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Ligand-induced transphosphorylation between different FGF receptors
F Bellot1, G Crumley, J M Kaplow
1Molecular Biology Department, Rhône-Poulenc Rorer Central Research, King of Prussia, PA 19406.
Abstract:
Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co-express a kinase-negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase-negative mutant of full length bek (bek K517A), (ii) cells which express wild-type bek together with kinase-negative flg (flg K514A) and (iii) cells co-expressing wild-type flg together with bek K517A. Immunoprecipitations with either bek-or flg-specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti-FGFR antibodies and immunoblotting with anti-phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase-negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase-negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek----bek) and heterologous (bek----flg and flg----bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.
Insights
Fibroblast growth factor receptors (FGFR) undergo transphosphorylation, where a kinase-negative FGFR mutant is activated by a co-expressed active FGFR. This mechanism, involving both homologous and heterologous receptor interactions, is crucial for cell signaling.
Area of Science:
- Molecular Biology
- Cell Signaling
- Receptor Tyrosine Kinases
Background:
- Fibroblast growth factors (FGFs) bind to fibroblast growth factor receptors (FGFRs) with high affinity.
- FGFR tyrosine autophosphorylation is critical for downstream signaling.
- The precise mechanism of FGFR autophosphorylation, especially in co-expression systems, requires further elucidation.
Purpose of the Study:
- To investigate the mechanism of fibroblast growth factor receptor (FGFR) tyrosine autophosphorylation.
- To determine if kinase-negative FGFR mutants can be phosphorylated by co-expressed active FGFRs.
- To explore homologous and heterologous transphosphorylation between FGFRs in living cells.
Main Methods:
- Generation of NIH 3T3 cell lines co-expressing different combinations of wild-type and kinase-negative FGFR mutants (bek and flg).
- Immunoprecipitation using FGFR-specific antisera to confirm receptor expression.
- Stimulation with acidic FGF (aFGF) followed by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibodies to detect phosphorylation.
Main Results:
- Co-expression of active and kinase-negative FGFR mutants was confirmed in transfected NIH 3T3 cells.
- Acidic FGF stimulation induced tyrosine phosphorylation of kinase-negative FGFR mutants in all tested cell lines.
- Both homologous (bek-bek, flg-flg) and heterologous (bek-flg, flg-bek) transphosphorylation of FGFRs were observed in living cells.
Conclusions:
- Tyrosine autophosphorylation of kinase-negative FGFR mutants occurs via a transphosphorylation mechanism.
- Heterologous transphosphorylation between different FGFRs (bek and flg) happens in living cells.
- Heterologous transphosphorylation may amplify cellular responses to various FGFs and their receptors.