Journal: bioRxiv

Article Title: PTK7 is a positive allosteric modulator of GPR133 (ADGRD1) signaling in GBM

doi: 10.1101/2022.06.15.496232

Figure Lengend Snippet: A) Validation assay to test whether 6 of the top interactors identified in the screen can be affinity co-purified with TwinStrep-tagged GPR133-H543R in HEK293T cells. All candidate interactors were tagged with the Myc epitope. Ai) Western blot stained against Myc-tag and GPR133 (antibody against the cytosolic C-terminus) of whole cell lysates. Aii) Western blots of eluates after StrepTactin purification. Note that PTK7, and to a lesser extent TFRC, co-purify with GPR133. A representative blot of three biological repeats is depicted. B) Membrane topology of PTK7. Note that the juxtamembrane portion of PTK7 is cleaved by MT1-MMP (membrane type 1 matrix metalloproteinase) and ADAMs (a disintegrin and metalloproteinases). C) PTK7 is identified as one of the top interactors on the same volcano plot as depicted in . D) Normalized intensities of peptides corresponding to GPR133 and PTK7 as detected in the mass spectrometry analysis in , detailing all biological replicates. E) Comparison of the GPR133 binding interaction of PTK7 vs. the previously reported ligand PLXDC2 using the StrepTactin purification paradigm. PTK7 and PLXDC2 (prey) were tagged with the Myc epitope for detection, while GPR133-H543R (bait) was tagged at the C-terminus with TwinStrep tag for purification. Note that PTK7 co-purifies with GPR133, while PLXDC2 does more co-purify at detectable amounts under the same experimental conditions.

Article Snippet: Ligand candidate cDNAs were obtained from the following commercially available plasmids: PTK7 (Addgene, Cat#65250), PCDHGC3 (SinoBiological, Cat#HG22064-UT), TFRC (Addgene, Cat#69610), NECTIN2 (SinoBiological, Cat#HG10005-G), CADM4 (SinoBiological, Cat#HG16033-G), MCAM (SinoBiological, Cat#HG10115-M), PLXDC2 (SinoBiological, Cat#HG23536-U).

Techniques: Purification, Western Blot, Staining, Mass Spectrometry, Binding Assay