TfR2 localizes in lipid raft domains and is released in exosomes to activate signal transduction along the MAPK pathway

Alessia Calzolari¹, Carla Raggi², Silvia Deaglio³, Nadia Maria Sposi¹, Marit Stafsnes¹, Katia Fecchi², Isabella Parolini¹, Fabio Malavasi³, Cesare Peschle¹, Massimo Sargiacomo², and Ugo Testa^1,*

¹ Department of Hematology, Oncology and Molecular Medicine,, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
² Department of Pharmacology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
³ Laboratory of Immunogenetics, Department of Genetics, Biology, and Biochemistry, University of Turin Medical School, Via Santena 19, 10125 Turin, Italy

^* Author for correspondence (e-mail: u.testa{at}iss.it)

Abstract: Transferrin receptor 2 (TfR2) possesses a YQRV motif similar to the YTRF motif of transferrin receptor 1 (TfR1) responsible for the internalization and secretion through the endosomal pathway. Raft biochemical dissection showed that TfR2 is a component of the low-density Triton-insoluble (LDTI) plasma membrane domain, able to co-immunoprecipitate with caveolin-1 and CD81, two structural raft proteins. In addition, subcellular fractionation experiments showed that TfR1, which spontaneously undergoes endocytosis and recycling, largely distributed to intracellular organelles, whereas TfR2 was mainly associated with the plasma membrane. Given the TfR2 localization in lipid rafts, we tested its capability to activate cell signalling. Interaction with an anti-TfR2 antibody or with human or bovine holotransferrin showed that it activated ERK1/ERK2 and p38 MAP kinases. Integrity of lipid rafts was required for MAPK activation. Co-localization of TfR2 with CD81, a raft tetraspanin exported through exosomes, prompted us to investigate exosomes released by HepG2 and K562 cells into culture medium. TfR2, CD81 and to a lesser extent caveolin-1, were found to be part of the exosomal budding vesicles. In conclusion, the present study indicates that TfR2 localizes in LDTI microdomains, where it promotes cell signalling, and is exported out of the cells through the exosome pathway, where it acts as an intercellular messenger.

Key Words: Transferrin • Iron metabolism • Lipid rafts • Cell signalling

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