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

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidénius, Nicolai

doi:10.1083/jcb.200702151

Cited by 81 publications

(107 citation statements)

References 41 publications

(79 reference statements)

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“…Ligandmediated integrin activation resulted in a dramatic increase in nanoclustering of GPI-anchored proteins, locally driving the formation of nascent adhesion sites ( 88 ), which can eventually stabilize into lipid ordered domains in stable focal adhesions ( 120 ). In the same context, ECM components can also modulate the dimerization and diffusion of uPAR, the GPI-linked receptor for urokinase ( 121 ). Interestingly, uPAR also interacts with integrin and uses integrin as a coreceptor for its signaling ( 122 ), pointing toward confi rmed that a model TM-ABD probe can exhibit actindependent clustering.…”

Section: Gpi-hotspots and Signal Transductionmentioning

confidence: 99%

GPI-anchored protein organization and dynamics at the cell surface

Saha

Anilkumar

Mayor

2016

Journal of Lipid Research

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of the protein called glycosylphosphatidylinositol (GPI)-anchored proteins. The basic structure of a GPI-anchored protein consists of phosphatidylinositol (PI) linked to an unusual non-N -acetyl glucosamine, which, in turn, is linked to three mannose residues followed by an ethanolamine covalently linked to the protein via an amide linkage (EtNP-6Man ␣ 1-2Man ␣ 1-6Man ␣ 1-4GlcN ␣ 1-6 myo inositolphospholipid, Fig. 1A ). Depending on the species and functional context, there may exist variations in the side chain associated with the glycan core. These have been summarized in ( 1 ). The lipid moiety is necessary for the incorporation of GPI-anchored proteins into so-called lipid rafts/microdomains ( 2, 3 ), which can serve as a sorting station for a number of cell signaling molecules, thereby functioning as a reaction center. GPI-anchored proteins can exist in different forms depending on the context and the tissue in which they are expressed. Alternate splicing can cause the same protein to exhibit TM, soluble, or GPI-anchored forms; for example, neural cell adhesion molecule (NCAM) can exist in its GPI-anchored and soluble form when expressed in muscles; whereas, it takes up a TM form instead of the soluble form in brain. GPI anchoring of proteins occurs at the luminal face of The plasma membrane of the cell is comprised of a diverse set of proteins, many of which are transmembrane (TM) proteins spanning the entire bilayer, but a significant proportion of proteins are also lipid tethered, containing a complex glycan core attached to the C terminus

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Section: Gpi-hotspots and Signal Transductionmentioning

confidence: 99%

GPI-anchored protein organization and dynamics at the cell surface

Saha

Anilkumar

Mayor

2016

Journal of Lipid Research

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“…At present, the factors regulating the dynamics and extent of MT4-MMP dimerization/oligomerization remain unknown. It will be interesting to determine whether monomeric and dimeric forms of MT4-MMP display a different substrate profile and whether fluctuations in the monomer/dimer ratio will alter the subcellular distribution (basal versus apical) of MT4-MMP, as reported with other GPIanchored proteins (35)(36)(37).…”

Section: Analyses Of Mt4-mmp Stem Peptide By Computationalmentioning

confidence: 99%

Characterization of the Dimerization Interface of Membrane Type 4 (MT4)-Matrix Metalloproteinase

Sohail

Marco

Zhao

et al. 2011

Journal of Biological Chemistry

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MT4-MMP (MMP17) belongs to a unique subset of membrane type-matrix metalloproteinases that are anchored to the cell surface via a glycosylphosphatidylinositol moiety. However, little is known about its biochemical properties. Here, we report that MT4-MMP is displayed on the cell surface as a mixed population of monomeric, dimeric, and oligomeric forms. Sucrose gradient fractionation demonstrated that these forms of MT4-MMP are all present in lipid rafts. Mutational and computational analyses revealed that Cys 564 , which is present within the stem region, mediates MT4-MMP homodimerization by forming a disulfide bond. Substitution of Cys 564 results in a more rapid MT4-MMP turnover, when compared with the wild-type enzyme, consistent with a role for dimerization in protein stability. Expression of MT4-MMP in Madin-Darby canine kidney cells enhanced cell migration and invasion of Matrigel, a process that requires catalytic activity. However, a serine substitution at Cys 564 did not reduce MT4-MMP-stimulated cell invasion of Matrigel suggesting that homodimerization is not required for this process. Deglycosylation studies showed that MT4-MMP is modified by N-glycosylation. Moreover, inhibition of N-glycosylation by tunicamycin diminished the extent of MT4-MMP dimerization suggesting that N-glycans may confer stability to the dimeric form. Taken together, the data presented here provide a new insight into the characteristics of MT4-MMP and highlight the common and distinct properties of the glycosylphosphatidylinositol-anchored membrane type-matrix metalloproteinases. Matrix metalloproteinases (MMPs)2 are zinc-dependent endopeptidases responsible for the hydrolytic cleavage of multiple proteins and thus play key roles in regulation of diverse physiological processes. Because uncontrolled MMP activity can lead to tissue damage and can contribute to pathological conditions, the functions of the various members of the MMP family are strictly regulated, so that their proteolytic tasks are accomplished only within a suitable spatiotemporal window meant to sustain normal cell function. As in many other proteolytic systems, substrate specificity by MMPs is achieved, in part, by specific localization of the protease and its substrate(s) at precise cellular sites. To cover a wide range of cellular locations, the MMP family includes both soluble and membraneanchored variants. The latter subclass of MMPs, known as membrane type-MMPs (MT-MMPs), is equipped with membrane-anchoring domains, which specifically target the proteases to plasma membranes. The MT-MMPs are classified as transmembrane or GPI-anchored MT-MMPs, a classification that is based on the presence of either a transmembrane domain or a GPI anchor (1). Although much information is available on the regulation and function of transmembrane MT-MMPs, there is a conspicuous paucity of knowledge on the GPI-anchored MT-MMPs. The GPI-MT-MMPs includes two enzymes, MT4-(MMP17) and MT6-MMP (MMP25), that share similar structural features and biochemical properties (2). The pr...

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“…Firstly, PAI-1 has been shown to act as an antagonist of cell adhesion to VN through its competitive inhibition of adhesion receptors binding to the RGD motif [39]. Secondly, both uPAR and uPA have been characterized as agonist of cell adhesion to VN, with uPAR acting as a bona fide VN adhesion receptor [40] and uPA as a soluble factor increasing the affinity and avidity of uPAR for VN [13,41]. The findings presented here, starting from the discovery of cleavage of the RGD motif by uPA and plasmin, clearly suggest a novel and radically different view on the function of the plasminogen activation system components in the regulation of cell adhesion to VN.…”

Section: Embo Reportsmentioning

confidence: 99%

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

et al. 2016

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Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR. Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

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

Cited by 81 publications

References 41 publications

GPI-anchored protein organization and dynamics at the cell surface

GPI-anchored protein organization and dynamics at the cell surface

Characterization of the Dimerization Interface of Membrane Type 4 (MT4)-Matrix Metalloproteinase

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin

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