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