The ALK2 interaction network in U2OS cells

Summary of ALK2 interactors identified in U2OS cells


Discussion: theintracellular ALK2 interaction network in U2OS cells

  • Apart from typical signaling components, the ALK2 interaction network shows a range of regulatory proteins involved in receptor sorting and turnover
  • These include classical chaperones, components of the ubiquitination pathway and the ERAD pathway
  • The presence of these interactors, together with the increase in regulatory interactions after BMP4 stimulations, suggest a tight regulation of ALK2 in U2OS cells
  • The increase in regulatory proteins which bind to ALK2 after BMP4 stimulation also suggests that ALK2 is rapidly internalized and degraded after activation
  • Contrary to wild type ALK2, the FOP (Fibrodysplasia ossificans progressiva) mutant variant R206H apparently does not interact with components of the ubiquitination and ERAD pathways
  • This suggests that ALK2 R206H may be mis-regulated and may not be removed from the cell surface after activation with BMP4

A modelfor ALK2 R206H hyperactivation in FOP

  • The commonly accepted model for FOP postulates a hyperactivation of ALK2 R206H, which leads to differentiation of inflamed tissues into bone
  • Our observation that the R206H mutant fails to interact with many components of the degradation machinery points to a possible explanation for receptor hyperactivity
  • Failure to remove the mutated ALK2 receptor from the surface after BMP4 stimulation may lead to accumulation of active receptor on the cell surface and therefore continued signaling into the downstream network
  • As the ALK2 R206H mutant is capable of correctly inducing downstream signaling in Hek293 cells via both the Smad and p38 pathways, accumulated active ALK2 R206H may „hyperactivate“ these pathways, pushing the cells towards differentiation into bone cells






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

Klonierung 34

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

Etablierung 37

  • 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

Etablierung 37

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


Introduction FOP

Introduction to Fibrodysplasia ossificans progressiva (FOP, Münchmeyer syndrome)

Fibrodysplasia ossificans progressiva (FOP, Münchmeyer syndrome)

  • First mentioned in England around 1740
  • Orphan disease, approx. one patient per 2 million individuals
  • Approx. 600 cases are documented
  • Autosomal dominant
  • Average life span: 45 years
  • Early indicator: deformed big toes


  • Progressive incidents of inflammation, followed by differentiation of inflamed tissue into bone
  • Patients evolve a “second skeleton”, leading to progressive immobilization and eventually, death
  • In 2006, a landmark study showed that a point mutation in the ALK2 receptor is linked to FOP (Shore et al., 2006)
  • ACVR1/ALK2 is part of the family of type I BMP receptors (BMPRs)
  • BMPRs play a crucial role in bone formation during development
  • The study suggests that the identified ALK2 R206H mutation leads to receptor hyperactivation