LB-OR-3 Roles of Insulin Receptor Substrate Complexes Containing Rabankyrin-5 in Insulin-Like Growth Factor-I-Induced Macropinocytosis

Program: Late-Breaking Abstracts
Session: LB-OR-Late-Breaking Oral Session
Saturday, June 15, 2013: 11:30 AM-1:00 PM
Presentation Start Time: 12:00 PM
Room 122 (Moscone Center)
Shin Ichiro Takahashi*1, Takayuki Yano2, Yosuke Yoneyama2, Toshiaki Matsui2, Hiroshi Okajima2, Kazuhiro Chida2 and Fumihiko Hakuno3
1Univ of Tokyo, Tokyo, Japan, 2The University of Tokyo, Graduate School of Agriculture and Life Sciences, Tokyo, Japan, 3Grad Sch Agric and Life Sci, Tokyo, Japan
Macropinocytosis, which is the major system of endocytosis responsible for liquid-phase uptake in a number of cell types, has been reported to be regulated by various growth factors. Binding of insulin-like growth factors (IGFs) to their specific receptors on the plasma membrane induces activation of receptor intrinsic tyrosine kinase. Activated receptor then phosphorylates insulin receptor substrates (IRSs), leading to downstream signal activation. These events exert a variety of IGF bioactivities. The present study is undertaken to elucidate the effects of IGFs on macropinocytosis and the molecular mechanism to regulate it. First, we examined whether IGF-I stimulation induces macropinocytosis in MCF-7 human breast cancer cells. Based on time-lapse microscopy analysis, we found that IGF-I treatment induced macropinocytosis. In addition, serum starved cells were incubated with Texas-Red conjugated-dextran in the presence or absence of IGF-I and uptake of dextran was analyzed. The results revealed that dextran uptake was enhanced by IGF-I stimulation. On the other hand, uptake of Alexa546 conjugated-transferrin, which represents receptor-mediated endocytosis was not enhanced by IGF-I stimulation, suggesting that IGF-I specifically activates macropinocytosis. To investigate the roles of IRS-1 in macropinocytosis, we repressed endogenous IRS-1 levels in MCF-7 cells using siRNA and assessed pinocytosis. We found that IRS-1 knockdown decreased macropinocytosis. Recently, we have shown that IRSs form  high-molecular-mass complexes and have identified Rabankyrin-5 as a novel IRS-1-associated protein by yeast-two hybrid screening using IRS-1 as a bait and placenta cDNA library as a prey. Rabankyrin-5 is an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We then examined the interaction between IRS and Rabankyrin-5 by co-immunoprecipitation assay. FLAG-IRS-1/2 and GFP-Rabankyrin-5 were co-transfected in HEK293T cells and the lysates were subjected to immunoprecipitation with anti-FLAG antibody. As a result, Rabankyrin-5 was co-immunoprecipitated with IRS-1, but not with IRS-2. IRS-1 was co-localized with Rabankyrin-5 around the plasma membrane where actin was polymerized in response to IGF-I stimulus. Using time-lapse microscopy, we also found that IGF-I induced formation of Rabankyrin-5-rich vesicles at protrusion and endocytosis of these vesicles. In cells overexpressing Rabankyrin-5 and the dominant negative form of Rab5, IGF-I caused actin polymerization, but not vesicle formation. Taken together, we concluded that IRS-1-Rabankyrin-5 complex plays some roles in IGF-I-induced vesicle formation through Rab5 activation leading to macropinocytosis.

Nothing to Disclose: SIT, TY, YY, TM, HO, KC, FH

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