Urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that plays an important role in several physiological and pathological processes. Research has shown that uPAR-binding proteins, such as urokinase plasminogen activator (uPA) and vitronectin(VN), influence uPAR-mediated signaling by modifying uPAR structure and dimerization levels. Interestingly, cellular studies also suggest that dimerization levels may influence uPAR affinity for lipid rafts, although the exact underlying mechanisms remain elusive. Here we report a sensitive model membrane approach, in which the influence of ligand binding on uPAR dimerization and sequestering is investigated in the presence of stable micron-size lipid microdomains of well-defined compositions. Herein receptor sequestering information is obtained using confocal fluorescence intensity analysis and corresponding receptor dimerization data are acquired using the photon counting histogram (PCH) method. According to our experiments, in the absence of ligands, uPAR has a preference for cholesterol-enriched lipid domains and forms a substantial amount of dimers. More importantly, ligand-mediated changes in uPAR dimerization level were found to alter the sequestration behavior of this GPI-anchored receptor in model membrane mixtures with coexisting liquid-ordered (cholesterol-enriched) and liquid-disordered (cholesterol-deficient) lipid domains. The role of different ligands and lipid compositions on uPAR sequestration and dimerization is discussed.

Ligand binding alters dimerization and sequestration of GPI-anchored urokinase plasminogen activator receptor (uPAR) in model membrane mixtures

246th National Meeting (2013)

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