Molecular characterization of the intracellular traffic of LRP1 and alpha 2 macroglobulin/LRP1 complex

JALDIN-FINCATI JR; BARCELONA PF; SÁNCHEZ MC; CHIABRANDO GA

Puerto Madryn

Congreso; XLVI Annual Meeting - Argentine Society for Biochemistry and Molecular Biology Research (SAIB); 2010

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

The LDL receptor-related protein 1 (LRP1) is an endocytic receptor involved in the activated α2-Macroglubulin (α2M*) internalization. Previously we demonstrated that LRP1 mediated the α2M* induced intracellular signaling activation. However, the molecular regulation of the LRP1 signaling and endocytosis activity is not well established. In this work we tried to characterize the LRP1 intracellular traffic with Alexa-Fluor α2M* using pull-chase experiments at 37 °C (0 to 60 min) with a previous binding step at 4 °C (30 min). The intracellular localization of Alexa-Fluor α2M* was examined by confocal microscopy using specific fluorescent antibodies against intracellular vesicles. The clatrin-mediated endocytosis of LRP1 and α2M*/LRP1-complex was compared with Transferring Receptor (TfR), using Alexa-Fluor Tf, and specifically blocked by a dominant-negative form of Eps15-GFP (DN-EPS15-GFP). Our data demonstrated that α2M* is clatrin-dependent internalized by LRP1, since it was fully blocked in cells transiently expressing DN-EPS15-GFP. Then, we show that α2M*/LRP1-complex is localized in early endosomes after 5 min up to 15 min of ligand internalization. After this time, Alexa-Fluor α2M* is localized in late endosomes/lysosomes, whereas LRP1 is in recycling endosomes. Our data suggest that the signaling activity of LRP1 induced by α2M* occur in the plasmatic membrane and/or in early endosomes.