Ubiquitin-interacting motifs of Epsin are involved in the regulation of insulin-dependent endocytosis.
Epsin is a key molecule in receptor-mediated endocytosis. Epsin is phosphorylated and ubiquitinated, and these post-translational modifications are necessary for the regulation of endocytosis. Since human Epsin (hEpsin) has two ubiquitin-interacting motifs (UIMs), we investigated the roles of these UIMs in endocytosis. hEpsin formed a complex with ubiquitinated proteins but did not bind to monoubiquitin. Neither of the two UIMs of hEpsin alone was sufficient to form a complex with ubiquitinated proteins: both UIMs were necessary. Mutations of Asp209 and Asp210 to Ala in UIM (hEpsinDA) abolished the binding activity of hEpsin to ubiquitinated proteins. However, hEpsinDA interacted with Eps15, POB1, and AP-2, which are involved in receptor-mediated endocytosis, as efficiently as wild-type hEpsin. Expression of hEpsinDA in CHO-IR cells affected neither the binding of insulin to nor insulin-dependent autophosphorylation of its receptor. Expression of wild-type hEpsin inhibited the internalization of insulin, whereas that of hEpsinDA did not. These results suggest that the UIM motifs of hEpsin interact with proteins modified with ubiquitin, and that the complex formation is involved in insulin-dependent receptor endocytosis.
Sugiyama
Shinichiro
S
Departments of Biochemistry and Medicine and Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551.
Kishida
Shosei
S
Chayama
Kazuaki
K
Koyama
Shinya
S
Kikuchi
Akira
A
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007328:Insulin / Q000502:physiology*; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C489722:REPS2 protein, human; D025801:Ubiquitin; D033921:Vesicular Transport Proteins; C114154:epsin