Ligand-dependent and independent internalization of the GPI anchor protein uPAR follow two different mechanisms.

SAHORES MACARENA; CORTESE KATIA; MADSEN CRIS; CHIABRANDO GUSTAVO; BLASI FRANCESCO

Iguazú

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB). (2004); 2004

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

LIGAND-DEPENDENT AND INDEPENDENT INTER-NALIZATION OF THE GPI ANCHOR PROTEIN UPAR FOLLOW TWO DIFFERENT MECHANISMS. Sahores M.1, 2, 3., Cortese K.1, 2, Madsen C.1, 2, Chiabrando G.3, and Blasi F.1, 2.1Università Vita Salute San Raffaele, Milano, Italy, 2FIRC Institute of Molecular Oncology, Milano, Italy, and 3Departamento de Bioquímica Clínica, CIBICI-CONICET, U.N.C., Córdoba, Argentina. msahores@bioclin.fcq.unc.edu.ar uPAR is a GPI anchored protein known to be internalized by the uPA-PAI-1 complex via a clathrin- and LRP-dependent pathway. We employed HT1080 cells, that express the en-dogenous receptor, uPA and PAI-1, and HEK293-uPAR cells, expressing uPAR but not its ligands. In HT1080 cells, uPAR was detected in the plasma membrane, in ruffles, co-localizing with clathrin, LRP and cholera-toxin. And also intracellularly, associated with early endosomes. In HEK293-uPAR cells, uPAR still located in early endosomes, but no-colocalization was observed with LRP or clathrin. Biochemical studies demonstrated that in these cells uPAR is internalized in the absence of ligands. EM identified uPAR in endocytic vesicles devoided of clathrin. FACS analysis showed that the endocytosis was partially inhibited by a dominant negative of Rac1. On the other hand no effect of LRP pathway inhibitors was observed. These results suggest the existence of at least two endocytic routes for uPAR internalization. One is ligand- and LRP-mediated and occurs via clathrin-coated pits. The second is ligand-independent, constitutive and appears to be connected to the ruffling mechanism mediated by Rac1. Despite the presence of the GPI anchoring, both mechanisms do not require the presence of uPAR in lipid rafts.