Tag Archive for: FOP

Analysis of BMP4 interactions using LRC

• The ligand-receptor capture technique allows identification of cell surface receptors which interact with a ligand of interest
• Importantly, LRC allows detection of these interactions on living cells or tissues
• The ligand is modified with a trifunctionalcrosslinker (TRICEPS) and incubated with target cells
• Interaction of the ligand with its target receptor(s) leads to specific crosslinking between ligand and receptor
• Ligand-receptor complexes are purified and subjected to tandem mass spectrometry to identify the receptor(s) bound by the ligand
• LRC was used in this study to identify potential novel receptors of BMP4 in the context of FOP (Fibrodysplasia ossificans progressiva)

Labeling of BMP4 with TRICEPS

• Purified BMP4 was labeled with TRICEPS
• To determine whether labeling was successful, the BMP4-TRICEPS conjugate was tested via Dot blotting using HRP-conjugated streptavidin
• As control,>INS-TRICEPS was used
• As shown, BMP4 was successfully labeled with TRICEPS

Cluster analysisof LRC datasets

• Triplicate LRC datasets are shown, either from BMP4 purifications or>INS control purifications

Volcanoplotofreceptorsidentifiedwith BMP4

Results: Analysis of BMP4 interactions using LRC

• LRC analysis using BMP4 yielded a total of 7 receptors
• ALK2 was not identified
• 4 receptors are involved in cell surface protein sorting and degradation:

Lysosome membrane protein 2 (SCRB2)
Lysosome-associated membrane glycoprotein 1 (LAMP1)
Endoplasmin (ENPL)
HYOU1

Analysis of ALK2 interactions in U2O Scells

• To characterize interactions involving ALK2 in U2OS cells, transient transfection was used to express wild type or R206H variants of ALK2
• Transiently expressing U2OS were either used unstimulated, or were stimulated with BMP4
• Datasets from unstimulated and BMP4 stimulated cells were compared to identify changes in the protein interaction network after activation of the BMP pathway

Pilot purification experiments using U2OS cells

• Cells were transiently transfected with ALK2 wild type expression constructs
• Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
• Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells

• Cells were transiently transfected with ALK2 R206H expression constructs
• Transiently expressing cells were either used as non-stimulated control or stimulated with BMP4
• Small scale pilot purifications were carried out according to the optimized protocol developed with Hek293 cells

 

Large scale purification of ALK2 from unstimulated U2OS cells

• Volcano-Plot of final merged datasets for ALK2 R206H and control
• Significantly enriched complex partners for ALK2 R206H are boxed

• Primary dataset of large scale purifications for ALK2 wild type
• Triplicate purifications are shown for ALK2 wild type and control
• Color coding shows variations in relative abundance of complex partners identified

• Volcano-Plot of final merged datasets for ALK2 wild type and control
• Significantly enriched complex partners for ALK2 wild type are boxed

• Scatter plots of triplicate purifications are shown
• Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

• Primary dataset of large scale purifications for ALK2 R206H
• Triplicate purifications are shown for ALK2 R206H and control
• Color coding shows variations in relative abundance of complex partners identified

• Volcano-Plot of final merged datasets for ALK2 R206H and control
• Significantly enriched complex partners for ALK2 R206H are boxed

• Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
• Control purifications and ALK2 wild type purifications are shown to demonstrate reproducibility

• Primary dataset of large scale purifications for ALK2 wild type from BMP4 stimulated cells
• Triplicate purifications are shown for ALK2 wild type and control
• Color coding shows variations in relative abundance of complex partners identified

• Volcano-Plot of final merged datasets for ALK2 wild type and control BMP4 stimulated cells
• Significantly enriched complex partners for ALK2 wild type are boxed

• Scatter plots of triplicate purifications from BMP4 stimulated cells are shown
• Control purifications and ALK2 R206H purifications are shown to demonstrate reproducibility

 

• Primary dataset of large scale purifications for ALK2 R206H from BMP4 stimulated cells
• Triplicate purifications are shown for ALK2 R206H and control
• Color coding shows variations in relative abundance of complex partners identified

• Volcano-Plot of final merged datasets for ALK2 R206H and control BMP4 stimulated cells
• Significantly enriched complex partners for ALK2 R206H are boxed

 

 Results: Purification of ALK2 complexes from U2OS cells

• For ALK2 wild type purified from unstimulated U2OS cells, 8 interactors were identified
• Interactors were mainly involved in receptor sorting, recycling and degradation
• Stimulation of U2OS cells with BMP4 led to the identification of 15 interactors
• 9 additional interactors were identified upon BMP4 stimulation, one interaction was lost
• The ALK2 R206H mutant showed a strongly reduced interaction network: only one interactor was identified
• Stimulation with BMP4 did not change the interaction network of ALK2 R206H

 Comparison of ALK2 wild type and R206H interactors identified

Graphical representation of ALK2 interaction networks

• Merged interaction network of all ALK2 complexes identified (wild type vs R206H; unstimulated vs stimulated)
• Graphical representation was done using Cytoscape

Summary: ALK2interaction network in U2OScells

• The interaction network of ALK2 in U2OS cells shows a large number of proteins involved in receptor sorting and turnover
• Interactors include chaperones, components of the ubiquitination pathway and components of the ERAD pathway
• Stimulation with BMP4 leads to additional interaction of ALK2 with several lysosomal proteins and E3 ubiquitin ligases
• In contrast, the mutant ALK2 R206H does not show interactions with components of either the ubiquitination or ERAD pathways
• The only identified interaction involving ALK2 R206H is with the calcium binding protein calnexin (CANX)

 

Investigation of ALK2 interactions in Hek293 cells

The following BMP4 signal cascade components were chosen for investigation:

• ALK2 wild type receptor
• ALK2 mutated receptors:
  R206H (causes FOP (Fibrodysplasia ossificans progressiva))
  Q207D (constitutive active)
  K235R (dominant negative)
• ALK6 wild type receptor
• ALK6 mutated receptor (I200K): causes brachydactyl (shortness of the fingers and toes)
• BMPRII: type II receptor
• FKBP12

Construction of novel expression vectors

Novel expression/purification vectors were generated to stably express the selected baits:

• Strep II and HA epitope tags for detection and purification
• Inducible tet promoter for regulated expression
• Selection marker for stable cell line generation

An additional expressionvector was constructedfortheexpressionof type I integral membraneproteinscarrying a cleavable N-terminal signalsequence:

Expression constructs

Transfection and establishment of stable Hek293 cells

• Hek293 cells were transfected with the following constructs using the CaPo method:
  ALK2wt (wild type)
  ALK2 Q207D
  ALK2 K236R
  ALK2 R206H
• Stable lines were selected using hygromycine
• Expressed bait proteins were detected by Western blotting using an antibody against the HA tag
• Extraction and purification were optimized using different detergent mixes to ensure optimal recovery of transmembrane receptors
• Establishment of 8 stable Hek293 lines expressing key components of the BMP signaling cascade
• Optimization of purification procedure to ensure reproducible capture of interactors
• Western blots of representative purifications are shown below

• Optimization of purification procedure to ensure reproducible capture of interactors
• Western blots comparing purification of ALK6 wild type (wt) and I200K mutant under different conditions from stable Hek293 lines

Assessing the BMP signaling pathway in stable Hek293 lines

• Stable Hek293 lines were stimulated with the ligand BMP4 for 30 mins
• Phosphorylation of Smad proteins was assessed using a phospho-specific antibody at the indicated time points
• Stimulation was compared across the ALK2 wild type and mutant receptors
• „Tet“ indicates whether expression of the receptor was induced by tetracycline (+) or whether uninduced cells were used (-)

Purification of receptor complexes from stable Hek293 lines

• Protein complexes around the bait proteins were purified from stable Hek293 lines using the optimized large scale affinity purification procedure
• All purifications were carried out in triplicate using the Strep tag fused to the baits
• Purified protein complexes were digested with trypsin and peptides were subjected to Nano-LC separation, followed by tandem mass spectrometry analysis on a LTQ Orbitrap
• Triplicate datasets were analyzed using a custom bioinformatics pipeline and interaction networks were generated using the software Cytoscape

Example of an interactor list generated from a purification of ALK2 wt

Summary of purifications carried out on 8 stable Hek293 lines

Identifiedinteractorsarelisted in thetablebelow

• ALK2 wild type purifications yielded several known and novel interactors
• ALK2 Q207D purifications yielded several interactors which overlap substantially with ALK2 wild type interactors
• No interactions were identified for ALK2 R206H and K236R, or the BMPRII and ALK6 receptors
• FKBP12 purifications yielded one novel interactor, TMX1
• Interactors identified for ALK2 are mostly components involved in receptor sorting, turnover and receptor internalization

Graphical representation of ALK2 interactions in Hek293 cells