N-ethylmaleimide uncouples the glucagon receptor from the regulatory component of adenylyl cyclase

125I-Glucagon binding to rat liver plasma membranes was composed of high- and low-affinity components. N-Ethylmaleimide (NEM) and several other alkylating agents induced a dose-dependent loss of high-affinity sites. This diminished the apparent affinity of glucagon receptors for hormone without decreasing the binding capacity of membranes. Solubilized hormone-receptor complexes were fractionated as high molecular weight (Kav = 0.16) and low molecular weight (Kav = 0.46) species by gel filtration chromatography; NEM or guanosine 5'-triphosphate (GTP) diminished the fraction of high molecular weight complexes, suggesting that NEM uncouples glucagon receptor-N-protein complexes. Exposure of intact hepatocytes to the impermeable alkylating reagent p-(chloromercuri)benzenesulfonic acid failed to diminish the affinity of glucagon receptors on subsequently isolated plasma membranes, indicating that the thiol that affects receptor affinity is on the cytoplasmic side of the membrane. Hormone binding to plasma membranes was altered by NEM even after receptors were uncoupled from N proteins by GTP. These data suggest that a sensitive thiol group that affects hormone binding resides in the glucagon receptor, which may be a transmembrane protein. Alkylated membranes were fused with wild-type or cyc- S49 lymphoma cells to determine how alkylation affects the various components of the glucagon-adenylyl cyclase system. Stimulation of adenylyl cyclase with fluoride, guanylyl 5'-imidodiphosphate, glucagon, or isoproterenol was observed after fusion of cyc- S49 cells [which lack the stimulatory, guanine nucleotide binding, regulatory protein of adenylyl cyclase (Ns)] with liver membranes alkylated with 1.5 mM NEM.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH Selectivity of N-Ethylmaleimide Reactions with Opiate Receptor Complexes in Rat Brain Membranes

N-Ethylmaleimide (NEM) decreases opiate agonist binding presumably by blocking crucial sulfhydryl (SH) groups at receptor binding sites. At physiological pH, NEM decreased GTP and manganese regulation but increased sodium effects on [3H]D-Ala2-Met5-enkephalinamide (D-Ala enk) binding to rat brain membranes. To determine the apparent pK values of putative SH groups in opiate receptors that react with NEM, rat brain membranes were incubated with 100-250 microM NEM in buffers ranging from pH 4.5 to 8.0. Results showed that lowering pH below 6.5 reduced the NEM effect on opiate receptor functions and that the apparent pK values of NEM-reacting SH groups in binding and regulatory sites ranged between 5.4 to 6.0. Most of the total SH groups in brain membranes continued to react with NEM at low pH, so that when nonspecific SH groups were blocked by incubating membranes at pH 4.5 with NEM, opiate receptors became sensitive to very low concentrations (1 microM) of NEM.     

Altered Transition Between Agonist-and Antagonist-Favoring States of μ-Opioid Receptor in Brain Membranes with Modified Microviscosity

Abstract: In unmodified synaptosomal brain membranes the presence of NaCl inhibited the binding to μ receptors of the tritiated opioid agonists etorphine, Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, and sufentanil by 53, 43, and 37%, respectively, and increased that of the antagonist [³H]naltrexone by 54%. On the other hand, in membranes whose microviscosity was increased by incorporation of cholesteryl hemi-succinate (CHS) the effects of sodium on opioid agonist and antagonist binding were abolished and strongly reduced, respectively. Furthermore, in the modified membranes the ability of sodium to protect the opioid receptor from inactivation by the sulfhydryl-reactive agent N -ethyl-maleimide (NEM) was diminished. In CHS-treated membranes whose elevated microviscosity was reduced by the incorporation of oleic acid, the effectiveness of sodium in modulating opioid binding and attenuating receptor inactivation by NEM was restored. The results implicate membrane microviscosity in the mechanism by which sodium modulates the conversion between agonist-and antagonist-favoring states of μ opioid receptor.     

Reconstitution of the rat liver vasopressin receptor coupled to guanine nucleotide-binding proteins

The V1 vasopressin receptor has been solubilized from rat liver membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate (CHAPS) and reconstituted into phospholipid vesicles. There is essentially complete solubilization of the receptor by 3% CHAPS at a protein concentration of 15 mg/ml. Reconstitution into soybean phospholipid vesicles is readily achieved either by gel filtration chromatography or by membrane dialysis. The binding of [3H]vasopressin to proteoliposomes is specific, saturable, reversible, and magnesium-dependent. In contrast, the detergent-soluble vasopressin receptor does not display specific binding. The apparent affinity of the reconstituted receptor for [3H]vasopressin is approximately 4-fold lower than that of the receptor in native membranes. In addition, the binding of [3H]vasopressin to reconstituted vesicles is not sensitive to 100 microM guanosine 5'-O-thiotriphosphate (GTP gamma S) as it is in native membranes. However, the apparent affinity of the reconstituted receptor for ligand approximates that of native membranes when membranes are prebound with vasopressin prior to solubilization and reconstitution into vesicles. Furthermore, vesicles reconstituted from membranes prebound with vasopressin show GTP gamma S sensitivity of [3H] vasopressin binding. This finding strongly suggests that vasopressin stabilizes a receptor-G-protein complex during solubilization. The rat liver vasopressin receptor is a glycoprotein, as shown by its specific binding to the lectin "wheat germ agglutinin." The vasopressin receptor can be reconstituted from the N-acetylglucosamine-eluted peak of a wheat germ agglutinin-Sepharose column, and [3H] vasopressin binding activity is purified 5-6-fold from membranes by this chromatographic procedure. The functionality of the partially purified receptor is indicated by its ability to bind ligand with high affinity and by its ability to functionally interact with a G-protein when vasopressin is bound prior to solubilization.     

Receptors for insulin and epidermal growth factor: interaction with organomercurial agarose

The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I-labeled insulin and EGF, can be absorbed to an organomercurial-agarose derivative (Affi-Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink-labeled membranes with N-ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial-agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl-containing receptor domains that may play a role in the control of receptor function.     

the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

An iodinated compound, [125I]-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol, has been recently reported [Sidhu, A., & Kebabian, J.W. (1985) Eur. J. Pharmacol. 113, 437-440] to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained their ability to functionally couple to the HeLa adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.     

Biosynthesis and posttranslational finishing of the estradiol receptor

The time course of subcellular receptor distribution in porcine endometrial epithelium was studied after intrauterine administration of estradiol alone or in combination with puromycin. In untreated cells, the major proportion of receptor is associated with cytoplasmic membranes. The solubilization of receptor from isolated nuclei is independent of their estradiol content. Smooth cytoplasmic membranes are the site of origin of receptor which is swiftly translocated into the nucleus in a 1:1 ratio with the hormone after exposure of the cells to estradiol. Simultaneously administered puromycin delays receptor synthesis and reveals that the nuclear passage of receptor is terminated by receptor degradation. The synthesis of receptor proceeds in rough endoplasmic membranes. A subsequent finishing and deposition in smooth membranes depends on intact protein synthesis.     

A chlorate-hypersensitive, high nitrate/chlorate uptake mutant of Arabidopsis thaliana

N-ethylmaleimide (NEM) Lit 10-100 μ M led to a strong inhibition of the auxin-induced elongation growth of colcoptile segments, while fusicoccin-enhanced growth was not affected. Growth inhibition occurred only if NEM and auxin were allowed to act simultaneously. Preincubation of plant segments with NEM in the absence of auxin caused no inhibition of a subsequent growth stimulation by auxin, whenever NEM was removed before the application of IAA. However, preincubation with NEM plus auxin led to a remaining growth inhibition, which could not be reversed by a second auxin incubation in the absence of NEM. Fusicoccin added to NEM- plus auxin-treated segments was able to restore growth. It is suggested that auxin causes the unmasking of essential SH-groups of a protein to which NEM links covalently. thus inhibiting the growth process. This assumption was further supported by labeling experiments wish [¹⁴C]-NEM using membranes of maize ( Zea mays L. cv. Inraplus) coleoptiles. Two membrane fractions (S₂= 480-1900 g; S₄= 4300-15000 g) revealed a significantly higher [¹⁴C]-NEM labeling in the presence of auxin (2,4-diehlorophe-noxyacctic acid compared to 2,6 dichlorophenoxyacetic acid). This effect disappeared when the membranes were previously washed with EGTA [ethyleneglycolbis-(β-aminoethylether)-N,N,Nr',N'-tetraacetic acid]. The auxin-induced sensitization of coleoptilc segments against thiol-reagents and the auxin-induced expression of SH-groups of proteins of isolated membranes from coleoptiles arc suggested to be events involved in the primary action of auxins.     

Cytochrome b556 complexes solubilized from Micrococcus lysodeikticus membranes by triton X-100]

The integral protein of cytochrome b556 after its solubilization with Triton X-100 from M. lysodeikticus membranes was studied. The cytochrome was found in complexes differing in charge and size during preparative gel electrophoresis and centrifugation in a sucrose concentration gradient. Cytochrome b556, being in complexes, retains its ability to be reduced by NADH dehydrogenase. The electron micrographs of the membranes after solubilization by Triton X-100 demonstrated the maintenance of the membrane structure. It is concluded that native protein complexes marked with cytochrome b556 are extracted from the membranes under their solubilization.     

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

Irreversible inactivation of beta-adrenergic receptors of C6 glioma cells. Synthesis and study of a thiol derivative of propranolol

The beta-adrenergic receptor of C6 glioma cells contains a disulfide bridge which can be reduced by dithiothreitol (DTT). On intact cells, N-ethylmaleimide (NEM) (5 mM) does not change the affinity of [3H] H2-alprenolol ([3H] DHA) but reduces the total number of beta-adrenergic cell receptors by 21 +/- 3 per cent ; (N = 3). After receptor reduction by DTT, NEM irreversibly blocks the accessibility of the beta-adrenergic receptors to [3H]DHA. On isolated membranes, incubation in the presence of either NEM (5 mM) or isoproterenol (5.10(-7) M) does not significantly modify the total number of beta-adrenergic receptors accessible to [3H]DHA. Incubation of membranes with both NEM and isoproterenol reduces the number of binding sites by 33 +/- 2 per cent ; (N = 3). A thiol derivative of propranolol was synthetized. Its affinity is 10 times lower than that of propranolol. This sulfur derivative reduces the total number of beta-adrenergic receptors by 22 +/- 3 per cent (N = 3) when incubated with the native receptor and by 55 +/- 4 per cent (N = 4) when incubated with the reduced receptor. DTT does not significantly reverse the blockade induced by propranolol-SH. A model is proposed for explaining these results.     

Solubilization of adenosine A1 binding sites from sheep cortex

[³H]N⁶-cyclohexyladenosine binds with high affinity to sheep brain membranes with a drug specificity indicating an association with A₁ adenosine receptors. The [³H]N⁶-cyclohexyladenosine binding site has been solubilized with sodium cholate being the only detergent able to maintain specific binding after solubilization. After solubilization, the kinetics and drug specificity of binding are virtually identical with those obtained in the intact membranes, indicating a conservation of the binding site after removal of the receptor from its lipid environment. Gel filtration experiments indicated an apparent molecular weight of 400,000 for the receptor-detergent complex and a Stokes radius of 6.2 nm.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Complex effects of sulfhydryl reagents on ligand interactions with nucleoside transporters: evidence for multiple populations of ENT1 transporters with differential sensitivities to N-ethylmaleimide

Functional studies have implicated cysteines in the interaction of ligands with the ENT1 nucleoside transporter. To better define these interactions, N-ethylmaleimide (NEM) and p-chloromercuribenzylsulfonate (pCMBS) were tested for their effects on ligand interactions with the [(3)H] nitrobenzylthioinosine (NBMPR) binding site of the ENT1 transporters of mouse Ehrlich ascites cells and human erythrocytes. NEM had biphasic, concentration-dependent effects on NBMPR binding to intact Ehrlich cells, plasma membranes, and detergent-solubilized membranes, with about 35% of the binding activity being relatively insensitive to NEM inhibition. NBMPR binding to human erythrocyte membranes also displayed heterogeneity in that about 33% of the NBMPR binding sites remained, albeit with lower affinity for NBMPR, even after treatment with NEM at concentrations in excess of 1 mM. However, unlike that seen for Ehrlich cells, no "reversal" in NBMPR binding to human erythrocyte membranes was observed at the higher concentrations of NEM. pCMBS inhibited 100% of the NBMPR binding to both Ehrlich cell and human erythrocyte membranes, but had no effect on the binding of NBMPR to intact cells. The effects of NEM on NBMPR binding could be prevented by coincubation of membranes with nonradiolabeled NBMPR, adenosine, or uridine. Treatment with NEM and pCMBS also decreased the affinity of other nucleoside transport inhibitors for the NBMPR binding site, but enhanced the affinities of nucleoside substrates. These data support the existence of at least two populations of ENT1 in both erythrocyte and Ehrlich cell membranes with differential sensitivities to NEM. The interaction of NEM with the mouse ENT1 protein may also involve additional sulphydryl groups not present in the human ENT1.     

Selective Protection of Benzomorphan Binding Sites Against Inactivation by N-Ethylmaleimide. Evidence for k-Opioid Receptors in Frog Brain

Selective binding of [3H]bremazocine and [3H]-ethylketocyclazocine to kappa-opioid receptor sites in frog (Rana esculenta) brain membranes is irreversibly inactivated by the sulfhydryl group alkylating agent N-ethylmaleimide (NEM). Pretreatment of the membranes with kappa-selective compounds [ethylketocyclazocine (EKC), dynorphin (1-13), or U-50,488H] but not with [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAGO; mu specific ligand) or [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DADLE; delta specific ligand) strongly protects the binding of the radioligands against NEM inactivation. These results provide more evidence for the existence of kappa-opioid receptors in frog brain. The relatively high concentrations of NEM that are needed to decrease the specific binding of [3H]bremazocine together with the observation of an almost complete protection of its binding sites by NaCl suggest that bremazocine may act as an opioid antagonist in frog brain.     

Evidence for two GTPases activated by thrombin in membranes of human platelets.

Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Functional Reconstitution of Purified Gi and Go with μ-Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N-Ethylmaleimide

Functional coupling between mu-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective mu-opioid agonists stimulated low-Km GTPase in striatal membranes, in a Na(+)-dependent manner. The same mu-opioid agonist [( D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAGO)] caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 microM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the mu-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The mu-agonist stimulation of low-Km GTPase activity in NEM-treated membranes was recovered by reconstitution with purified Gi or Go. The mu-agonist stimulation of low-Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the mu-opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.