Dataset Information

Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

ABSTRACT: 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.

SUBMITTER: Ge Y

PROVIDER: S-EPMC4223190 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

Ge Yifan Y Siegel Amanda P AP Jordan Rainer R Naumann Christoph A CA

Biophysical journal 20141101 9

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 ...[more]

PMID: 25418095

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Project description: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 (?(v)?(3) and ?(5)?(1) 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 ?(v)?(3) and ?(5)?(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both ?(v)?(3) and ?(5)?(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, ?(v)?(3) sequesters strongly into raft-like domains and ?(5)?(1) 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.

Dataset Information

Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

Publications

Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

Similar Datasets

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