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The mitogenic effects of the B beta chain of fibrinogen are mediated through cell surface calreticulin
A J Gray1, P W Park, T J Broekelmann
1University College London Medical School, Division of Cardiopulmonary Biochemistry, United Kingdom.
Abstract:
We have previously shown that soluble partially degraded fibrin(ogen) remains in solution after fibrin clot formation and is a potent fibroblast mitogen (Gray, A.J., Bishop, J.E., Reeves J.T., Mecham, R.P., and Laurent, G.J. (1995) Am. J. Cell Mol. Biol. 12, 684-690). Mitogenic sites within the fibrin(ogen) molecule are located on the A alpha and B beta chains of the protein (Gray, A.J., Bishop, J. E., Reeves, J.T., and Laurent, G.J. (1993) J. Cell Sci. 104, 409-413). However, receptor pathways through which mitogenic effects are mediated are unknown. The present study sought to determine the nature of fibrin(ogen) receptors expressed on human fibroblasts which interact with the fibrinogen B beta chain. Receptor complexes were isolated from 125I-surface-labeled fibroblasts and purified on a fibrinogen B beta chain affinity column. Subsequent high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis analysis indicated fibrinogen B beta chain bound specifically to a 60-kDa surface protein. Sequence analysis of the amino terminus of this protein indicated 100% homology to human calreticulin. Immunoprecipitation experiments employing a polyclonal anti-calreticulin antibody provided further evidence that the 60-kDa protein isolated in this study was calreticulin. Further, polyclonal antibodies to human calreticulin significantly inhibited the mitogenic activity of fibrinogen B beta chain on human fibroblasts. The present study has shown that cell surface calreticulin binds to the B beta chain of fibrinogen mediating its mitogenic activity.
Insights
Soluble fibrinogen fragments stimulate fibroblast growth. Researchers identified cell surface calreticulin as the receptor that binds the fibrinogen B beta chain, mediating this mitogenic effect.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Soluble, partially degraded fibrinogen acts as a potent fibroblast mitogen.
- Mitogenic activity is localized to the A alpha and B beta chains of fibrinogen.
- The specific cell surface receptors and pathways mediating fibrinogen's mitogenic effects were previously unknown.
Purpose of the Study:
- To identify the fibrinogen receptor on human fibroblasts that interacts with the B beta chain.
- To elucidate the mechanism by which fibrinogen mediates fibroblast mitogenesis.
Main Methods:
- Isolation and purification of receptor complexes from 125I-surface-labeled human fibroblasts using a fibrinogen B beta chain affinity column.
- Analysis of purified proteins using high-performance liquid chromatography and SDS-polyacrylamide gel electrophoresis.
- Amino-terminal sequencing, immunoprecipitation with anti-calreticulin antibodies, and functional inhibition assays.
Main Results:
- A 60-kDa surface protein specifically bound the fibrinogen B beta chain.
- Sequence analysis revealed 100% homology to human calreticulin.
- Immunoprecipitation confirmed the 60-kDa protein as calreticulin.
- Anti-calreticulin antibodies significantly inhibited the mitogenic activity of the fibrinogen B beta chain.
Conclusions:
- Cell surface calreticulin binds to the B beta chain of fibrinogen.
- Calreticulin mediates the mitogenic activity of fibrinogen on human fibroblasts.