This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Investigating receptor dynamics and ligand interaction in living cells at the single molecule level. In particular, we focus on the Urokinase Receptor (uPAR) and its interaction with the extracellular Urokinase Plasminogen Activator (uPA) and Vitronectin. uPAR is a GPI-anchored protein which is found also in lipid rafts. Recent published data show that, in addition to the monomer, a dimeric uPAR is found in rafts (by floatation experiments). We have expressed a uPAR-GFP-GPI chimeras in HEK293 cells and subcloned cells populations expressing uPAR-GFP-GPI at different levels.
We aim at answering the following questions: 1) does uPA binds with different affinities to the the monomer and to dimeric receptor? with what stoichiometry? 2)Does the binding to Vitronectin occur to the dimeric receptor? 3)do lipid rafts localize and orient the receptor on the cell membrane? Therefore, we will carry out FCS and attempt FCCS to distinguish monomer versus dimer-ligand interaction, using fluorescently labeled uPA and vitronectin fragments, such as SMB.
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