Insulin-like growth factor-binding protein 5 (IGFBP-5) interacts with a four and a half LIM protein 2 (FHL2).
Recent studies using insulin-like growth factor I (IGF-I) knockout mice demonstrate that IGF-binding protein (IGFBP)-5, an important bone formation regulator, itself is a growth factor with cellular effects not dependent on IGFs. Because IGFBP-5 contains a nuclear localization sequence that mediates transport of IGFBP-5 into the nucleus, we propose that IGFBP-5 interacts with nuclear proteins to affect transcription of genes involved in bone formation. We therefore undertook studies to identify proteins that bind to IGFBP-5 using IGFBP-5 as bait in a yeast two-hybrid screen of a U2 human osteosarcoma cDNA library. Five related clones that interacted strongly with the bait corresponded to the FHL2 gene, which contains four and a half LIM domains. Co-immunoprecipitation studies with lysates from U2 cells overexpressing FHL2 and IGFBP-5 confirmed that interaction between IGFBP-5 and FHL2 occurs in whole cells. In vitro interaction studies revealed that purified FHL2 interacted with IGFBP-5 but not with IGFBP-3, -4, or -6. Northern blot analysis showed that FHL2 was strongly expressed in human osteoblasts. Nuclear localization of both FHL2 and IGFBP-5 was evident from Western immunoblot analysis and immunofluorescence. The role of FHL2 as an intracellular mediator of the effects of IGFBP-5 and other osteoregulatory agents in osteoblasts will need to be verified in future studies.
Amaar
Yousef G
YG
Musculoskeletal Disease Center, Jerry L. Pettis Veterans Affairs Medical Center, Loma Linda, California 92357, USA.
Thompson
Garrett R
GR
Linkhart
Thomas A
TA
Chen
Shin-Tai
ST
Baylink
David J
DJ
Mohan
Subburaman
S
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000378:metabolism; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000737:chemistry* / Q000378:metabolism*; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000378:metabolism; D060850:LIM-Homeodomain Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; C105987:FHL2 protein, human; C491382:Fhl2 protein, mouse; D018398:Homeodomain Proteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D018975:Insulin-Like Growth Factor Binding Protein 5; D018976:Insulin-Like Growth Factor Binding Protein 6; D060850:LIM-Homeodomain Proteins; D009124:Muscle Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors