Related Experiment Videos
An active site of transforming growth factor-beta(1) for growth inhibition and stimulation
S S Huang1, M Zhou, F E Johnson
1Departments of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA. huangjs@slu.edu
Abstract:
Transforming growth factor-beta (TGF-beta) is a bifunctional growth regulator. It inhibits growth of many cell types, including epithelial cells, but stimulates growth of others (e.g. fibroblasts). The active site on the TGF-beta molecule, which mediates its growth regulatory activity, has not been defined. Here, we show that antibody to a TGF-beta(1) peptide containing the motif WSLD (52nd to 55th amino acid residues) completely blocked both (125)I-TGF-beta(1) binding to TGF-beta receptors and TGF-beta(1)-induced growth inhibition in mink lung epithelial cells. Site-directed mutagenesis analysis revealed that the replacement of Trp(52) and Asp(55) by alanine residues diminished the growth inhibitory activity of TGF-beta(1) by approximately 90%. Finally, while wild-type TGF-beta(1) was able to stimulate growth of transfected NIH 3T3 cells, the double mutant TGF-beta(1) W52A/D55A was much less active. These results support the hypothesis that the WSLD motif is an active site of TGF-beta(1), which is important for growth inhibition of epithelial cells and growth stimulation of fibroblasts.
Insights
The WSLD motif in transforming growth factor-beta 1 (TGF-beta 1) acts as an active site. This motif is crucial for TGF-beta 1
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Transforming growth factor-beta (TGF-beta) is a key regulator of cell growth, exhibiting bifunctional activity.
- TGF-beta inhibits epithelial cell proliferation but stimulates fibroblast growth.
- The specific active site on TGF-beta responsible for its regulatory functions remained undefined.
Purpose of the Study:
- To identify the active site of TGF-beta 1 responsible for its growth regulatory functions.
- To investigate the role of the WSLD motif in TGF-beta 1 binding and activity.
Main Methods:
- Antibody blockade using a peptide containing the WSLD motif.
- Radioiodinated TGF-beta 1 binding assays to TGF-beta receptors.
- Site-directed mutagenesis of the WSLD motif (W52A/D55A).
- Cell proliferation assays using mink lung epithelial cells and NIH 3T3 fibroblasts.
Main Results:
- Antibody against the WSLD motif blocked TGF-beta 1 binding to its receptors and inhibited TGF-beta 1-induced growth inhibition in epithelial cells.
- Mutagenesis of Trp52 and Asp55 to alanine significantly reduced TGF-beta 1's growth inhibitory activity (~90%).
- The TGF-beta 1 W52A/D55A double mutant showed markedly reduced ability to stimulate NIH 3T3 cell growth compared to wild-type TGF-beta 1.
Conclusions:
- The WSLD motif (amino acid residues 52-55) represents a critical active site of TGF-beta 1.
- This motif is essential for both the growth inhibitory effects of TGF-beta 1 on epithelial cells and its growth-stimulatory effects on fibroblasts.