6 research outputs found
Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4
Fibroblast growth
factor 1 (FGF1) binds to specific FGF receptors
(FGFRs) at the surface of target cells to initiate intracellular signaling.
While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors,
it is uncertain whether there are additional binding sites for FGF1
at the cell surface. To address this, we devised and tested a method
to identify novel binding sites for FGF1 at the cell surface, which
may also be applicable for other protein ligands. We constructed an
APEX2–FGF1 fusion protein to perform proximal biotin labeling
of proteins following binding of the fusion protein to the cell surface.
After functional validation of the fusion protein by a signaling assay,
we used this method to identify binding sites for FGF1 on cell surfaces
of living cells. We confirmed the feasibility of our approach by detection
of FGFR4, a well-known and specific receptor for FGF1. We subsequently
screened for novel interactors using RPE1 cells and identified the
proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44
through its heparin-binding moiety. Moreover, we found that FGF1 was
colocalized with both CSPG4 and CD44 at the cell surface, suggesting
that these receptors act as storage molecules that create a reservoir
of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2
fusion proteins can be used to identify novel receptor interactions
on the cell surface
Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4
Fibroblast growth
factor 1 (FGF1) binds to specific FGF receptors
(FGFRs) at the surface of target cells to initiate intracellular signaling.
While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors,
it is uncertain whether there are additional binding sites for FGF1
at the cell surface. To address this, we devised and tested a method
to identify novel binding sites for FGF1 at the cell surface, which
may also be applicable for other protein ligands. We constructed an
APEX2–FGF1 fusion protein to perform proximal biotin labeling
of proteins following binding of the fusion protein to the cell surface.
After functional validation of the fusion protein by a signaling assay,
we used this method to identify binding sites for FGF1 on cell surfaces
of living cells. We confirmed the feasibility of our approach by detection
of FGFR4, a well-known and specific receptor for FGF1. We subsequently
screened for novel interactors using RPE1 cells and identified the
proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44
through its heparin-binding moiety. Moreover, we found that FGF1 was
colocalized with both CSPG4 and CD44 at the cell surface, suggesting
that these receptors act as storage molecules that create a reservoir
of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2
fusion proteins can be used to identify novel receptor interactions
on the cell surface
Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4
Fibroblast growth
factor 1 (FGF1) binds to specific FGF receptors
(FGFRs) at the surface of target cells to initiate intracellular signaling.
While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors,
it is uncertain whether there are additional binding sites for FGF1
at the cell surface. To address this, we devised and tested a method
to identify novel binding sites for FGF1 at the cell surface, which
may also be applicable for other protein ligands. We constructed an
APEX2–FGF1 fusion protein to perform proximal biotin labeling
of proteins following binding of the fusion protein to the cell surface.
After functional validation of the fusion protein by a signaling assay,
we used this method to identify binding sites for FGF1 on cell surfaces
of living cells. We confirmed the feasibility of our approach by detection
of FGFR4, a well-known and specific receptor for FGF1. We subsequently
screened for novel interactors using RPE1 cells and identified the
proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44
through its heparin-binding moiety. Moreover, we found that FGF1 was
colocalized with both CSPG4 and CD44 at the cell surface, suggesting
that these receptors act as storage molecules that create a reservoir
of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2
fusion proteins can be used to identify novel receptor interactions
on the cell surface
Proximity Labeling by a Recombinant APEX2–FGF1 Fusion Protein Reveals Interaction of FGF1 with the Proteoglycans CD44 and CSPG4
Fibroblast growth
factor 1 (FGF1) binds to specific FGF receptors
(FGFRs) at the surface of target cells to initiate intracellular signaling.
While heparan sulfate proteoglycans (HSPGs) are well-described coreceptors,
it is uncertain whether there are additional binding sites for FGF1
at the cell surface. To address this, we devised and tested a method
to identify novel binding sites for FGF1 at the cell surface, which
may also be applicable for other protein ligands. We constructed an
APEX2–FGF1 fusion protein to perform proximal biotin labeling
of proteins following binding of the fusion protein to the cell surface.
After functional validation of the fusion protein by a signaling assay,
we used this method to identify binding sites for FGF1 on cell surfaces
of living cells. We confirmed the feasibility of our approach by detection
of FGFR4, a well-known and specific receptor for FGF1. We subsequently
screened for novel interactors using RPE1 cells and identified the
proteoglycans CSPG4 (NG2) and CD44. We found that FGF1 binds CD44
through its heparin-binding moiety. Moreover, we found that FGF1 was
colocalized with both CSPG4 and CD44 at the cell surface, suggesting
that these receptors act as storage molecules that create a reservoir
of FGF1. Importantly, our data demonstrate that recombinant ligand–APEX2
fusion proteins can be used to identify novel receptor interactions
on the cell surface
Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport
The fibroblast growth factor receptors
(FGFRs) are important oncogenes
promoting tumor progression in many types of cancer, such as breast,
bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma.
However, little is known about how these receptors are internalized
and down-regulated in cells. We have here applied proximity biotin
labeling to identify proteins involved in FGFR4 signaling and trafficking.
For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal
tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed
in U2OS cells. Upon addition of biotin to these cells, proteins in
proximity to the FGFR4-BirA* fusion protein became biotinylated and
could be isolated and identified by quantitative mass spectrometry.
We identified in total 291 proteins, including 80 proteins that were
enriched in samples where the receptor was activated by the ligand
(FGF1), among them several proteins previously found to be involved
in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly,
many of the identified proteins were implicated in endosomal transport,
and by precise annotation we were able to trace the intracellular
pathways of activated FGFR4. Validating the data by confocal and three-dimensional
structured illumination microscopy analysis, we concluded that FGFR4
uses clathrin-mediated endocytosis for internalization and is further
sorted from early endosomes to the recycling compartment and the trans-Golgi
network. Depletion of cells for clathrin heavy chain led to accumulation
of FGFR4 at the cell surface and increased levels of active FGFR4
and PLCγ, while AKT and ERK signaling was diminished, demonstrating
that functional clathrin-mediated endocytosis is required for proper
FGFR4 signaling. Thus, this study reveals proteins and pathways involved
in FGFR4 transport and signaling that provide possible targets and
opportunities for therapeutic intervention in FGFR4 aberrant cancer
Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport
The fibroblast growth factor receptors
(FGFRs) are important oncogenes
promoting tumor progression in many types of cancer, such as breast,
bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma.
However, little is known about how these receptors are internalized
and down-regulated in cells. We have here applied proximity biotin
labeling to identify proteins involved in FGFR4 signaling and trafficking.
For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal
tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed
in U2OS cells. Upon addition of biotin to these cells, proteins in
proximity to the FGFR4-BirA* fusion protein became biotinylated and
could be isolated and identified by quantitative mass spectrometry.
We identified in total 291 proteins, including 80 proteins that were
enriched in samples where the receptor was activated by the ligand
(FGF1), among them several proteins previously found to be involved
in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly,
many of the identified proteins were implicated in endosomal transport,
and by precise annotation we were able to trace the intracellular
pathways of activated FGFR4. Validating the data by confocal and three-dimensional
structured illumination microscopy analysis, we concluded that FGFR4
uses clathrin-mediated endocytosis for internalization and is further
sorted from early endosomes to the recycling compartment and the trans-Golgi
network. Depletion of cells for clathrin heavy chain led to accumulation
of FGFR4 at the cell surface and increased levels of active FGFR4
and PLCγ, while AKT and ERK signaling was diminished, demonstrating
that functional clathrin-mediated endocytosis is required for proper
FGFR4 signaling. Thus, this study reveals proteins and pathways involved
in FGFR4 transport and signaling that provide possible targets and
opportunities for therapeutic intervention in FGFR4 aberrant cancer