The adhesion G-protein-coupled receptor GPR133 (ADGRD1) supports growth of the brain malignancy glioblastoma. How the extracellular interactome of GPR133 in glioblastoma modulates signaling remains unknown. Here, we use affinity proteomics to identify the transmembrane protein PTK7 as an extracellular binding partner of GPR133 in glioblastoma. PTK7 binds the autoproteolytically generated N-terminal fragment of GPR133 and its expression in trans increases GPR133 signaling. This effect requires the intramolecular cleavage of GPR133 and PTK7's anchoring in the plasma membrane. PTK7's allosteric action on GPR133 signaling is additive with but topographically distinct from orthosteric activation by soluble peptide mimicking the endogenous tethered Stachel agonist. GPR133 and PTK7 are expressed in adjacent cells in glioblastoma, where their knockdown phenocopies each other. We propose that this ligand-receptor interaction is relevant to the pathogenesis of glioblastoma and possibly other physiological processes in healthy tissues.