ENDOCYTIC RECYCLING OF LRP1 IN ALPHA 2-MACROGLOBULIN-STIMULATED CELLS

JALDIN FINCATI JR; BARCELONA PF; SANCHEZ MC; CHIABRANDO GA

San Luis

Congreso; SAIB 2011 (XLVII Reunión Anual de Sociedad Argentina en Bioquímica y Biología Molecular).; 2011

SAIB 2011 (XLVII Reunión Anual de Sociedad Argentina en Bioquímica y Biología Molecular).

The LDL receptor-related protein 1 (LRP1) is an endocytic and signaling receptor, which play an key role in the cellular migration and proliferation. Previously we demonstrated that a2- Macroglubulin (a2M*) induced intracellular signaling activation via LRP1, which is characterized by PKC and MAPK activation. Our hypothesis is that the cellular function of LRP1 involves the endocytic recycling and cell surface sorting of this receptor ina2- Macroglubulin (a2M*) induced intracellular signaling activation via LRP1, which is characterized by PKC and MAPK activation. Our hypothesis is that the cellular function of LRP1 involves the endocytic recycling and cell surface sorting of this receptor ina2M*) induced intracellular signaling activation via LRP1, which is characterized by PKC and MAPK activation. Our hypothesis is that the cellular function of LRP1 involves the endocytic recycling and cell surface sorting of this receptor in a2M*-stimulated cells. Hence, in this work we tried to characterize the endocytic recycling and cell membrane sorting of LRP1 in MIOM1 cells stimulated with a2M*. Using confocal microscopy, flow cytometry and a recombinant mini-receptor version of LRP1 (mLRP4/GFP) we demonstrated that a2M* induced the LRP1 localization in Rab11-recycling compartments between 15-30 min after a2M* stimulation. Then, by TIRF microscopy and LRP1 immunoprecipitation techniques of biotin-labeled cell surface proteins we showed that a2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptor2M*-stimulated cells. Hence, in this work we tried to characterize the endocytic recycling and cell membrane sorting of LRP1 in MIOM1 cells stimulated with a2M*. Using confocal microscopy, flow cytometry and a recombinant mini-receptor version of LRP1 (mLRP4/GFP) we demonstrated that a2M* induced the LRP1 localization in Rab11-recycling compartments between 15-30 min after a2M* stimulation. Then, by TIRF microscopy and LRP1 immunoprecipitation techniques of biotin-labeled cell surface proteins we showed that a2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptora2M*. Using confocal microscopy, flow cytometry and a recombinant mini-receptor version of LRP1 (mLRP4/GFP) we demonstrated that a2M* induced the LRP1 localization in Rab11-recycling compartments between 15-30 min after a2M* stimulation. Then, by TIRF microscopy and LRP1 immunoprecipitation techniques of biotin-labeled cell surface proteins we showed that a2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptora2M* induced the LRP1 localization in Rab11-recycling compartments between 15-30 min after a2M* stimulation. Then, by TIRF microscopy and LRP1 immunoprecipitation techniques of biotin-labeled cell surface proteins we showed that a2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptora2M* stimulation. Then, by TIRF microscopy and LRP1 immunoprecipitation techniques of biotin-labeled cell surface proteins we showed that a2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptora2M* promoted (after 15 min) the intracellular sorting of the constitutive LRP1 and mLRP4 to the cell membrane. This sorting was partially blocked by the negative dominant mutant form of Rab11. However, other Rab forms, probably Rab8 and Rab6, could be involved in this sorting process. Our data suggest that the LRP1 function ina2M*-stimulated cells is dependent on the endocytic recycling of this receptora2M*-stimulated cells is dependent on the endocytic recycling of this receptor