Lipid raft-dependent FcepsilonRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors.
The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcepsilonRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcepsilonRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcepsilonRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.
Department of Experimental Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, Sapienza University, Rome, Italy.
Rosa
R
Molfetta
Francesca
F
Gasparrini
Giovanna
G
Peruzzi
Laura
L
Vian
Mario
M
Piccoli
Luigi
L
Frati
Angela
A
Santoni
Rossella
R
Paolini
D000818:Animals; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D047468:Immunoprecipitation; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding / Q000502:physiology; D034741:RNA, Small Interfering; D051381:Rats; D017455:Receptors, IgE / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
D034741:RNA, Small Interfering; D017455:Receptors, IgE; D025801:Ubiquitin