A proteomic approach for the elucidation of the specificity of ectodomain shedding.
Ectodomain shedding (shedding) is a posttranslational modification mechanism, which liberates extracellular domains of membrane proteins through juxtamembrane processing. Because shedding alters cell characteristics in a rapid and irreversible manner, it must be strictly regulated. However, the regulatory mechanisms of shedding in response to environmental changes remain obscure. To evaluate the regulatory mechanisms of endogenous shedding, we previously developed a proteomic screening system to identify shedding targets. This system revealed a comprehensive picture of membrane proteins shed under defined conditions. In this study, we have improved the screening system to compare the shedding patterns in a mouse macrophage cell line treated with two different shedding inducers, lipopolysaccharide (LPS) and 12-O-tetradecanoylphorbol 13-acetate (TPA). We show here that LPS simultaneously activates the shedding of multiple membrane proteins. We further show that TPA specifically activates the shedding of αM/β2 integrin (Mac-1), which was not shed upon LPS-stimulation of macrophages. These results clearly demonstrate that the regulation of endogenous membrane protein shedding is both stimulus- and substrate-specific.


CONCLUSIONS
The shedding targets reported to date play pivotal roles in a variety of biological phenomena, including the immunological response, cell growth, cell adhesion and cell movement. In addition, several disease-related membrane proteins are shedding targets. Thus, understanding the regulation of shedding is important for the elucidation of pathogenesis and the development of therapeutic strategies. We submit that a comprehensive characterization of endogenous shedding is indispensable for understanding the regulatory mechanisms of shedding, and thus have developed a proteomic screening system to identify shedding targets. In this study, using our screening system, we demonstrate that different extracellular stimuli activate different types of shedding, even in a single cell. Our results prove that this proteomic approach is quite effective for the elucidation of the regulatory mechanisms of shedding.
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: kshira@z8.keio.jp.
Kyoko
K
Shirakabe
Division of Biochemistry, School of Pharmaceutical Science, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Yoshio
Y
Shibagaki
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
Akihiko
A
Yoshimura
Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; Laboratory for Immune Cell System, RCAI, RIKEN Center for Integrative Medical Sciences (IMS), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.
Shigeo
S
Koyasu
Division of Biochemistry, School of Pharmaceutical Science, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
Seisuke
S
Hattori
D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D002460:Cell Line; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017434:Protein Structure, Tertiary; D040901:Proteomics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002273:Carcinogens; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D013755:Tetradecanoylphorbol Acetate
2D-DIGE; ADAMs; Ectodomain shedding; Mac-1; Macrophage