Native Plasma Membrane Research Articles

Functional Reconstitution of Ehrlich Cell Plasma Membrane Ferricyanide Reductase

Native plasma membrane fractions isolated from Ehrlich ascites tumor cells were used as the starting point for two easy and efficient procedures to functionally reconstitute ferricyanide reductase activity. The obtained proteoliposomes showed high specific activities. The reconstituted ferricyanide reductase seemed to have one or more cysteine essential residues, as judged by inhibition with sulfhydryl reagents.

Modulation of GABA flux across rat brain membranes resolved by a rapid quenched incubation technique

The progress and inhibition of [ 3H]GABA influx in native plasma membrane vesicles from the rat cerebral cortex was studied on a subsecond to minute time scale under different conditions by applying a rapid quenched incubation technique. In the absence of Ca 2+ ([Ca 2+] free = 10 −8 M), the progress of influx followed by the addition of 10 nM [ 3H]GABA to the membrane vesicle suspension with time (500 ms to 15 min) can be described by a first-order rate equation giving an overall rate constant, k, of 3.93 ± 0.48 × 10 −3 s −1 and equilibrium influx value, INF e, of 8.84 ± 0.41 pmol [ 3 H]GABA/mg protein . In the presence of Ca 2+ ([Ca 2+] free = 2.4 × 10 −3 M) a significant increase in the INF e value was observed (k = 4.64 ± 0.41 × 10 −3 s −1 and INF e = 13.9 ± 0.40 pmol[ 3 H]GABA/mg protein ). Multiplicity of GABA transporters was indicated in the time-dependent inhibition of [ 3H]GABA influx by different uptake blockers. In the absence of Ca 2+, depolarization (75 mM KCl) inhibited the influx of [ 3H]GABA into the vesicles by ∼70% and initiated the efflux from vesicles loaded with [ 3H]GABA. Different uptake blockers inhibited the Ca 2+-independent translocation of [ 3H]GABA in both directions with similar specificities.

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.

Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes

We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubation of cells did not interfere with the insulin effect to stimulate glucose transport rate in either intact cells or in native PMs; it did, however, reduce PM GTer levels by 27-30%, but not affecting those in the intracellular pool. However, CHX additively increased the insulin-stimulated intrinsic activity of PM GTers by 67%. Relative reconstitution efficiencies, assessed by immunoblotting both native and reconstituted membranes against specific antibodies, were similar for GLUT 1 and GLUT 4. Although insulin did not alter this efficiency, CHX slightly decreased it for GLUT 4. Our data suggest that insulin stimulation of glucose transport may involve, as part of its mechanism, modulation of the GTer intrinsic activity. We further hypothesize that CHX effects on increasing this activity state of GTer may involve as yet unknown protein synthesis-dependent regulator(s).

Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes.

We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubation of cells did not interfere with the insulin effect to stimulate glucose transport rate in either intact cells or in native PMs; it did, however, reduce PM GTer levels by 27-30%, but not affecting those in the intracellular pool. However, CHX additively increased the insulin-stimulated intrinsic activity of PM GTers by 67%. Relative reconstitution efficiencies, assessed by immunoblotting both native and reconstituted membranes against specific antibodies, were similar for GLUT 1 and GLUT 4. Although insulin did not alter this efficiency, CHX slightly decreased it for GLUT 4. Our data suggest that insulin stimulation of glucose transport may involve, as part of its mechanism, modulation of the GTer intrinsic activity. We further hypothesize that CHX effects on increasing this activity state of GTer may involve as yet unknown protein synthesis-dependent regulator(s).

Aquaporin CHIP: the archetypal molecular water channel.

Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling

We have quantitated and studied the topology of isoforms of the Na+/K(+)-ATPase and of the glucose transporter in rat adipocyte plasma membranes. Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/K(+)-ATPase isoforms alpha 1 and alpha 2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K(+)-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting. We found that the Na+/K(+)-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations. The vast majority of the Na+/K(+)-ATPase molecules in the adipocyte plasma membrane were of the alpha 2 isoform; GLUT4 was the dominating glucose transporter isoform. The total number of Na+/K(+)-ATPase molecules labeled in the plasma membrane was 3.5 x 10(5) per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5 x 10(5) per cell.

Bradykinin inhibits cyclic AMP accumulation in D384-human astrocytoma cells via a calcium-dependent inhibition of adenylyl cyclase

Bradykinin causes a concentration-dependent, transient rise in intracellular Ca 2+ and a sustained inhibition of forskolin-, dopamine- and 5′- N-ethyl-carboxamidoadenosine (NECA)-stimulated cAMP accumulation in D384 astrocytoma cells. Chelation of intracellular calcium abolished bradykinin's inhibitory effect on cAMP accumulation. Chelating extracellular Ca 2+ did not block the initial, but eliminated the sustained inhibition of cAMP accumulation. Increasing Ca 2+ influx by calcium ionophore A23187 caused a concentration-dependent inhibition of stimulated cAMP accumulation. A hydroquinone derivative 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), which inhibits microsomal Ca 2+ sequestration, did not mimic the effect of bradykinin, although it increased [Ca 2+] i even more than A23187 did. The inhibitory effect of bradykinin was not mediated by Ca 2+/CaM-dependent stimulation of phosphodiesterase (PDE). Forskolin-stimulated adenylyl cyclase activity was inhibited by Ca 2+ (10 −7 to 10 −3 M), both in ethyleneglycol-bis-( β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) washed and native D384 plasma membranes. This effect was not altered by calmodulin (CaM) or CaM-antagonists. Bradykinin treatment, which attenuates cAMP accumulation in intact cells, did not do so in plasma membranes. These findings suggest that bradykinin-induced inhibition of cAMP formation in D384 cells requires mobilization of [Ca 2+] i and subsequent entry of Ca 2+ which directly interacts with a component of the adenylyl cyclase system.

Ca(2+)-activated K+ channels from an insulin-secreting cell line incorporated into planar lipid bilayers.

This study evaluates the use of the planar lipid bilayer as a functional assay of Ca(2+)-activated K+ channel activity for use in purification of the channel protein. Ca(2+)-activated K+ channels from the plasma membrane of an insulin-secreting hamster Beta-cell line (HIT T15) were incorporated into planar lipid bilayers. The single channel conductance was 233 picoSiemens (pS) in symmetrical 140 mmol/l KCl and the channel was strongly K(+)-selective (PCl/PK = 0.046; PNa/PK = 0.027). Channels incorporated into the bilayer with two orientations. In 65% of cases, the probability of the channel being open was increased by raising calcium on the cis side of the bilayer (to which the membrane vesicles were added) or by making the cis side potential more positive. At a membrane potential of + 20 mV, which is close to the peak of the Beta-cell action potential, channel activity was half-maximal at a Ca2+ concentration of about 15 mumol/l. Charybdotoxin greatly reduced the probability of the channel being open when added to the side opposite to that at which Ca2+ activated the channel. These results resemble those found for Ca(2+)-activated K+ channels in native Beta cell membranes and indicate that the channel properties are not significantly altered by incorporation in a planar lipid bilayer.

Cyclic AMP-dependent protein kinase activation of cystic fibrosis transmembrane conductance regulator chloride channels in planar lipid bilayers.

Membrane vesicles, prepared from mouse NIH-3T3 fibroblasts and Chinese hamster ovary cells expressing high levels of cystic fibrosis transmembrane conductance regulator (CFTR), were fused with Mueller-Rudin planar lipid bilayers. Upon addition of the catalytic subunit of cAMP-dependent protein kinase and ATP, low conductance Cl(-)-selective ion channels were observed in 10 of 16 experiments. The channels had a linear current-voltage relationship and a unitary conductance of approximately 6.5 pS. The channels were more permeable to Cl- than to I- and showed no appreciable time-dependent voltage activation. In contrast, addition of cAMP-dependent protein kinase and ATP to lipid bilayers fused with vesicles prepared from mock transfected (n = 14) cells failed to activate Cl- channels. These data support the conclusion that CFTR is a Cl- channel. They indicate that it can be reconstituted in a planar lipid bilayer and that the biophysical and regulatory properties are very similar to those observed in the native cell membrane. These data also argue against the requirement for loosely associated factors for regulation or function of the channel.

Effects of dicyclohexylcarbodiimide (DCCD) treatment on coupled activities of vanadate‐sensitive ATPase from plasma membrane of maize roots

The presence of dicyclohexylcarbodiimide (DCCD) inhibited the activities of vanadate‐sensitive H+ ‐ATPase in both native and reconstituted plasma membrane of maize (Zea mays L. cv. WF9 × Mo 17) roots. Concentration dependence of DCCD inhibition on adenosine triphosphate (ATP) hydrolysis of native plasma membrane vesicles suggested that the molar ratio of effective DCCD binding to ATPase was close to 1. The DCCD inhibition of ATP hydrolysis could be slightly reduced by the addition of ATP, Mg:ATP, adenosine monophosphate (AMP), Mg:AMP and adenosine diphosphate (ADP). More hydrophilic derivatives of DCCD such as l‐ethyl‐N?‐3‐trimethyl ammonium carbodiimide (EDAC) or 1‐ethyl‐3‐3‐dimethyl‐aminopropyl carbodiimide (EDC) gave no inhibition, indicating that the effective DCCD binding site was located in a hydrophobic region of the protein. The proton transport activity of reconstituted plasma membrane at a temperature below 20°C or above 25°C was much sensitive to DCCD treatment. Build‐up of the proton gradient was analyzed according to a kinetic model, which showed that proton leakage across de‐energized reconstituted plasma membranes was not affected by DCCD, but was sensitive to the method employed to quench ATP hydrolysis. Reconstituted plasma membrane vesicles treated with DCCD exhibited a differential inhibition of the coupled H+‐transport and ATP hydrolysis. The presence of 50 μM DCCD nearly abolished transport but inhibited less than 50% of ATP hydrolysis. The above results suggest that the link between proton transport and vanadate‐sensitive ATP hydrolysis is indirect in nature.

Examination of a lens 'native' plasma membrane fraction and its associated crystallins.

A bovine lens "native" plasma membrane fraction containing its full compliment of extrinsic proteins was prepared by sucrose density centrifugation of the water insoluble fraction. The major membrane fraction was found at the 25/45% sucrose interface. This fraction contained 73% of the total water insoluble phospholipid, 74% of the total water insoluble cholesterol and 58% of the total urea-insoluble protein. Only 9% of the total urea-soluble protein was membrane associated (extrinsic protein), most (75%) was recovered from the pellet. The major intrinsic protein (8 M-urea-insoluble) of the membrane fraction was MIP28, with lesser amounts of MP17. Extrinsic proteins (8 M-urea-soluble) were examined by SDS-PAGE, isoelectric focusing, immunoblotting and amino acid composition analysis. Approximately 70% of the total extrinsic protein appeared to be alpha A-crystallins and modified alpha A-crystallins. About 20% of the extrinsic protein was apparently beta- and gamma-crystallins. The remainder contained presumed cytoskeletal proteins and perhaps other unidentified polypeptides. The native plasma membrane was found distributed throughout the lens with only minor differences in the quantitative composition of the membrane fraction. We have concluded that the native membrane fraction represents the lens plasma membrane with its extrinsic proteins which exist in vivo. These extrinsic proteins appeared to be primarily acidic alpha-crystallin polypeptides with minor amounts of beta- and gamma-crystallins, and presumed cytoskeletal elements. We speculate that these extrinsic proteins may serve as a nucleation site for the association of other water insoluble protein through protein-protein interactions such as those found in the non-membrane associated urea-soluble protein. Together, these interactions may form a structured cytoplasmic matrix important for the maintenance of lens transparency.

Endogenous phosphorylation and dephosphorylation of rat liver plasma membrane proteins, suggesting a 18 kDa phosphoprotein as a potential substrate for alkaline phosphatase

Purified rat liver plasma membranes were incubated for 0-60 min with [gamma-32P]ATP and analysis of 32P-labeled proteins by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the presence of two shifted kinetic phenomena. The use of 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinases, allowed the identification of one as the endogenous protein phosphorylation. The other was shown to be the labeling of two phospho-intermediate forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1.], which have apparent molecular masses of 151 and 135 kDa. Bromolevamisole, a potent inhibitor of the enzyme, stabilized these phospho-intermediates, and consequent on this inhibition the labelling of a 18 kDa phosphoprotein was augmented. So, when alkaline phosphatase was studied in its native plasma membrane environment, a specificity of this enzyme over the endogenous phosphoproteins was established.

Inhibition of subunit dissociation and release of the stimulatory G-protein, Gs, by βγ-subunits and somatostatin in S49 lymphoma cell membranes

We examined the interaction between the stimulatory guanine-nucleotide-binding protein, Gs, and the inhibitory guanine-nucleotide-binding protein, Gi, in cell membranes of S49 lymphoma cells. In these cells, beta-adrenergic receptors stimulate the activity of adenylate cyclase via Gs, whereas inhibition via somatostatin receptors is transduced by an inhibitory G-protein, Gi. Using an antibody that selectively recognizes alpha s, the monomeric, but not the heterotrimeric, alpha-subunit of Gs, we quantified the extent of dissociation of Gs in a competitive e.l.i.s.a. Incubation of S49-cell plasma membranes with 0.1 microM-isoprenaline, 100 microM free Mg2+ and 100 microM-GTP produced substantial subunit dissociation of Gs, which was reversible by addition of purified beta gamma-subunit dimer or somatostatin. Somatostatin produced an immediate (without a lag) time- and concentration-dependent decrease in the concentration of dissociated Gs (kinhib. for somatostatin = 51 +/- 12 nM) and in the activity of adenylate cyclase (kinhib. = 121 +/- 20 nM). By contrast, after addition of a 10-fold molar excess of beta gamma-dimer relative to alpha s, there was a 2-3 min lag, after which the beta gamma-dimer re-associated Gs. Isoprenaline-induced dissociation of Gs was accompanied by a release of alpha s from the incubated membranes to a post-100,000 g supernatant, and somatostatin could reverse this release. Immunoblot analysis with both a C-terminal anti-peptide antibody and an antibody directed against a sequence near the N-terminal also showed release of alpha s by the beta-agonist and reversal by somatostatin. Membrane release of Gs by isoprenaline that could be blocked by somatostatin was also confirmed in reconstitution studies of supernatant fraction into cyc- S49-cell membranes. We conclude that in native cell membranes somatostatin-induced activation of Gi dissociates Gi and interferes with the Gs activation cycle by providing beta gamma-dimer, which acts to prevent or reverse formation of monomeric alpha s. Because alpha s can be released from the cell membrane, regulation of the local concentration of GTP-liganded dissociated alpha s is likely to be an important factor in modulating the activity of adenylate cyclase.

High-Molecular-Weight Alkaline Phosphatase in Serum Has Properties Similar to the Enzyme in Plasma Membranes of the Liver

Partially purified high-molecular-weight alkaline phosphatase from serum was compared with two other forms of the enzyme from the human liver, enzyme in native plasma membranes and purified alkaline phosphatase as a hydrophilic dimer. In a high-molecular-weight form from serum and plasma membranes, and when treated with 1% (v/v) Triton X-100, alkaline phosphatase showed a major band on gradient gel electrophoresis with a mobility equivalent to 400 kD. Nondetergent-treated material from both sources did not enter the gel and was in the voided volume of a gel permeation column. Stimulation of catalytic activity by four different phospholipids and by albumin yielded similar results for high-molecular-weight alkaline phosphatase and for the enzyme in plasma membranes, but these were different from the hydrophilic form. Inhibitors of alkaline phosphatase had similar effects on all forms. Of the three forms of the enzyme, only the hydrophilic dimer did not become incorporated into liposomes or adsorb to octyl-Sepharose after solubilization with Triton X-100 and removal of the detergent. Km (substrate concentration to give half maximal velocity) values with p-nitrophenylphosphate and heat and sodium dodecyl sulfate stabilities were similar for all forms. In the high-molecular-weight form from serum and in plasma membranes, alkaline phosphatase and 5'-nucleotidase showed similar rates of release by phosphatidylinositol phospholipase C. Three preparations of phospholipase D failed to release alkaline phosphatase from either the high-molecular-weight form or from plasma membranes. Based on these similarities, it is probable that the complex of high-molecular-weight alkaline phosphatase in serum most often originates from fragments of hepatic plasma membranes.

Signal amplification in HL‐60 granulocytes

Receptors for the chemotactic peptide fMet-Leu-Phe (fMet, N-formylmethionine) are present in membranes of myeloid differentiated human leukemia (HL-60) cells and stimulate phospholipase C via a pertussis-toxin-sensitive guanine-nucleotide-binding regulatory protein(s) [G-protein(s)]. We have developed methods for the assessment of formyl-peptide-receptor-stimulated binding of radiolabeled guanosine 5'-[gamma-thio]triphosphate ([35S]GTP[S]) to native HL-60 membranes. Agonist stimulation of [35S]GTP[S] association with the membrane was minimal (less than or equal to 20%) when GTP[S] was the sole nucleotide present in the incubation medium. In contrast, receptor activation led to a marked (up to sixfold) stimulation of [35S]GTP[S] binding when GDP or GTP were present in high (greater than 100-fold) excess of [35S]GTP[S]. The increase in [35S]GTP[S] binding caused by the chemotactic agonist was strictly dependent on the presence of Mg2+ and was significantly increased by Na+. Agonist-independent binding of [35S]GTP[S] and the increase due to the chemotactic agonist were markedly attenuated by both pertussis and cholera toxin. Comparison of the number of chemotactic-peptide-sensitive [35S]GTP[S]-binding sites to the number of chemotactic peptide receptors present in HL-60 membranes provided direct evidence that a single formyl-peptide receptor is capable of catalyzing the binding of [35S]GTP[S] to, and thus the activation of, multiple (up to 20) G-proteins in native plasma membranes.

Evidence for the Binding of Calmodulin to Endogenous B‐50 (GAP‐43) in Native Synaptosomal Plasma Membranes

The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.

Distinctive regulation of the functional linkage between the human cation-independent mannose 6-phosphate receptor and GTP-binding proteins by insulin-like growth factor II and mannose 6-phosphate.

The rat insulin-like growth factor II (IGF-II) receptor develops transmembrane signaling functions by directly coupling to a guanine nucleotide-binding protein (G protein) having a 40-kDa alpha subunit, Gi-2, whereas recent studies have indicated that the IGF-II receptor is a molecule identical to the cation-independent mannose 6-phosphate receptor (CI-MPR), a receptor implicated in lysosomal enzyme sorting. In this study, by using vesicles reconstituted with the clonal human CI-MPR and G proteins, we indicated that the CI-MPR could stimulate guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding and GTPase activities of Gi proteins in response to IGF-II. The stimulatory effect of IGF-II on Gi-2 depended on the reconstituted amount of the CI-MPR; it could not be found in vesicles reconstituted with Gi-2 alone; and it was also observed on Gi-1 reconstituted with the CI-MPR in phospholipid vesicles. Of interest, such stimulatory effect was not reproduced by Man-6-P in CI-MPR vesicles reconstituted with either G protein. Furthermore, the affinity for Man-6-P-mediated beta-glucuronidase binding to several kinds of native cell membranes was not reduced by 100 microM GTP gamma S. Instead, however, Man-6-P dose-dependently inhibited IGF-II-induced Gi-2 activation with an IC50 of 6 microM in vesicles reconstituted with the CI-MPR and Gi-2. The action of 100 nM IGF-II was completely abolished by 1 mM Man-6-P. Such an inhibitory effect of Man-6-P was reproduced by 4000 times lower concentrations of beta-glucuronidase or similar concentrations of fructose 1-phosphate, but not by mannose or glucose 6-phosphate. These results indicate that the human CI-MPR has two distinct signaling functions that positively or negatively regulate the activity of Gi-2 in response to the binding of IGF-II or Man-6-P.

Insulin-like growth factor-II/mannose 6-phosphate receptor is incapable of activating GTP-binding proteins in response to mannose 6-phosphate, but capable in response to insulin-like growth factor-II

We previously reported that insulin-like growth factor-II (IGF-II) stimulates both calcium influx and DNA synthesis by acting on the cell surface IGF-II receptor (IGF-IIR) in a manner sensitive to pertussis toxin, and recently demonstrated that IGF-II binding to the IGF-IIR gives rise to functional changes of purified Gi-2, a GTP-binding protein (G protein) in phospholipid vesicles as well as in broken cell membranes. On the other hand, a variety of evidence indicates that the IGF-IIR binds mannose 6-phosphate (man6P) with high affinity probably at a receptor extracellular region different from the IGF-II-binding site. In the present study, we examined whether man6P stimulation of the IGF-IIR evokes the activation of Gi-2 in phospholipid vesicles and in native cell membranes. In vesicles reconstituted with purified rat IGF-IIR and bovine Gi-2, man6P did not stimulate GDP dissociation from Gi-2 even in concentrations up to 10 mM, while IGF-II dose-dependently facilitated GDP release from Gi-2 with an EC50 of 6 nM. The stimulatory effect of IGF-II was not observed in vesicles reconstituted with Gi-2 alone. In addition, also in a native environment of cell membranes, man6P did not affect an endogenous 40-kDa protein or exogenously added purified Gi-2 as assessed with reduction of the pertussis toxin-catalyzed ADP-ribosylation. These results indicate that the IGF-IIR does not activate Gi-like proteins upon man6P binding in phospholipid vesicles and in native cellular membranes, whereas the receptor activates Gi-like proteins upon IGF-II binding in both environments. Thus, we postulate that the IGF-IIR dissimilarly responds to the two structurally unrelated ligands, IGF-II and man6P, in the linkage function with G proteins.

Correct insertion of a simple eukaryotic plasma-membrane protein into the cytoplasmic membrane of Escherichia coli

A genetic system for directly synthesizing eukaryotic membrane proteins in Escherichia coli and assessing their ability to insert into the bacterial cytoplasmic membrane is described. The components of this system are the direct expression vector, pYZ4, and the mature β-lactamase (BlaM) cassette plasmid, pYZ5, that can be used to generate translational fusions of BlaM to any synthesized membrane protein. The β-subunit of sheep-kidney Na,K-ATPase (βNKA), a class-II plasma membrane protein, was synthesized in E. coli using pYZ4, and BlaM was fused to a normally extracellular portion of it. The fusion protein conferred ampicillin resistance on individual host cells, indicating that the BlaM portion had been translocated to the bacterial periplasm, and that, by inference, the eukaryotic plasma-membrane protein can insert into the bacterial cytoplasmic membrane. A series of 31 βNKA :: BlaM fusion proteins was isolated and characterised to map the topology of the eukaryotic plasma membrane protein with respect to the bacterial cytoplasmic membrane. This analysis revealed that the organisation of the βNKA in the E. coli cytoplasmic membrane was indistinguishable from that in its native plasma membrane.