title: 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.
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University, Indianapolis, Indiana.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Ge
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University, Indianapolis, Indiana; Integrated Nanosystems Development Institute, Indiana University-Purdue University, Indianapolis, Indiana.
authors.forename: Amanda P
authors.initials: AP
authors.lastname: Siegel
authors.affiliation: Makromolekulare Chemie, TU Dresden, Dresden, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Jordan
authors.affiliation: Department of Chemistry and Chemical Biology, Indiana University-Purdue University, Indianapolis, Indiana; Integrated Nanosystems Development Institute, Indiana University-Purdue University, Indianapolis, Indiana. Electronic address: canauman@iupui.edu.
authors.forename: Christoph A
authors.initials: CA
authors.lastname: Naumann
mesh_terms: D019281:Dimerization; D008051:Lipid Bilayers / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D011485:Protein Binding; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*; D013057:Spectrum Analysis
chemical_list: D008051:Lipid Bilayers; D010743:Phospholipids; D055293:Receptors, Urokinase Plasminogen Activator