DIP2 disco-interacting protein 2 homolog A (Drosophila) is a candidate receptor for follistatin-related protein/follistatin-like 1--analysis of their binding with TGF-β superfamily proteins.
Follistatin-related protein (FRP)/follistatin-like 1 (FSTL1) is a member of the follistatin protein family, all of which share a characteristic structure unit found in follistatin, called the FS domain. Developmental studies have suggested that FRP regulates organ tissue formation in embryos. Immunological studies showed that FRP modifies joint inflammation in arthritic disease, and modulates allograft tolerance. However, the principle physiological function of FRP is currently unknown. To address this issue, we cloned four FRP-associated proteins using a two-hybrid cloning method: disco-interacting protein 2 homolog A from Drosophila (DIP2A), CD14, glypican 1 and titin. Only DIP2A was expected to be a membrane receptor protein with intracellular regions. Over-expression of FLAG epitope-tagged DIP2A augmented the suppressive effect of FRP on FBJ murine osteosarcoma viral oncogene homolog (FOS) expression, and the Fab fragment of IgG to FLAG blocked this effect. Knockdown of Dip2a leaded to Fos gene up-regulation, and this was not affected by exogenous FRP. These in vitro experiments confirmed that DIP2A could be a cell-surface receptor protein and mediate a FOS down-regulation signal of FRP. Moreover, molecular interaction analyses using Biacore demonstrated that FRP bound to DIP2A and CD14, and also with proteins of the TGF-β superfamily, i.e. activin, TGF-β, bone morphogenetic protein 2/4 (BMP-2/4), their receptors and follistatin. FRP binding to DIP2A was blocked by CD14, follistatin, activin and BMP-2. FRP blocked the ligand-receptor binding of activin and BMP-2, but integrated itself with that of BMP-4. This multi-specific binding may reflect the broad physiological activity of FRP.
Tanaka
Masao
M
Division of Hematology and Immunology, Department of Internal Medicine, Kanazawa Medical University, Ishikawa, Japan. masatana@kanazawa-med.ac.jp
Murakami
Kosaku
K
Ozaki
Shoichi
S
Imura
Yoshitaka
Y
Tong
Xiao-Peng
XP
Watanabe
Takuo
T
Sawaki
Toshioki
T
Kawanami
Takafumi
T
Kawabata
Daisuke
D
Fujii
Takao
T
Usui
Takashi
T
Masaki
Yasufumi
Y
Fukushima
Toshihiro
T
Jin
Zhe-Xiong
ZX
Umehara
Hisanori
H
Mimori
Tsuneyo
T
D002352:Carrier Proteins / Q000378:metabolism*; D018076:DNA, Complementary; D038702:Follistatin-Related Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos; D011956:Receptors, Cell Surface; D055411:TGF-beta Superfamily Proteins / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; C481541:DIP2A protein, human; D018076:DNA, Complementary; D038702:Follistatin-Related Proteins; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D011956:Receptors, Cell Surface; D055411:TGF-beta Superfamily Proteins