Ligand Binding Alters Dimerization and Sequestering of Urokinase Receptors in Raft-Mimicking Lipid Mixtures

Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.     

Changes in Cholesterol Level Alter Integrin Sequestration in Raft-Mimicking Lipid Mixtures

The influence of cholesterol (CHOL) level on integrin sequestration in raft-mimicking lipid mixtures forming coexisting liquid-ordered (l_o) and liquid-disordered (l_d) lipid domains is investigated using complementary, single-molecule-sensitive, confocal detection methods. Systematic analysis of membrane protein distribution in such a model membrane environment demonstrates that variation of CHOL level has a profound influence on l_o-l_d sequestration of integrins, thereby exhibiting overall l_d preference in the absence of ligands and l_o affinity upon vitronectin addition. Accompanying photon-counting histogram analysis of integrins in the different model membrane mixtures shows that the observed changes of integrin sequestration in response to variations of membrane CHOL level are not associated with altering integrin oligomerization states. Instead, our experiments suggest that the strong CHOL dependence of integrin sequestration can be attributed to CHOL-mediated changes of lipid packing and bilayer thickness in coexisting l_o and l_d domains, highlighting the significance of a biophysical mechanism of CHOL-mediated regulation of integrin sequestration. We envision that this model membrane study may help clarify the influence of CHOL in integrin functionality in plasma membranes, thus providing further insight into the role of lipid heterogeneities in membrane protein distribution and function in a cellular membrane environment.     

Microsecond simulations indicate that ethanol binds between subunits and could stabilize an open-state model of a glycine receptor

Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α_vβ₃ and α₅β₁ integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α_vβ₃ and α₅β₁, respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α_vβ₃ and α₅β₁ sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α_vβ₃ sequesters strongly into raft-like domains and α₅β₁ loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.     

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.     

SAMP14, a novel,acrosomal membrane-associated,glycosylphosphatidylinositol-anchored member of the Ly-6/urokinase-type plasminogen activator receptor superfamily with a role in sperm-egg interaction

We report a new member of the Ly-6/urokinase-type plasminogen activator receptor (uPAR) superfamily of receptors, SAMP14, which is retained on the inner acrosomal membrane of the human spermatozoan following the acrosome reaction and may play a role in fertilization. The SAMP14 sequence predicted a glycosylphosphatidylinositol (GPI)-anchored protein with a signal peptide, a transmembrane domain near the carboxyl terminus, and a putative transamidase cleavage site in the proprotein. Attachment of SAMP14 to the membrane by a lipid anchor was confirmed by its sensitivity to phosphatidylinositol phospholipase C. SAMP14 has a single functional domain similar to the Ly-6 and urokinase plasminogen activator receptor superfamily of proteins, and the gene mapped to 19q13.33, near the PLAUR locus for uPAR at 19q13.2. Northern and dot blotting showed that SAMP14 expression was testis-specific. Indirect immunofluorescence and immunoelectron microscopy with antisera to purified recombinant SAMP14 localized the protein to outer and inner acrosomal membranes as well as the acrosomal matrix of ejaculated human sperm. Acrosome-reacted sperm demonstrated SAMP14 immunofluorescence, indicating its retention on the inner acrosomal membrane following the acrosome reaction. However, SAMP14 localized to the entire sperm when unwashed swim-up sperm from the ejaculate were stained, indicating that some SAMP14 is loosely associated with the plasma membrane. Antibodies against recombinant SAMP14 inhibited both the binding and the fusion of human sperm to zona free hamster eggs, suggesting that SAMP14 may have a role in sperm-egg interaction. SAMP14 represents a GPI-anchored putative receptor in the Ly-6/uPAR family that is exposed on the inner acrosomal membrane after the acrosome reaction.     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

MConfocal Fluorescence Microscopy of Urokinase Plasminogen Activator Receptor and Cathepsin D in Human MDA-MB-231 Breast Cancer Cells Migrating in Reconstituted Basement Membrane

Using confocal fluorescence microscopy with a monoclonal antibody, we have localized the receptor for urokinase plasminogen activator (uPAR) in MDA-MB-231 human breast cancer cells migrating into a reconstituted basement membrane. Patchy and polarized uPAR immunoreactivity was found at the cell membrane, and strong staining was found both in the ruffled border or leading edge of the cells and at pseudopodia penetrating into the membrane. Intracellular uPAR staining was localized in the paranuclear region and in rounded granule-like structures: some of these were identified as lysosomes by double staining for uPAR and the lysosomal enzyme cathepsin D. Urokinase plasminogen activator (uPA) activity has previously been shown to play a role in migration of cells into basement membranes, and it has been proposed that uPAR also is involved in this process. uPA is known to be internalized and degraded after complex formation with the inhibitor PAI-1. Lysosomal uPAR immunoreactivity may result from concomitant internalization of the receptor.     

Urokinase-type Plasminogen Activator Receptor (uPAR) Ligation Induces a Raft-localized Integrin Signaling Switch That Mediates the Hypermotile Phenotype of Fibrotic Fibroblasts

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.     

Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.     

bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo–electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2–20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2–0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid–protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.     

Monomer dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents.     

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.     

Real-time analysis of protein and protein mixture interaction with lipid bilayers

Artificial lipid bilayers in the form of planar supported or vesicular bilayers are commonly used as models for studying interaction of biological membranes with different substances such as proteins and small molecule pharmaceutical compounds. Lipid membranes are typically regarded as inert and passive scaffolds for membrane proteins, but both non-specific and specific interactions between biomolecules and lipid membranes are indeed ubiquitous; dynamic exchange of proteins from the environment at the membrane interface can strongly influence the function of biological membranes. Such exchanges would either be of a superficial (peripheral) or integrative (penetrating) nature. In the context of viral membranes (termed envelopes), this could contribute to the emergence of zoonotic infections as well as change the virulence and/or pathogenicity of viral diseases. In this study, we analyze adsorption/desorption patterns upon challenging tethered liposomes and enveloped virus particles with proteins – or protein mixtures - such as bovine serum albumin, glycosylphosphatidylinositol anchored proteins and serum, chosen for their different lipid-interaction capabilities. We employed quartz crystal microbalance and dual polarization interferometry measurements to measure protein/membrane interaction in real time. We identified differences in mass uptake between the challenges, as well as differences between variants of lipid bilayers. Tethered viral particles showed a similar adsorption/desorption behavior to liposomes, underlining their value as model system. We believe that this methodology may be developed into a new approach in virology and membrane research by enabling the combination of biophysical and biochemical information.     

Electrically controlling and optically observing the membrane potential of supported lipid bilayers

Supported lipid bilayers are a well-developed model system for the study of membranes and their associated proteins, such as membrane channels, enzymes, and receptors. These versatile model membranes can be made from various components, ranging from simple synthetic phospholipids to complex mixtures of constituents, mimicking the cell membrane with its relevant physiochemical and molecular phenomena. In addition, the high stability of supported lipid bilayers allows for their study via a wide array of experimental probes. In this work, we describe a platform for supported lipid bilayers that is accessible both electrically and optically, and demonstrate direct optical observation of the transmembrane potential of supported lipid bilayers. We show that the polarization of the supported membrane can be electrically controlled and optically probed using voltage-sensitive dyes. Membrane polarization dynamics is understood through electrochemical impedance spectroscopy and the analysis of an equivalent electrical circuit model. In addition, we describe the effect of the conducting electrode layer on the fluorescence of the optical probe through metal-induced energy transfer, and show that while this energy transfer has an adverse effect on the voltage sensitivity of the fluorescent probe, its strong distance dependency allows for axial localization of fluorescent emitters with ultrahigh accuracy. We conclude with a discussion on possible applications of this platform for the study of voltage-dependent membrane proteins and other processes in membrane biology and surface science.     

Role of sterols in modulating the human mu-opioid receptor function in Saccharomyces cerevisiae

This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems. Restoring the properties of the human mu-opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes. The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor. Ergosterol was found to constrain the mu-opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it. These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes. This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another. As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function. This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.     

Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles

GUVs have been widely used for studies on lipid mobility, membrane dynamics and lipid domain (raft) formation, using single molecule techniques like fluorescence correlation spectroscopy. Reports on membrane protein dynamics in these types of model membranes are by far less advanced due to the difficulty of incorporating proteins into GUVs in a functional state. We have used sucrose to prevent four distinct membrane protein(s) (complexes) from inactivating during the dehydration step of the GUV-formation process. The amount of sucrose was optimized such that the proteins retained 100% biological activity, and many proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid mixtures composed of neutral and anionic lipids, an alternate current electric field was required for GUV formation from neutral lipids. Distribution, lateral mobility, and function of an ATP-binding cassette transport system, an ion-linked transporter, and a mechanosensitive channel in GUVs were determined by confocal imaging, fluorescence correlation spectroscopy, patch-clamp measurements, and biochemical techniques. In addition, we show that sucrose slows down the lateral mobility of fluorescent lipid analogs, possibly due to hydrogen-bonding with the lipid headgroups, leading to larger complexes with reduced mobility.     

Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures

There is growing recognition that lipid heterogeneities in cellular membranes play an important role in the distribution and functionality of membrane proteins. However, the detection and characterization of such heterogeneities at the cellular level remains challenging. Here we report on the poorly understood relationship between lipid bilayer asymmetry and membrane protein sequestering in raft-mimicking model membrane mixtures using a powerful experimental platform comprised of confocal spectroscopy XY-scan and photon-counting histogram analyses. This experimental approach is utilized to probe the domain-specific sequestering and oligomerization state of α_vβ₃ and α₅β₁ integrins in bilayers, which contain coexisting liquid-disordered/liquid-ordered (l_d/l_o) phase regions exclusively in the top leaflet of the bilayer (bottom leaflet contains l_d phase). Comparison with previously reported integrin sequestering data in bilayer-spanning l_o-l_d phase separations demonstrates that bilayer asymmetry has a profound influence on α_vβ₃ and α₅β₁ sequestering behavior. For example, both integrins sequester preferentially to the l_o phase in asymmetric bilayers, but to the l_d phase in their symmetric counterparts. Furthermore, our data show that bilayer asymmetry significantly influences the role of native ligands in integrin sequestering.     

Differential proteome expression associated with urokinase plasminogen activator receptor (uPAR) suppression in malignant epithelial cancer

Dysregulation of the plasminogen activation cascade is a prototypic feature in many malignant epithelial cancers. Principally, this is thought to occur through activation of overexpressed urokinase plasminogen activator (uPA) concomitant with binding to its high specificity cell surface receptor urokinase plasminogen activator receptor (uPAR). Up-regulation of uPA and uPAR in cancer appears to potentiate the malignant phenotype, either (i) directly by triggering plasmin-mediated degradation or activation of uPA's or plasmin's proteolytic targets (e.g., extracellular matrix zymogen proteases or nascent growth factors) or indirectly by simultaneously altering a range of downstream functions including signal transduction pathways ( Romer, J. ; Nielsen, B. S. ; Ploug, M. The urokinase receptor as a potential target in cancer therapy Curr. Pharm. Des. 2004, 10 ( 19), 235976 ). Because many malignant epithelial cancers express high levels of uPAR, uPA or other components of the plasminogen activation cascade and because these are often associated with poor prognosis, characterizing how uPAR changes the downstream cellular "proteome" is fundamental to understanding any role in cancer. This study describes a carefully designed proteomic study of the effects of antisense uPAR suppression in a previously studied colon cancer cell line (HCT116). The study utilized replicate 2DE gels and two independent gel image analysis software packages to confidently identify 64 proteins whose expression levels changed (by > or =2 fold) coincident with a moderate ( approximately 40%) suppression of cell-surface uPAR. Not surprisingly, many of the altered proteins have previously been implicated in the regulation of tumor progression (e.g., p53 tumor suppressor protein and c-myc oncogene protein among many others). In addition, through a combination of proteomics and immunological methods, this study demonstrates that stathmin 1alpha, a cytoskeletal protein implicated in tumor progression, undergoes a basic isoelectric point shift (p I) following uPAR suppression, suggesting that post-translational modification of stathmin occur secondary to uPAR suppression. Overall, these results shed new light on the molecular mechanisms involved in uPAR signaling and how it may promulgate the malignant phenotype.     

Physical association of uPAR with the alphaV integrin on the surface of human NK cells

The urokinase-type plasminogen activator receptor (uPAR) serves as a receptor for urokinase plasminogen activator (uPA) and plays a role in invasion and migration of certain immune cells, including NK cells. Although uPAR is anchored to the plasma membrane via a glycosylphosphatidylinositol lipid moiety, we have previously shown that uPAR crosslinking results in MAP kinase signaling and increased integrin expression on the surface of the human NK cell line, YT. We report, herein, that the binding of uPA to uPAR also activates the MAP kinase signaling cascade. Furthermore, we show the physical association between uPAR and integrins on YT cells using cocapping and fluorescence microscopy. These results suggest that signaling initiated by either uPAR binding to uPA or by uPAR clustering may depend on the physical association of uPAR with integrins, a process that may be a prerequisite for NK cell accumulation within established tumor metastases during adoptive therapy.     

Urokinase-type plasminogen activator binding to its receptor stimulates tumor cell migration by enhancing integrin-mediated signal transduction.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) participate in matrix degradation and cell migration by focusing proteolysis and functioning as a signaling ligand/receptor complex. uPAR, anchored by a lipid moiety in the membrane, is thought to require a transmembrane adapter to transduce signals into the cytoplasm. To study uPAR signaling, we transfected the prostate carcinoma cell line LNCaP, which does not express endogenous uPA or uPAR, with a uPAR encoding cDNA, resulting in high-level surface expression. We studied migration of these cells on fibronectin, which is mediated by the integrin alpha5beta1. Ligation of uPAR with uPA or its amino-terminal fragment enhanced haptotactic migration to fibronectin. In cells on fibronectin, but not on poly-l-lysine, ligation of uPAR also resulted in tyrosine phosphorylation of several proteins, including two proteins involved in integrin signaling, focal adhesion kinase and the crk-associated substrate p130(Cas). Furthermore, after uPAR ligation, uPAR was co-immunoprecipitated with beta1 integrins from the detergent-insoluble fraction of cell lysates. Thus, our data suggest that uPAR occupancy results in an interaction between uPAR and integrins and a potentiation of integrin-mediated signaling, which leads to enhanced cell migration.