Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.     

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.     

In vitro characterization of radioiodinated (+)-2-[4-(4-iodophenyl) piperidino]cyclohexanol [(+)-pIV] as a sigma-1 receptor ligand

We investigated the binding characteristics of a (+)-enantiomer of radioiodinated 2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[125I]pIV], radioiodinated at the para-position of the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (+)-pIV (Ki=1.30 nM) had more than 10 times higher affinity to the sigma-1 (sigma-1) receptor than (+)-pentazocine (Ki=19.9 nM) or haloperidol (Ki=13.5 nM) known as sigma ligands. Also, the binding affinity of (+)-pIV for the sigma-1 receptor (Ki=1.30 nM), was about 16 times higher than the sigma-2 (sigma-2) receptor (Ki=20.4 nM). (+)-pIV (Ki=1260 nM) had a much lower affinity for VAChT than (-)-vesamicol (Ki=13.0 nM) or (-)-pIV (Ki=412 nM). (+)-[125I]pIV had low affinity for the dopamine, serotonin, adrenaline, and acetylcholine receptors. Furthermore, in a saturation binding study, (+)-[125I]pIV exhibited a K) of 6.96 nM with a Bmax of 799 fmol/mg of protein. These results showed that (+)-pIV binds to the sigma-1 receptor with greater affinity than sigma receptor ligands such as (+)-pentazocine or haloperidol, and that radioiodinated (+)-pIV is suitable as radiotracer for sigma-1 receptor studies in vitro.     

The Purified Gaba/Benzodiazepine/Barbiturate Receptor Complex: Four Types of Ligand-Binding Sites,and the Interactions between them,are Preserved in a Single Isolated Protein Complex

Abstract

A GABA / benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABA_A agonist [³H]muscimol and for the chloride ion channel blocking agent [³⁵S]t-butylbicyclophosphoro-thionate (TBPS), whereas the binding properties for the benzodiazepine [³H] flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53000 and Mr 57000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [³H] flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [³H] flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires > 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments < M_r 10000.     

High-affinity folate receptor in human ovary, serous ovarian adenocarcinoma, and ascites: radioligand binding mechanism, molecular size, ionic properties, hydrophobic domain, and immunoreactivity.

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.     

The gamma-aminobutyric-acid/benzodiazepine-receptor protein from rat brain. Large-scale purification and preparation of antibodies

The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

Molecular Properties of 5-Hydroxytryptamine3 Receptor-Type Binding Sites Purified from NG1O8-15 Cells

5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.     

Interaction of Strychnine-Insensitive Glycine Binding with MK-801 Binding in Brain Synaptic Membranes

Strychnine-insensitive [3H]glycine binding was detected in brain synaptic membranes treated with Triton X-100 using a filtration assay method. The binding was a time-dependent, inversely temperature-dependent, and reversible process with a relatively high affinity for the neuroactive amino acid. Scatchard analysis revealed that Triton treatment doubled both the affinity and density of the binding sites, which consisted of a single component. The binding was not only displaced by structurally-related amino acid such as D-serine and D-alanine, but also inhibited by some peptides containing glycine, including glycine methylester and N-methylglycine. These ligands invariably potentiated the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine ([3H]MK-801), a noncompetitive antagonist for the N-methyl-D-aspartate-sensitive subclass of the central excitatory amino acid receptors, in a concentration-dependent manner. Among various endogenous tryptophan metabolites, kynurenic acid significantly inhibited the strychnine-insensitive [3H]glycine binding. The Triton treatment did not affect the pharmacological profile of [3H]MK-801 binding sites. These results suggest that brain synaptic membranes treated with Triton X-100 are useful in evaluating the strychnine-insensitive and kynurenate-sensitive binding sites of glycine, which are functionally linked to N-methyl-D-aspartate- sensitive receptor channels.     

Molecular weight of the acetylcholine receptors of electric organs and the effect of Triton X-100.

Studies are reported on the influence of Triton X-100 on the molecular weight and functional properties of the acetylcholine receptor. Results are presented principally for receptors purified from Torpedo californica and Torpedo marmorata with a limited number of observations on the receptor from Electrophorus electricus. In equilibrium dialysis measurements Trito, X-100 greatly reduced acetylcholine binding to the high affinity sites of the receptor from T. californica, but had only a small effect on the sites of lower affinity. Sedimentation equilibrium experiments on receptor in the absence of added Triton X-100 revealed average apparent molecular weight values of 510,000 for receptor from T. californica and 665,000 for T. marmorata. Under those conditions 0.113 mg of residual Triton X-100 were found per mg of protein as determined by using 3H-labeled Triton X-100. The sedimentation data indicated the presence of more than one molecular species, involving a unit with an apparent molecular weight of 330,000 and higher aggregates. Upon addition of Triton X-100, the higher aggregates were reduced, and above 0.1 percent Triton X-100 the 330,000 unit was the principal component present for receptor from all three species examined. Various structural models are considered in the light of this value, the polypeptide size from Na dodecyl sulfate-gel electrophoresis, and the protomer size determined by the molecular weight of an acetylcholine binding site.     

Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose

Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.     

Disclosure by triton X-100 of NMDA-sensitive [3H] glutamate binding sites in brain synaptic membranes

Pretreatment of brain synaptic membrane homogenates with Triton X-100 resulted in a drastic disclosure of [3H] glutamate (Glu) binding activity which was sensitive to one of the central Glu receptor agonists, N-methyl-D-aspartic acid (NMDA). The NMDA-sensitive binding was inversely dependent on the incubation temperature, and was a reversible and saturable process. Scatchard analysis revealed that Triton X-100 treatment yielded in a significant enhancement of the affinity with a concomitant increment of the density of binding sites. Electrophysiologically identified agonists and antagonists for the NMDA receptors all significantly inhibited the binding to Triton-treated membranes. These results suggest that Triton-treatment may disclose NMDA-sensitive [3H] Glu binding sites in brain synaptic membranes.     

Molecular mechanisms of olfactory reception. IV. Some biochemical characteristics of the camphor receptor from rat olfactory epithelium.

Some parameters of the receptor element from the rat olfactory epithelium are evaluated; it is characterized by high affinity for camphor (KD = 1.5. x 10(-9) M). Triton X-100 has no marked effect on the binding of [3H]camphor. Neither RNAase nor phospholipase C affected [3H]camphor-binding activity. Pronase and trypsin abolished [3H]camphor binding activity by 65 and 40%, respectively. Sulfhydryl reagents decrease the binding of [3H]camphor by a factor of 5--8. The isoelectric point of the receptor solubilized with Triton X-100 is 4.8, as determined by isoelectric focusing. The molecular weight of the receptor as determined by gel electrophoresis is about 120 000. It is proposed that the camphor receptor is a membrane protein containing sulfhydryl groups and playing a key role in olfactory reception.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

[3H]GABA binding in developing rabbit retina

We have studied the developmental sequence of the GABA system in the rabbit retina using an in vitro binding assay to monitor developmental changes in the post-synaptic receptor. A variety of tissue treatments including perchlorate and Triton X-100 were employed to optimize binding and remove endogenous factors which inhibit binding. Pre-treatment of the tissue with 0.05% Triton X-100 revealed high affinity binding for [³H]GABA which increased in a sigmoidal fashion with the post-natal age of the animal. A constant level of binding, at about 16% of adult levels, was noted until day 8, at which time a rapid increase occurred. At 16 days post-natal, the amount of specific binding reached a plateau near adult levels. Kinetic analysis of the GABA receptor showed an increase in the number of receptors (B_max) with little or no change in the apparent affinity (K_D). Our results suggest that the onset of post-synaptic receptor activity is delayed approximately 1 to 2 days, relative to the pre-synaptic components, and the period of rapid increase in GABA receptor binding coincides with the period of maximum increase in retinal synaptic density.     

Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells

A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular mass of greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10% of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.     

Isolation and purification of morphine receptor by affinity chromatography

Brain membranes were solubilized by sonication and Triton X-100 extraction and applied to an affinity column consisting of a 6-succinyl morphine derivative of Affi Gel-102. A fraction exhibiting high opiate binding was eluted by tris-buffer containing naloxone, CHAPS and NaCl. This fraction consisted of both proteins and acidic lipids. The opiate binding properties of this purified material exhibited many properties similar to those of membrane bound receptors of the u-type, including high affinity, stereospecificity, Na-effect and rank order in affinity for opiates. This opiate binding material was highly sensitive to both trypsin and N-ethylmaleimide. Based on the protein content of the isolated membrane receptor, a 3200-fold purification over the original brain P2 fraction was achieved.     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Prevention of the agonist binding to gamma-aminobutyric acid B receptors by guanine nucleotides and islet-activating protein, pertussis toxin, in bovine cerebral cortex. Possible coupling of the toxin-sensitive GTP-binding proteins to receptors

Using the membranes treated with Triton X-100, we studied the interaction between gamma-aminobutyric acid (GABA)B receptors and the GTP-binding proteins which are the substrates for ADP-ribosylation by the islet-activating protein (IAP), pertussis toxin. The addition of guanine nucleotides to the membranes markedly decreased the binding of GABA to GABAB receptors. Preincubation of the membranes with IAP plus NAD caused ADP-ribosylation of the 41,000- and 39,000-Da proteins selectively and decreased GABA binding to GABAB receptors in a time- and dose-dependent manner. This decrease of binding appeared to be due to the reduction of receptor affinity for agonist. The GTP-binding proteins which are ADP-ribosylated by IAP were purified from the membrane fraction of bovine cerebral cortex. The addition of the purified GTP-binding proteins to IAP-treated membranes restored the high affinity binding of GABA to GABAB receptor. The two GTP-binding proteins which were resolved by octyl-Sepharose column chromatography showed similar efficacy in restoring GABA binding. Thus, GABAB receptors are coupled to GTP-binding proteins, IAP-specific substrates, in the brain membranes.     

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.