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Abstract: SA-PO997

Angiotensin II Type 1 Receptor-Associated Protein Interacts With Transferrin Receptor 1 and Promotes Its Internalization

Session Information

  • CKD: Pathobiology - II
    November 05, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Abe, Eriko, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Wakui, Hiromichi, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Hirota, Keigo, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Takahiro, Yamaji, Duke-NUS Medical School, Singapore, Singapore
  • Urate, Shingo, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Suzuki, Toru, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Tanaka, Shohei, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Taguchi, Shinya, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Tsukamoto, Shunichiro, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Azushima, Kengo, Yokohama City University Graduate School of Medicine, Yokohama, Japan
  • Tamura, Kouichi, Yokohama City University Graduate School of Medicine, Yokohama, Japan

Group or Team Name

  • Department of Medical Science and Cardiorenal Medicine
Background

Angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP) was originally identified as a binding protein of AT1R. ATRAP promotes constitutive internalization of AT1R so as to inhibit the pathological activation of its downstream signaling. Also, we reported that genetic knockdown of ATRAP exacerbates kidney fibrosis in mice along with functional mitochondrial abnormalities and subsequent increases in ROS production. These effects of ATRAP were suggested to be AT1R-independent actions. Thus,this study aimed to explore a novel interacting protein involved in the mechanism that ATRAP protects against kidney fibrosis independent of the interaction with AT1R.

Methods

We established Human Embryonic Kidney 293 cells which were able to induce the expression of Flag-ATRAP. In this cell line, after immunoprecipitation with anti-Flag antibodies, the Flag-ATRAP complex was analyzed with a mass spectrometer. Among identified proteins, we focused on transferrin receptor1(TfR1). To confirm the molecular interaction, co-immunoprecipitation was performed. Additionally, to validate functional interactions, we analyzed intracellular iron concentrations using fluorescent probe of iron. Furthermore, to verify TfR1 expression and localization, immunofluorescence staining of TfR1 in the whole cell or on the cell surface only.

Results

Mass spectrometry analysis revealed various proteins associated with vesicular trafficking including TfR1.We confirmed the molecular interaction between ATRAP and TfR1 by co-immunoprecipitation.Enhanced ATRAP expression decreased the cellular iron levels and downregulate TfR1 expression on the cell surface despite no significant difference in whole cell.

Conclusion

We propose a molecular and functional link between ATRAP and TfR1. ATRAP would regulate TfR1 availability via downregulation of cell surface TfR1 via promotion of its internalization. TfR1 promotes intracellular localization of the iron-bound transferrin. Iron is a key factor in the process of kidney fibrosis via production of ROS in relation to deterioration of mitochondrial function. Taken together, this novel ATRAP-TfR1 axis might be the mechanism relevant to the ROS/mitochondrial dysfunction-mediated process of kidney fibrosis.