD-95 complex was verified by coimmunoprecipitation from transfected cells and solubilized brain membranes. In neurons, dendritically localized SSTR4 partially colocalizes with postsynaptic PSD-95. As functional parameters of the receptor, such as coupling to potassium channels, are not affected by the interaction with PSD-95, the association may serve to localize the receptor to postsynaptic sites. 相似文献
9.
Regulation of the solubilized bovine cerebral cortex muscarinic receptor by GTP and Na+ 总被引:2,自引:0,他引:2
T Kuno O Shirakawa C Tanaka 《Biochemical and biophysical research communications》1983,112(3):948-953
The muscarinic acetylcholine receptor was solubilized, in a sensitive form for GTP and Na+, from bovine cerebral cortex using a zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The solubilized muscarinic receptor displayed characteristics as follows: (1) high affinity to nanomolar concentration of Z-[3H]quinuclidinyl benzilate; (2) muscarinic agonists and antagonists had similar inhibitory potencies as on the membrane-bound receptor; (3) without Na+, GTP did not significantly alter the binding affinity of muscarinic agonists and antagonists; (4) GTP in the presence of Na+, selectively decreased the affinity of muscarinic agonists, carbamylcholine and oxotremoline, but not the antagonist binding affinity; (5) Na+ in the absence or presence of GTP, reduced both muscarinic agonist and antagonist affinities. 相似文献
10.
Several studies suggest, but do not prove directly, that muscarinic receptors may be able to form dimeric or oligomeric arrays. To address this issue in a more direct fashion, we designed a series of biochemical experiments using a modified version of the rat m3 muscarinic receptor (referred to as m3') as a model system. When membrane lysates prepared from m3' receptor-expressing COS-7 cells were subjected to Western blot analysis under non-reducing conditions, several immunoreactive species were observed corresponding in size to putative receptor monomers, dimers, and oligomers. However, under reducing conditions, the monomeric receptor species represented the only detectable immunoreactive protein, consistent with the presence of disulfide-linked m3 receptor complexes. Similar results were obtained when native m3 muscarinic receptors present in rat brain membranes were analyzed. Control experiments carried out in the presence of high concentrations of the SH group alkylating agent, N-ethylmaleimide, suggested that disulfide bond formation did not occur artifactually during the preparation of cell lysates. The formation of m3' receptor dimers/multimers was confirmed in coimmunoprecipitation studies using differentially epitope-tagged m3' receptor constructs. In addition, these studies showed that m3' receptors were also able to form non-covalently associated receptor dimers and that m3' receptor dimer formation was receptor subtype-specific. Immunological studies also demonstrated that m3' receptor dimers/multimers were abundantly expressed on the cell surface. Site-directed mutagenesis studies indicated that two conserved extracellular Cys residues (Cys-140 and Cys-220) play key roles in the formation of disulfide-linked m3' receptor dimers. These results provide the first direct evidence for the existence of muscarinic receptor dimers and highlight the specificity and molecular diversity of G protein-coupled receptor dimerization/oligomerization. 相似文献
11.
Moshe Gavish Mordechai Sokolovsky 《Biochemical and biophysical research communications》1982,109(3):819-824
The muscarinic acetylcholine receptor was solubilized from rat brain cortex by zwitterionic detergent 3-[(3-chloramidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). About 15% of the binding activity was solubilized and 40% of the activity was destroyed by the detergent. Binding of the muscarinic antagonist [3H]-N-methyl-4-piperidyl benzilate (4NMPB) was saturable. Scatchard analysis revealed a single population of binding sites with KD value of 0.7 nM and a Bmax value of 340 fmoles/mg protein. The homogenate and the CHAPS treated pellet and soluble receptors showed similar affinity for the agonists oxotremorine and carbamylcholine and for the antagonists QNB and atropine. The dissociation of 4NMPB from the soluble receptors appears slightly slower than from the membrane bound receptors. 相似文献
12.
Dopamine D2 receptors , detected by [3H]spiperone Dopamine D2 receptors , detected by [3H]spiperone binding, were solubilized from bovine caudate nucleus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmaco-logical properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D2 dopamine receptor is therefore a glycoprotein. binding, were solubilized from bovine caudate nucJeus by cholate/sodium chloride and were found to bind to wheat germ agglutinin immobilized on agarose. Specific elution could be achieved with N-acetylglucosamine whereas other sugars tested were inactive in this regard . The eluted preparation was enriched in solubilized receptors about sevenfold. The pharmacological properties of the preparation were essentially unchanged by the lectin affinity purification procedure. The D2 dopamine receptor is therefore a glycoprotein. 相似文献
13.
The effectiveness of several detergents and salts in solubilizing the muscarinic acetylcholine receptor (identified by its atropine-sensitive [3H]3-quinuclidinyl benzilate (QNB) binding) from bovine striatal membranes is reported. The highest density of receptor is obtained by extraction with 1% digitonin-0.1 mM EDTA. Although the total solubilized muscarinic receptors (sites/ml) are increased and the nonspecific binding is decreased when 1 M NaCl is included in this extraction medium, the receptor density (sites/mg protein) is lower. The solubilized receptors have the same specific QNB binding affinity, and sensitivity to a variety of drugs, as the membrane-bound muscarinic receptors. 相似文献
14.
Muscarinic receptors purified from porcine atria and devoid of G protein underwent a 9-27-fold decrease in their apparent affinity for the antagonists quinuclidinyl benzilate, N-methylscopolamine, and scopolamine when treated with the thiol-selective reagent N-ethylmaleimide. Their apparent affinity for the agonists carbachol and oxotremorine-M was unchanged. Conversely, the rate of alkylation by N-ethylmaleimide, as monitored by the binding of [(3)H]quinuclidinyl benzilate, was decreased by antagonists while agonists were without effect. The receptor also underwent a time-dependent inactivation that was hastened by N-ethylmaleimide but slowed by quinuclidinyl benzilate and N-methylscopolamine. The destabilizing effect of N-ethylmaleimide was counteracted fully or nearly so at saturating concentrations of each antagonist and the agonist carbachol. Similar effects occurred with human M(2) receptors differentially tagged with the c-Myc and FLAG epitopes, coexpressed in Sf9 cells, and extracted in digitonin/cholate. The degree of coimmunoprecipitation was unchanged by N-ethylmaleimide, which therefore was without discernible effect on oligomeric size. The data are quantitatively consistent with a model in which the purified receptor from porcine atria interconverts spontaneously between two states (i.e. R R*). Antagonists favor the R state; agonists and N-ethylmaleimide favor the comparatively unstable R* state, which predominates after purification. Occupancy by a ligand stabilizes both states, and antagonists impede alkylation by favoring R over R*. Similarities with constitutively active receptors suggest that R and R* are akin to the inactive and active states, respectively. Purified M(2) receptors therefore appear to exist predominantly in their active state. 相似文献
15.
Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions. 相似文献
16.
gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues. 相似文献
17.
A H Lin P L Ruppel R R Gorman 《Biochemical and biophysical research communications》1985,128(2):878-883
Specific leukotriene B4 (LTB4) receptors in human neutrophils were solubilized by treatment of "receptor fraction" membranes with the zwitterionic detergent (3-[(3-cholamidopropyl)-dimethylammonio]1-propane sulfonate (CHAPS). The soluble receptors were assayed by polyethylene glycol (PEG) precipitation coupled with Millipore filtration. The solubilized receptors retained all of the characteristics of the receptor sites in intact neutrophils. The binding of LTB4 was rapid, reversible and stereospecific. Mathematical modeling analysis revealed biphasic binding of [3H] LTB4 indicating two classes of binding sites. The high affinity binding site had a dissociation constant of 1.93 nM and Bmax of 281 fmoles/mg protein; the low affinity binding site had a dissociation constant of 78.92 nM and Bmax of 2522 fmoles/mg protein. Competitive binding experiments with structural analogs of LTB4 demonstrate that the interaction between LTB4 and its binding site is stereospecific and correlates with the relative biological activity of the analogs. These data suggest that it may be possible to purify the LTB4 receptor from human neutrophil membranes. 相似文献
18.
Takeshi Nakano Michele Muscillo† Kousaku Ohno rew J. Hoffman Kunihiko Suzuki† 《Journal of neurochemistry》1988,51(3):984-987
The M1-selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N-[3H]methylscopolamine [( 3H]NMS) and [3H]quinuclidinylbenzilate from intact human SK-N-SH neuroblastoma cells with a low affinity (Ki = 869-1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF-DX 116 [11-2[[2-[(diethylamino)methyl]-1-piperidinyl]- acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) bound to the mAChRs with a very low affinity (Ki = 6.0 microM), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki = 7.2 nM). 4-DAMP was also 1,000-fold more effective than AF-DX 116 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilycholine mustard) suggest that the size of the SK-N-SH mAChR (Mr = 81,000-98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr = 66,000), an M1-enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover. 相似文献
19.
Evidence for the presence of muscarinic acetylcholine receptors in bovine brain coated vesicles 总被引:6,自引:0,他引:6
Evidence obtained from quinuclidinylbenzilate binding determinations suggested that muscarinic acetylcholine receptor molecules are present in purified bovine brain coated vesicles. Immunoprecipitates formed from coated vesicles with polyclonal antibodies to clathrin bound on the surface of fixed Staphylococcus aureus cells also showed quinuclidinylbenzilate binding activity. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Muscarinic receptor sites for quinuclidinylbenzilate binding to coated vesicles displayed a Kd of 25 pM and a Bmax of about 191 fmol/mg of protein. Binding competition experiments using atropine, N-methylscopolamine, oxotremorine, and carbamylcholine confirmed the typical muscarinic nature of the binding site. Ranking order of potency for the receptors was: atropine greater than N-methylscopolamine greater than oxotremorine greater than carbachol Analysis of data using a two-site model revealed 13% high-affinity sites for oxotremorine, 66% high-affinity sites for carbachol, and 62% for the antagonist N-methylscopolamine. Heterogeneity of binding affinities for muscarinic drugs detected in the coated vesicles may be related to the presence of coated vesicle subpopulations in brain tissue, (Kohtz, D. S., Kohtz, J. D., Schook, W. J., and Puszkin, S. (1985) J. Cell Biol. 101, 48a; Pfeffer, S. R., and Kelly, R. B. (1985) Cell 40, 949-957). 相似文献
20.
M Maletti M Andersson J C Marie G Rosselin V Mutt 《The Journal of biological chemistry》1992,267(22):15620-15625
Somatostatin-28 (SRIF-28) preferring receptors were solubilized from hamster beta cell insulinoma using the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. The binding of the iodinated [Leu8-D-TRP22-Tyr25]SRIF-28 analog (referred to as 125I[LWY] SRIF-28) to the solubilized fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the solubilized extract contained two classes of SRIF-28-binding sites: a high affinity site (Kd = 0.3 nM and Bmax = 1 pmol/mg protein) and a low affinity site (Kd = 13 nM and Bmax = 4.7 pmol/mg protein). The binding of 125I[LWY]SRIF-28 to solubilized SRIF-28 receptors was sensitive to the GTP analog guanosine-5'-O-thiotriphosphate, suggesting that receptors are functionally linked to a G-protein. By anion-exchange chromatography of the solubilized extract followed by chromatography on wheat germ agglutinin, a 46-fold purification of SRIF-28 receptors was obtained. At this stage of purification, only high affinity sites were found (Kd = 1 nM) and the GTP effect was not maintained. A specific protein of 37 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling. We suggest that this protein is the putative SRIF-28 receptor or a subunit thereof. 相似文献