Phospho-FGFR1 (Y766) Antibody

What is FGFR1 and what is the significance of Y766 phosphorylation?

FGFR1 (Fibroblast Growth Factor Receptor 1) is a tyrosine-protein kinase that acts as a cell-surface receptor for fibroblast growth factors and plays essential roles in embryonic development, cell proliferation, differentiation, and migration. It is widely expressed in various tissues including the brain, skeletal muscle, and the cardiovascular system .

Y766 is a specific phosphorylation site located in the kinase tail of FGFR1. When phosphorylated, this site serves as a critical binding site for Phospholipase C gamma (PLCγ) . This phosphorylation event is significant because it activates PLCγ, leading to the production of important cellular signaling molecules: diacylglycerol and inositol 1,4,5-trisphosphate . Notably, Y766 is the fourth site to be phosphorylated in the ordered sequence of FGFR1 transphosphorylation events (Y653, Y583, Y463, Y766, Y585, and Y654) .

What signaling pathways are activated by phosphorylation of FGFR1 at Y766?

Phosphorylation of FGFR1 at Y766 primarily activates the PLCγ pathway. When PLCγ binds to phosphorylated Y766, it becomes activated and catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) . These second messengers trigger distinct downstream events:

• DAG activates protein kinase C (PKC), influencing various cellular processes

• IP3 stimulates calcium release from intracellular stores

Additionally, Y766 phosphorylation contributes to MAPK pathway activation. Studies using Y766F mutation (which prevents phosphorylation at this site) have shown reduced FGF2-induced activation of PLCγ and diminished FGFR1 activation of ERK1/2 . This indicates that while PLCγ activation is fully dependent on Y766 phosphorylation, MAPK pathway activation is partially mediated through this site but also involves other phosphorylation sites.

How can researchers detect and validate FGFR1 Y766 phosphorylation in experimental systems?

Detecting FGFR1 Y766 phosphorylation requires specific methodological approaches:

Antibody-based detection methods:

• Western blotting (WB): The primary method for quantitatively assessing Y766 phosphorylation levels in cell or tissue lysates

• Immunohistochemistry on paraffin-embedded sections (IHC-P): For visualizing spatial distribution of phosphorylated FGFR1 in tissues

Validation approaches:

• Y766F mutant expression: The most definitive control is expressing FGFR1 with a Y766F mutation, which eliminates the phosphorylation site. A specific antibody should show no signal in cells expressing this mutant after FGF stimulation

• Stimulation controls: Phospho-Y766 signal should increase with FGF concentration in a predictable manner

• Time-course experiments: Signal should follow expected kinetics, with rapid increases after stimulation followed by decreases due to receptor downregulation or dephosphorylation

Experimental design considerations:

• Include both positive controls (FGF-stimulated cells) and negative controls (unstimulated cells or Y766F mutant-expressing cells)

• Use multiple detection methods when possible to confirm findings

• Consider cross-reactivity with other phosphorylated tyrosine residues in FGFR1 or related receptors

How does mutation of Y766F affect downstream signaling pathways in different cell types?

The Y766F mutation of FGFR1 eliminates the phosphorylation site necessary for PLCγ binding and activation, with distinct effects across different experimental systems:

In urothelial cells (TERT-NHUC):

• Complete elimination of FGF2-induced activation of PLCγ

• Reduced FGFR1 activation of ERK1/2 compared to wild-type FGFR1, but not complete elimination

In BCR-FGFR1 fusion proteins (relevant to hematological malignancies):

In developmental contexts:

• Y766F mutations are compatible with survival but lead to alterations in anterior-posterior patterning of the vertebral column

• These alterations are in the opposite direction to hypomorphic FGFR1 alleles, suggesting phosphorylation of Y766 may play a role in negative regulation of certain FGFR1 functions during development

This variability in phenotypic outcomes suggests that Y766 phosphorylation has context-dependent functions that may differ between cell types, developmental stages, and disease states.

What are the differences in Y766 phosphorylation kinetics in response to different FGF ligands?

Different FGF ligands induce distinct patterns of FGFR1 phosphorylation at Y766, revealing ligand-specific signaling biases:

FGF8 shows a distinctive phosphorylation profile:

• It is biased against phosphorylation of Y653/654 (activation loop) and Y766

• Shows stronger bias toward phosphorylation of the adaptor protein FRS2

• Exhibits a smaller decrease in phosphorylation over time compared to FGF4 and FGF9

FGF4 displays unusual dose-response behavior:

• At high concentrations, FGF4 can lead to decreased Y766 phosphorylation

• The de-phosphorylation kinetics in response to FGF4 are similar to FGF9

• This suggests the decrease in phosphorylation at high FGF4 concentrations is not due to faster de-phosphorylation

FGF9 shows more typical dose-response patterns:

• Progressive increase in Y766 phosphorylation with increasing concentration

• Similar de-phosphorylation kinetics to FGF4 over time

These differences in phosphorylation patterns demonstrate that different FGF ligands can bias FGFR1 signaling toward specific downstream pathways, a concept known as ligand bias. This may explain how a single receptor can mediate diverse biological responses depending on which FGF ligand is present.

How does the Y766F mutation influence developmental phenotypes compared to hypomorphic FGFR1 mutations?

The Y766F mutation in FGFR1 produces developmental phenotypes that differ significantly from those caused by hypomorphic FGFR1 mutations:

Y766F mutation effects:

• Compatible with survival (unlike complete knockout)

• Leads to alterations in anterior-posterior patterning of the vertebral column

• These alterations are in the opposite direction to those observed with hypomorphic alleles

• Suggests a role for FGFR1 in patterning the embryonic anterior-posterior axis via regulation of Hox gene activity

Hypomorphic FGFR1 mutations:

This comparison reveals the complex roles of specific phosphorylation sites in FGFR1 function during development. The Y766 site appears to have dual roles - mediating positive signaling through PLCγ activation while also contributing to negative regulation of other FGFR1 functions, particularly those involved in axial skeletal patterning.

What critical factors should be considered when designing experiments to study FGFR1 Y766 phosphorylation?

When studying FGFR1 Y766 phosphorylation, researchers should consider several important methodological factors:

Temporal dynamics:

• Phosphorylation patterns change rapidly after stimulation

• Time course experiments (e.g., 0, 1, 5, 10, 20, and 60 min after stimulation) are essential to capture transient phosphorylation events

• Both fast phosphorylation and subsequent de-phosphorylation/downregulation should be monitored

Dose-response relationships:

• Different FGF ligands show distinct dose-response curves

• A broad range of ligand concentrations should be tested to fully characterize responses

• Some FGFs (like FGF4) show unexpected decreases in phosphorylation at high concentrations

Multiple readouts:

• Measure multiple phosphorylation sites simultaneously (e.g., Y653/654, Y766)

• Monitor both receptor phosphorylation and downstream effector phosphorylation (e.g., FRS2, PLCγ)

• Assess receptor downregulation by measuring total FGFR1 levels alongside phosphorylation

Normalization and quantification:

• When comparing different ligands, samples should be re-run on common gels for accurate scaling

• Quantification should involve multiple independent experiments (at least three)

• Standard errors should be calculated to assess variability

How can phospho-FGFR1 (Y766) antibodies be used to investigate FGFR1 signaling in disease models?

Phospho-FGFR1 (Y766) antibodies provide valuable tools for investigating FGFR1 signaling in various disease models:

In cancer research:

• Urinary bladder cancer: Studies have shown that FGFR1 has significant effects on urothelial cell phenotype and may represent a therapeutic target in some cases of urinary bladder cancer

• BCR-FGFR1 fusion proteins in hematological malignancies: These fusion proteins show altered signaling properties that can be characterized using phospho-Y766 antibodies

• Glioblastoma: Somatic gain-of-function mutations like R576W have been identified in glioblastoma that may affect FGFR1 phosphorylation patterns

Methodological approaches:

• Western blotting: For quantitative assessment of phosphorylation levels and pathway activation

• Immunohistochemistry: For spatial analysis of FGFR1 activation in tumor tissues

• Phospho-proteomics: For broader analysis of how Y766 phosphorylation impacts the global phospho-proteome

Experimental designs:

• Drug response studies: Monitoring Y766 phosphorylation before and after treatment with FGFR inhibitors

• Genetic manipulation: Comparing phosphorylation patterns in cells with wild-type FGFR1 versus disease-associated mutations

• Time-course analyses: Examining how disease states alter the dynamics of FGFR1 phosphorylation and downstream signaling

What is known about the structural basis of Y766 phosphorylation and PLCγ binding?

The structural aspects of Y766 phosphorylation and PLCγ binding reveal important mechanistic insights:

Receptor dimerization and phosphorylation:

• FGFR1 forms asymmetric dimers during activation, which appears important for transphosphorylation of sites including Y766

• Crystal structures show direct interactions between R577 of one molecule and D519 of another

• Mutations in these interface residues affect receptor function (D519N causes loss-of-function; R576W is a gain-of-function mutation found in glioblastoma)

Sequential phosphorylation:

• Y766 is the fourth site phosphorylated in the sequence: Y653, Y583, Y463, Y766, Y585, Y654

• This ordered process suggests hierarchical activation of different pathways

• The preceding phosphorylation events may be prerequisites for Y766 phosphorylation

PLCγ binding interaction:

• Phosphorylated Y766 serves as a docking site for the SH2 domains of PLCγ

• This interaction positions PLCγ for phosphorylation by the active FGFR1 kinase domain

• Crystal structures have helped elucidate these interaction details

How does FGFR1 Y766 phosphorylation interact with other signaling pathways to coordinate cellular responses?

FGFR1 Y766 phosphorylation interfaces with multiple signaling networks to coordinate complex cellular responses:

Integration with MAPK signaling:

• Y766 phosphorylation and PLCγ activation contribute to full MAPK pathway activation

• Inhibition of PLCγ activation by Y766F mutation reduces MAPK activation levels

• This creates a situation where PLCγ and MAPK pathways are partially interdependent

Cross-talk with FRS2-initiated signaling:

• While Y766 phosphorylation activates PLCγ, FRS2 phosphorylation triggers recruitment of GRB2, GAB1, PIK3R1, and SOS1

• These parallel pathways together coordinate RAS, MAPK, and AKT signaling

• Different FGF ligands show biases for either Y766 or FRS2 phosphorylation, enabling diverse cellular responses

Negative feedback mechanisms:

• Y766 phosphorylation may contribute to negative regulation of FGFR1 in certain contexts

• Y766F mutations cause developmental phenotypes opposite to those from hypomorphic mutations

• This suggests that in addition to activating PLCγ, signals from Y766 may downregulate other FGFR1 functions

• Such dual positive/negative roles could allow for precise regulation of signal duration and intensity