Pretreatment Of Plasma Membranes Research Articles

Recruitment of the cross-linked opsonic receptor CD32A (FcgammaRIIA) to high-density detergent-resistant membrane domains in human neutrophils.

We have previously shown that CD32A (or FcgammaRIIA), one of the main opsonin receptors, was rapidly insolubilized and degraded in intact neutrophils after its cross-linking. In view of these experimental difficulties, the early signalling steps in response to CD32A activation were studied in purified plasma membranes of neutrophils. After CD32A cross-linking in these fractions, the tyrosine phosphorylation of two major substrates, the receptor itself and the tyrosine kinase Syk, was observed. Phosphorylation of these two proteins was observed only in the presence of orthovanadate, indicating the presence, in the membranes, of one or more tyrosine phosphatases that maintain CD32A dephosphorylation. The tyrosine phosphorylation of these two proteins was inhibited by the Src kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). The ligation of CD32A led to its recruitment to a previously uncharacterized subset of high-density flotillin-1-positive DRMs (detergent-resistant membranes). The changes in the solubility properties of CD32A were observed in the absence of added ATP; therefore, they were probably not secondary to the tyrosine phosphorylation of the receptor, rather they preceded it. Src kinases as well as Syk were constitutively present in DRMs of high and low density and no evident changes in their distribution were detected after cross-linking of CD32A. Pretreatment of plasma membranes with methyl-beta-cyclodextrin did not inhibit the recruitment of CD32A to DRMs, although it led to the loss of the Src kinase Lyn from these fractions. In addition, methyl-beta-cyclodextrin inhibited the tyrosine phosphorylation of CD32A and Syk induced by cross-linking of CD32A. This membrane model allowed us to observe a movement of CD32A from detergent-soluble regions of the membranes to DRMs, where it joined Src kinases and Syk and became tyrosine-phosphorylated.

Dehydroepiandrosterone Activates Endothelial Cell Nitric-oxide Synthase by a Specific Plasma Membrane Receptor Coupled to Gαi2,3

The adrenal steroid dehydroepiandrosterone (DHEA) has no known cellular receptor or unifying mechanism of action, despite evidence suggesting beneficial vascular effects in humans. Based on previous data from our laboratory, we hypothesized that DHEA binds to specific cell-surface receptors to activate intracellular G-proteins and endothelial nitric-oxide synthase (eNOS). We now pharmacologically characterize a putative plasma membrane DHEA receptor and define its associated G-proteins. The [3H]DHEA binding to isolated plasma membranes from bovine aortic endothelial cells was of high affinity (K(d) = 48.7 pm) and saturable (B(max) = 500 fmol/mg protein). Structurally related steroids failed to compete with DHEA for binding. The putative DHEA receptor was functionally coupled to G-proteins, because guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) inhibited [3H]DHEA binding to plasma membranes by 69%, and DHEA increased [35S]GTPgammaS binding by 157%. DHEA stimulated [35S]GTPgammaS binding to Galpha(i2) and Galpha(i3), but not to Galpha(i1) or Galpha(o). Pretreatment of plasma membranes with antibody to Galpha(i2) or Galpha(i3), but not to Galpha(i1), inhibited the DHEA activation of eNOS. Thus, DHEA receptors are expressed on endothelial cell plasma membranes and are coupled to eNOS activity through Galpha(i2) and Galpha(i3). These novel findings should allow us to isolate the putative receptor and reevaluate the physiological role of DHEA activity.

Downregulation of Phospholipase D by Protein Kinase A in a Cell-Free System of Human Neutrophils

Agents which elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD) in human neutrophils. The PLD activity of human neutrophils requires protein factors in both membrane and cytosolic fractions. We have studied the regulation of PLD by the catalytic subunit of protein kinase A (cPKA) in a cell-free system. cPKA significantly inhibited GTPγS-stimulated PLD activity but had no effect on phorbol ester-activated PLD activity. Pretreatment of plasma membranes with cPKA and ATP inhibited subsequent PLD activation upon reconstitution with untreated cytosol. RhoA, which is known to be a plasma membrane activator of PLD, was dissociated from PKA-treated plasma membrane by addition of cytosol. Plasma membrane-associated RhoA in human neutrophils was phosphorylated by cPKA. The PKA-phosphorylated form of RhoA was more easily extracted from membranes by RhoGDI than the unphosphorylated form. These results suggest that inhibition of neutrophil PLD by PKA may be due to phosphorylation of RhoA on the plasma membrane.

Catalytic Activation of Guanylate Cyclase Atrial/Natriuretic Factor Receptor by Combined Effects of ANF and GTPγS in Plasma Membranes of Leydig Tumor Cells: Involvement of G-Proteins

Guanosine 5′-(γ-thio) triphosphate (GTPγS) exhibited a modulatory role in the catalytic activation of guanylate cyclase-A/atrial natriuretic factor receptor (GC-A/ANF-R) in the plasma membrane preparations of murine Leydig tumor (MA-10) cells. Both atrial natriuretic factor (ANF) and GTPγS synergistically stimulated the guanylate cyclase (GC) activity of GC-A/ANF-R in a dose- and time-related manner. Other nucleotides and their analogs such as ATP, adenosine 5′(γ-thio)triphosphate, adenosine 5′-(β,γ-imino)triphosphate, GDP, and guanosine 5′-(2-O-thiodiphosphate) (100 μM each) did not show any discernible effect on GC catalytic activity of GC-A/ANF-R. A significant stimulation of GC activity was observed in the presence of mastoparan, AlF−4 , and benzalkonium chloride. The saturation binding assay of [35S]GTPγS showed the dissociation constant (Kd) of 2.3 × 10−9 M and the binding capacity (Bmax) of 76 pmol/mg protein in the plasma membrane preparations of MA-10 cells. ANF increased the [35S]GTPγS-binding capacity, however, without affecting its affinity constant. Pretreatment of plasma membranes with antibodies against Gsα subunit attenuated the GTPγS-stimulated GC activity, whereas antibodies against Giα subunit enhanced the stimulatory effect of GTPγS on GC catalytic activity of GC-A/ANF-R. However, the antibodies against Goα subunit did not show any effect on GC activity, These results provide the evidence that both Gs and Gi subunits of G-proteins seem to be involved in the regulation of GC catalytic activity of GC-A/ANF-R in the plasma membranes of MA-10 cells.

NADH Oxidase Activity of Plasma Membranes of Soybean Hypocotyls Is Activated by Guanine Nucleotides.

The activity of an auxin-stimulated NADH oxidase of the plasma membrane of hypocotyls of etiolated soybean (Glycine max Merr.) seedlings responded to guanine and other nucleotides, but in a manner that differed from that of enzymes coupled to the classic trimeric and low molecular weight monomeric guanine nucleotide-binding proteins (G proteins). In the presence and absence of either auxin or divalent ions, both GTP and GDP as well as guanosine-5[prime]-O-(3-thiotriphosphate) (GTP-[gamma]-S) and other nucleoside di- and triphosphates stimulated the oxidase activity over the range 10 [mu]M to 1 mM. GTP and GTP-[gamma]-S stimulated the activity at 10 nM in the absence of added magnesium and at 1 nM in the presence of added magnesium ions. Other nucleotides stimulated at 100 nM and above. The NADH oxidase was stimulated by 10 [mu]M mastoparan and by 40 [mu]M aluminum fluoride. Neither cholera nor pertussis toxins, tested at a concentration sufficient to block mammalian G protein function, inhibited the activity. Guanosine 5[prime]-O-(2-thiodi-phosphate) (GDP-[beta]-S) did not stimulate activity, suggesting that the stimulation in response to GDP may be mediated by a plasma membrane nucleoside diphosphate kinase through conversion of GDP to GTP. Auxin stimulation of the NADH oxidase was unaffected by nucleotides at either high or low nucleotide concentrations in the absence of added divalent ions. However, pretreatment of plasma membranes with auxin increased the apparent affinity for nucleotide binding. This increased affinity, however, appeared not to be the mechanism of auxin stimulation of the oxidase, since auxin stimulation was similar with or without low concentrations of guanine nucleotides. The stimulation by nucleotides was observed after incubating the membranes with 0.1% Triton X-100 prior to assay. The results suggest a role of guanine (and other) nucleotides in the regulation of plasma membrane NADH oxidase that differs from the interactions with G proteins commonly described for animal models.

In vitro binding of isolated synaptic vesicles to presynaptic plasma membranes: Activation by Ca 2+ and protein kinase C

An in vitro model to study the molecular control of binding of highly purified synaptic vesicles to presynaptic plasma membranes has been developed. Presynaptic plasma membranes were immobilized by dotting onto nitrocellulose, and binding of iodinated synaptic vesicle membranes was studied under varying experimental conditions. Synaptic vesicles bind to presynaptic plasma membranes in the presence of Ca 2+ and ATP. Binding is reduced in the presence of EGTA and abolished by the calmodulin antagonist trifluoperazine. Vesicle binding is stimulated 5-fold after incubation—prior to dotting—of presynaptic plasma membranes with ATP in the presence of the phorbolester 12- O-tetradecanoylphorbol-13-acetate (1 μM) and 2.5-fold after preincubation with Ca 2+ (50 μM). Pretreatment of plasma membranes with alkaline phosphatase strongly reduces vesicle binding. Microsomes prepared from bovine liver did not bind to presynaptic plasma membranes. Our results suggest that activation of protein kinase C and Ca 2+ stimulate binding of synaptic vesicles to the presynaptic membrane. In the intact nerve terminal this interaction may represent an initial step in synaptic vesicle exocytosis.

Photoaffinity labeling with GTP-γ-azidoanilide of a cholera toxin-sensitive 40 kDa protein from pancreatic acinar cells

In isolated pancreatic acinar plasma membranes a 40 kDa protein was labeled with the photoreactive GTP-analogue [alpha 32P] GTP-gamma-azidoanilide. Increased incorporation of the photolabel into the 40 kDa protein was obtained in the presence of increasing concentrations of cholecystokinin-octapeptide (10(-8) - 10(-5) M) but not with carbachol. Adenylyl cyclase activating hormones such as vasoactive intestinal polypeptide and secretin had no effect. Pretreatment of plasma membranes with cholera toxin reduced incorporation of GTP-gamma-azidoanilide into the 40 kDa protein by about 30%. This reduction was reversed if ADP-ribosylation by cholera toxin was performed in the presence of cholecystokinin, whereas carbachol had no effect. The data indicate that a cholera toxin-sensitive 40 kDa GTP-binding protein is involved in functionally coupling cholecystokinin- but not muscarinic acetylcholine-receptors to phospholipase C.

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect.

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.16 M (NH4)2SO4 and was maximal at 0.6-1.0 M. The maximal activation of the enzyme was observed within 3 min of pretreatment and was not readily reversible. The effect was specific for the NH+4 ion since pretreatment of membranes with other NH+4 salts could activate the enzyme, whereas pretreatment with NaCl or KCl could not. Pretreatment of plasma membranes with (NH4)2SO4 eliminated the sensitivity of the enzyme to the inhibitory effect of guanine nucleotides. (NH4)2SO4 pretreatment also significantly attenuated the inhibition by guanine nucleotides of cAMP binding to its plasma membrane receptor. The effect of (NH4)2SO4 on GTP inhibition of cAMP binding to its receptor was even more dramatic when the salt was present in the binding assay. (NH4)2SO4 also increased the ADP-ribosylation by cholera toxin of a 39,000-Da membrane protein. The data support the hypothesis that (NH4)2SO4-induced changes in adenylate cyclase and the cAMP receptor are due to an alteration of a putative G protein.

Interaction of Pseudomonas aeruginosa cytotoxin with plasma membranes from Ehrlich ascites tumor cells.

Biologically active 125I-cytotoxin from Pseudomonas aeruginosa binds to plasma membranes from Ehrlich ascites tumor cells in a saturable manner. The Scatchard plot indicated a single binding site with a capacity of 260 pmoles/mg of membrane protein and a KD of 2 X 10(-8) M. Specific binding was dependent on temperature, pH and ionic strength. Thus constant levels of bound 125I-cytotoxin were attained either within 30 min at 30 degrees C or within 3 h at 4 degrees C. Binding was 30-fold higher at 4 degrees C vs 30 degrees C and 2-6-fold higher at pH 5.3 vs pH 8.3. Binding was not effected by 50 mM sugar or sialic acid. 300 mM sucrose, however, instead of phosphate buffer, reduced binding by 50%. Pretreatment of plasma membranes with trypsin or papain led to a significant decrease in 125I-cytotoxin binding. A pretreatment with phospholipase C or D had no effect, whereas phospholipase A2 induced a decrease by 34%. The collected data suggest that the binding site for 125I-cytotoxin within the plasma membrane from Ehrlich ascites tumor cells is a membrane protein. Correlation of 125I-cytotoxin binding and membrane action of the unlabelled cytotoxin can be observed through (a) increased lowering of the cellular K+ and Na+ gradient by decrease of medium pH, (b) decreased toxicity after substitution of ions by sugar, and (c) increased breakdown of cellular cationic gradient after temperature shift from 4 degrees C to 37 degrees C.

The CCK receptor on pancreatic plasma membranes: binding characteristics and covalent cross-linking

The cholecystokinin (CCK) receptor in purified plasma membranes prepared from mouse pancreatic acini had a binding affinity of 1.8 nM, an acid pH optimum between 6.0 and 6.5, and an analog specificity of CCK 8 > CCK 33 > desulphated CCK 8 > CCK 4. Binding of CCK to its receptor was abolished by pretreatment of plasma membranes with trypsin. When [ 125I]CCK was cross-linked to its receptors with disuccinimidyl suberate, and the preparation solubilized and subjected to gel electrophoresis and autoradiography, the hormone was associated with M r 80000 protein in both the presence and absence of the reducing agent dithiothretol.

Receptor-mediated gonadotropin action in ovary. Possible regulatory role of cell-surface sialic acid in gonadotropin interaction to purified bovine corpus luteum plasma membranes.

The role of cell‐surface sialic acid in the interaction of 125I‐labeled choriogonadotropin with gonadotropin receptors from plasma membranes of bovine corpus luteum was investigated. Pretreatment of plasma membranes with neuraminidases purified from Clostridium perfringens or Vibrio cholera enhanced the binding activity of 125I‐choriogonadotropin in a concentration‐dependent manner. C. perfringens neuraminidase (100 mU/ml) and V. cholerae neuraminidase (1.0 I.U./ml) maximally stimulated gonadotropin‐binding activity. The neuraminidase stimulation of binding was due to unmasking of gonadotropin‐binding sites and not due to a change in affinity of the receptor for 125I‐choriogonadotropin. The activity of plasma‐ membrane 5′‐nucleotidase, a glycoprotein enzyme, however, was not affected by neuraminidase treatment. The stimulatory effect of C. perfringens enzyme was abolished by co‐incubation of plasma membrane with glycoproteins, fetuin and mucin. Furthermore, the stimulatory effect of neuraminidase was due to an intrinsic property of the enzyme and was accompanied by a parallel dose‐dependent loss of plasma‐membrane‐associated sialic acid residues. Other neuraminidase preparations including that purified from Arthrobacter ureafaciens also enhanced 125I‐choriogonadotropin binding with concomitant hydrolysis of plasma‐membrane sialic acid. By contrast, enzyme purified from influenza virus did not release plasma‐membrane sialic acid and consequently failed to modulate receptor activity. Treatment of plasma membrane with neuraminidase and subsequent fractionation demonstrated that the enzyme hydrolyzed 50–60% sialic acid from glycoprotein and ganglioside components. Preincubation of plasma membrane with wheat‐germ agglutinin partially blocked the hydrolysis of glycoprotein sialic acid as well as stimulation of 125I‐choriogonadotropin‐binding activity. Incubation with other lectins, concanavalin A and peanut agglutinin, neither affected the hydrolysis of glycoprotein/ganglioside sialic acid by neuraminidase nor prevented the stimulatory action of enzyme on 125I‐choriogonadotropin‐binding activity. These studies demonstrate a possible involvement of plasma‐membrane‐associated sialic acid (sialoglycoproteins) in the activity of gonadotropin receptors.

Studies on the Molecular Architecture and the Composition of the GH Receptor from Rabbit Liver,

Binding of human growth hormone (hGH) to plasma membranes from rabbit liver is slightly inhibited by β-mercaptoethanol (β-MSH), dithiothreitol (DTT), and N-ethylmaleimide (NEM). L-Cys has a marked stimulating effect at 20 mM. Pretreatment of plasma membranes with phospholipase A2-C, and D does not influence hGH binding. In the presence of DNase and RNase a slight but distinct elevation of hormone binding is seen. HGH-receptor interaction is totally abolished in the presence of trypsin. This effect is inhibited by trasylol and trypsin inhibitor from soya bean. Neuraminidase has no effect on hGH binding, whereas α or β-galactosidase lead to a complete abolition of hGH-receptor interaction. Analysis of the receptor complex in the presence and absence of hGH leads to at least two distinct polypeptide chains with molecular weights 68 000 and 76 000 daltons on the SDS-gel electrophoresis. The significance of a peptide with 56 000 dalton is not clear. It is concluded that -SH groups and phospholipids are not involved in hGH binding. The effect of nucleases is not understood. Within the hormone recognition area peptide chains carrying galactose residues in α and β-glycosidic bonding are essential for hGH-receptor interaction. Two or three proteins seem to represent integral constituents of the hGH receptor complex.