Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

Modification of lipid phase behavior with membrane-bound cryoprotectants

Several derivatives of cholesterol containing oxyethylene headgroups with and without a terminal galactose have been synthesized in order to examine the effects of immobilizing a cryoprotectant at a membrane surface. In this work, we have studied the ability of the triethoxycholesterol (TEC) and triethoxycholesterol galactose (TEC-Gal) derivatives to modulate the phase behavior of phosphatidylcholine and phosphatidylethanolamine membranes. Methods of fluorescence polarization, 31P-NMR and freeze-fracture electron microscopy were employed to monitor these changes in lipid phase behavior. Fluorescence polarization data demonstrated the ability of the derivatives to fluidize gel state and rigidify liquid-crystalline state phosphatidylcholines in a manner similar to that observed for cholesterol. Unlike cholesterol, however, the Tm of dipalmitoylphosphatidylcholine (DPPC) was reduced in a concentration-dependent manner with each of the derivatives. Freeze-fracture electron microscopy and 31P-NMR of DOPE dispersions indicate an increase in the lamellar to hexagonal phase-transition temperature on the order of 10-20 C degrees above room temperature for mixtures with 20 mol% of the derivatives. These results are discussed in terms of the properties exhibited by compounds such as carbohydrates, which are known to serve as cryoprotectants for synthetic and biological membranes.     

Proton-induced phase separation in phosphatidylserine/phosphatidylcholine membranes

Effects of ph and ionic strength on phosphatidylserine/phosphatidylcholine mixed membranes prepared on Millipore filter pore surfaces have been studied using spin-labeled phosphatidylcholine. Lowering pH at constant ionic strength and lowering ionic strength at constant pH caused a lateral reorganization of the membrane. The trigger was protonation of the serine carboxyl group which caused solidification of phosphatidylserine molecules in the membrane, leaving a fluid phase consisting mainly of phosphatidylcholine. The appearent pK for the proton-induced phase separation was measured in a wide range of salt concentrations. The ionic strength dependence was satisfactorily explained based on the electrostatic free energy of proton in the field of membrane surface potential. The Gouy-Chapman theory gave a good approximation for the surface potential. The surface pK of phosphatidylserine and phosphatidic acid vesicles was directly measured in various salt concentrations by 31P-NMR and the results confirmed validity of the Gouy-Chapman-type analysis. The lateral reorganization was triggered by electrostatic interaction but the bulk of the stabilization energy for the structural changes would be the gains in intermolecular van der Waals energy due to closer packing of phosphatidylserine on solidification.     

Effects of temperature variation and phenethyl alcohol addition on acyl chain order and lipid organization in Escherichia coli derived membrane systems. A 2H- and 31P-NMR study.

Using 2H- and 31P-NMR techniques the effects of temperature variation and phenethyl alcohol addition were investigated on lipid acyl chain order and on the macroscopic lipid organization of membrane systems derived from cells of the Escherichia coli fatty acid auxotrophic strain K1059, which was grown in the presence of [11,11-2H2]oleic acid. Membranes of intact cells showed a gel to liquid-crystalline phase transition in the range of 4-20 degrees C, which was similar to that observed for the total lipid extract and for the dominant lipid species phosphatidylethanolamine (PE). Phosphatidylglycerol (PG) remained in a fluid bilayer throughout the whole temperature range (4-70 degrees C). At 30 degrees C acyl chain order was highest in PE, followed by the total lipid extract, PG, intact cells, and isolated inner membrane vesicles. Acyl chain order in E. coli PE and PG was much higher than in the corresponding dioleoylphospholipids. E. coli PE was found to maintain a bilayer organization up to about 60 degrees C, whereas in the total lipid extract as well as in intact E. coli cells bilayer destabilization occurred already at about 42 degrees C. It is proposed that the regulation of temperature at which the bilayer-to-non-bilayer transition occurs may be important for membrane functioning in E. coli. Addition of phenethyl alcohol did not affect the macroscopic lipid organization in E. coli cells or in the total lipid extract, but caused a large reduction in chain order of about 70% at 1 mol% of the alcohol in both membrane systems. It is concluded that while both increasing temperature and addition of phenethyl alcohol can affect membrane integrity, in the former case this is due to the induction of non-bilayer lipid structures, whereas in the latter case this is caused by an increase in membrane fluidity.     

Influence of stigmastanol and stigmastanyl-phosphorylcholine, two plasma cholesterol lowering substances, on synthetic phospholipid membranes. A 2H- and 31P-NMR study.

Cholesterol, stigmastanol, and stigmastanyl-phosphorylcholine (ST-PC) were incorporated into model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). POPC and ST-PC were deuterated at the lipid headgroup, DOPC at the cis-double bonds. The influence of the three sterols on the motion and conformation of the lipid headgroups and the hydrocarbon chains was monitored with 2H- and 31P-NMR. All three sterols were freely miscible with the lipid matrix in concentrations of up to 50 mol% without inducing phase separations or nonbilayer structures. However, the molecules exert quite different effects on the phospholipid bilayer. Cholesterol and stigmastanol are largely buried in the hydrocarbon part of the membrane, distinctly restricting the flexing motions of the fatty acyl chains whereas the conformation of the phospholipid headgroups is little affected. In contrast, ST-PC is anchored with its headgroup in the layer of phospholipid dipoles, preventing an extensive penetration of the sterol ring into the hydrocarbon layer. Hence ST-PC has almost no effect on the hydrocarbon chains but induces a characteristic conformational change of the phospholipid headgroups. The 2H- and 31P-NMR spectra of mixed phospholipid/ST-PC membranes further demonstrate that the PC headgroup of ST-PC has a similar orientation as the surrounding phosphatidylcholine headgroups. For both types of molecules the -P-N+ dipole is essentially parallel to the membrane surface. Addition of ST-PC induces a small rotation of the POPC headgroup towards the water phase.     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.     

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

Tetracaine-membrane interactions: effects of lipid composition and phase on drug partitioning, location, and ionization

Interactions of the local anesthetic tetracaine with unilamellar vesicles made of dimyristoyl or dipalmitoyl phosphatidylcholine (DMPC or DPPC), the latter without or with cholesterol, were examined by following changes in the drug's fluorescent properties. Tetracaine's location within the membrane (as indicated by the equivalent dielectric constant around the aromatic fluorophore), its membrane:buffer partition coefficients for protonated and base forms, and its apparent pK(a) when adsorbed to the membrane were determined by measuring, respectively, the saturating blue shifts of fluorescence emission at high lipid:tetracaine, the corresponding increases in fluorescence intensity at this lower wavelength with increasing lipid, and the dependence of fluorescence intensity of membrane-bound tetracaine (TTC) on solution pH. Results show that partition coefficients were greater for liquid-crystalline than solid-gel phase membranes, whether the phase was set by temperature or lipid composition, and were decreased by cholesterol; neutral TTC partitioned into membranes more strongly than the protonated species (TTCH(+)). Tetracaine's location in the membrane placed the drug's tertiary amine near the phosphate of the headgroup, its ester bond in the region of the lipids' ester bonds, and associated dipole field and the aromatic moiety near fatty acyl carbons 2-5; importantly, this location was unaffected by cholesterol and was the same for neutral and protonated tetracaine, showing that the dipole-dipole and hydrophobic interactions are the critical determinants of tetracaine's location. Tetracaine's effective pK(a) was reduced by 0.3-0.4 pH units from the solution pK(a) upon adsorption to these neutral bilayers, regardless of physical state or composition. We propose that the partitioning of tetracaine into solid-gel membranes is determined primarily by its steric accommodation between lipids, whereas in the liquid-crystalline membrane, in which the distance between lipid molecules is larger and steric hindrance is less important, hydrophobic and ionic interactions between tetracaine and lipid molecules predominate.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Triton promotes domain formation in lipid raft mixtures

Biological membranes are supposed to contain functional domains (lipid rafts) made up in particular of sphingomyelin and cholesterol, glycolipids, and certain proteins. It is often assumed that the application of the detergent Triton at 4 degrees C allows the isolation of these rafts as a detergent-resistant membrane fraction. The current study aims to clarify whether and how Triton changes the domain properties. To this end, temperature-dependent transitions in vesicles of an equimolar mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, egg sphingomyelin, and cholesterol were monitored at different Triton concentrations by differential scanning calorimetry and pressure perturbation calorimetry. Transitions initiated by the addition of Triton to the lipid mixture were studied by isothermal titration calorimetry, and the structure was investigated by (31)P-NMR. The results are discussed in terms of liquid-disordered (ld) and -ordered (lo) bilayer and micellar (mic) phases, and the typical sequence encountered with increasing Triton content or decreasing temperature is ld, ld + lo, ld + lo + mic, and lo + mic. That means that addition of Triton may create ordered domains in a homogeneous fluid membrane, which are, in turn, Triton resistant upon subsequent membrane solubilization. Hence, detergent-resistant membranes should not be assumed to resemble biological rafts in size, structure, composition, or even existence. Functional rafts may not be steady phenomena; they might form, grow, cluster or break up, shrink, and vanish according to functional requirements, regulated by rather subtle changes in the activity of membrane disordering or ordering compounds.     

Coupled changes between lipid order and polypeptide conformation at the membrane surface. A 2H NMR and Raman study of polylysine-phosphatidic acid systems

Thermotropism and segmental chain order parameters of sn-2-perdeuteriated dimyristoyl-phosphatidic acid (DMPA)-water dispersions, with and without poly(L-lysine) (PLL) of different molecular weights, have been investigated by solid-state deuterium NMR spectroscopy. The segmental chain order parameter profile of this negatively charged lipid is similar to that already found for other lipids. Addition of long PLL (MW = 200,000) increases the temperature, Tc, of the lipid gel-to-fluid phase transition, whereas short PLL (MW = 4000) has practically no effect on Tc. In the fluid phase both varieties of PLL increase the "plateau" character of segmental order parameters up to carbon position 10. At the same reduced temperature, long PLL more significantly increases the segmental ordering, especially at the methyl terminal position. This leads to the conclusion that polar head-group capping and charge neutralization by PLL induce severe changes in lipid chain ordering, even down to the bilayer core. The structure of PLL bound to the lipid bilayer surface was monitored by Raman spectroscopy, following the amide I bands. Results show that the lipid gel-to-fluid phase transition triggers a conformational transition from ordered beta-sheet to random structure of short PLL, while it does not affect the strongly stabilized beta-sheet structure of long PLL. It is concluded that both short and long PLL can efficiently cap and neutralize lipid head groups, whatever their structure, and that peptide length is a key parameter in whether lipids or peptides are the driving force in conformationally coupled changes of both partners in the membrane.     

Evidence that 31P NMR is a sensitive indicator of small conformational changes in the coenzyme of aspartate aminotransferase

The pH dependence of 31P-NMR spectra of pig cytosolic aspartate aminotransferase, containing either N-(5'-phosphopyridoxyl)-L-aspartate or pyridoxal 5'-deoxymethylenephosphonate in place of the normal coenzyme pyridoxal 5'-phosphate, has been analysed. The chemical shifts of phosphopyridoxylaspartate and of pyridoxal 5'-deoxymethylenephosphonate model Schiff base in free solution show pK values of 6.3 and 7.4, attributable to the second deprotonation step of phosphate and phosphonate, respectively. However, these compounds behave very differently when bound to apoaspartate aminotransferase. 31P-NMR spectra of these enzyme derivatives indicate that the phosph(on)ate group remains dianionic throughout the pH range 4-8.5. A clear correlation between apparent pK values obtained from spectrophotometric titration of the coenzyme chromophore and those obtained by 31P NMR indicates that the same ionisation is being reported by both methods. The data are interpreted, on the basis of available crystallographic structures of chicken mitochondrial aspartate aminotransferase, to indicate that in each case the alteration in 31P chemical shift results from a conformational change in the coenzyme 5' side chain, in which one of the structures involves a near-eclipsed pair of bonds. Such a stressed conformation produces slight alterations in bond angles around the phosphorus atom, which in turn cause the observed change in 31P chemical shift. The evidence is taken to indicate that in this case 31P NMR is a sensitive reporter of stress in enzyme-bound pyridoxal 5'-phosphate and its derivatives.     

Restriction of lipid motion in membranes triggered by beta-sheet aggregation of the anti-apoptotic BH4 domain

The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state (2)H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by (31)P-NMR at 30 degrees C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static (14)N and (31)P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated beta-sheet secondary structure of BH4 with a 42 degrees tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.     

Lipid bilayers in the gel phase become saturated by triton X-100 at lower surfactant concentrations than those in the fluid phase

It has been repeatedly observed that lipid bilayers in the gel phase are solubilized by lower concentrations of Triton X-100, at least within certain temperature ranges, or other nonionic detergents than bilayers in the fluid phase. In a previous study, we showed that detergent partition coefficients into the lipid bilayer were the same for the gel and the fluid phases. In this contribution, turbidity, calorimetry, and 31P-NMR concur in showing that bilayers in the gel state (at least down to 13-20°C below the gel-fluid transition temperature) become saturated with detergent at lower detergent concentrations than those in the fluid state, irrespective of temperature. The different saturation may explain the observed differences in solubilization.     

Synthesis and polymorphic phase behaviour of polyunsaturated phosphatidylcholines and phosphatidylethanolamines

A series of phosphatidylcholines and phosphatidylethanolamines was synthesized containing two acyl chains of the following polyunsaturated fatty acids: linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4) and docosahexaenoic acid (22:6). In addition two phospholipids with mixed acid composition were synthesized: 16:0/18:1_c phosphatidylcholine and 16:0/18:1_c phosphatidylethanolamine. The structural properties of these lipids in aqueous dispersions in the absence and in the presence of equimolar cholesterol were studied using ³¹P-NMR, freeze fracturing and differential scanning calorimetry (DSC).The phosphatidylcholines adopt a bilayer configuration above 0°C. Incorporation of 50 mol% of cholesterol in polyunsaturated species induces a transition at elevated temperatures into structures with ³¹P-NMR characteristics typical of non-bilayer organizations. When the acyl chains contain three or more double bonds, this non-bilayer organization is most likely the hexagonal H_II phase, 16:0/15:1_c phosphatidylethanolamine shows a bilayer to hexagonal transition temperature of 75°C. The polyunsaturated phosphatidylethanolamines exhibit a bilayer to hexagonal transition temperature below 0°C which decreases with increasing unsaturation and which is lowered by approximately 10°C upon incorporation of 50 mol% of cholesterol. Finally, it was found that small amounts of polyunsaturated fatty acyl chains in a phosphatidylethanolamine disproportionally lower its bilayer to hexagonal transition temperature.     

Retinal and retinol promote membrane fusion.

Disk membranes from the bovine retinal rod outer segments (ROS) were found to fuse with vesicles made of lipids extracted from unbleached ROS disk membranes, using a lipid mixing assay for membrane fusion (relief of self-quenching of R18, octadecylrhodamine B chloride). If the retinal chromophore of rhodopsin was reductively linked to opsin before lipid extraction, the vesicles made of the extracted lipids were not suitable targets for fusion of the disk membranes. The addition of retinal and retinol to these vesicles restored their ability to fuse. Therefore, the presence of all-trans retinal was implicated in promoting membrane fusion in this system. To test this possibility, the ability of retinal and retinol to influence the phase behavior and the fusion capability of large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl-DOPE) was examined. Both retinal and retinol stimulated the fusion of vesicles of N-methyl-DOPE (contents mixing with ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; DPX, p-xylylene bis(pyridinium bromide)). Both compounds reduced the onset temperature for isotropic resonances in the 31P-NMR spectra of N-methyl-DOPE dispersions and the onset temperature, TH, for formation of hexagonal II phase. These results were consistent with previous studies in which the onset temperature for the 31P-NMR isotropic resonances were correlated with stimulation of membrane fusion. These data suggested that both retinal and retinol may stimulate membrane fusion by destabilizing the bilayers of membranes.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-β-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.