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Analysis using LRC BMP4

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, insulin-TRICEPS was used
  • As shown, BMP4 was successfully labeled with TRICEPS

fop-65Cluster analysisof LRC datasets

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

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Volcanoplotofreceptorsidentifiedwith BMP4

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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