PTK7 is a positive allosteric modulator of GPR133 signaling in glioblastoma

Abstract

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.

Keywords: ADGRD1; CP: Cancer; CP: Molecular biology; G protein-coupled receptor; GPR133; PTK7; adhesion GPCR; allosteric signal activation; glioblastoma; ligand discovery.

Figure 1.. Co-purification/mass spectrometry-based ligand discovery in patient-derived GBM cells detects GPR133’s extracellular and membrane-bound interactome

(A) Schematic overview of the affinity co-purification approach. Full-length cleavage-deficient GPR133-H543R with C-terminal intracellular TwinStrep tag was overexpressed in patient-derived GBM cultures. GPR133 and endogenous binding partners were crosslinked with DTSSP, followed by affinity enrichment and analysis by mass spectrometry. GAIN, G-protein-coupled receptor autoproteolysis-inducing domain; PTX, pentraxin-like domain. (B) Eluates after Strep-Tactin purification from three different GBM cultures (with three independent biological repeats each) transduced with either TwinStrep-tagged uncleavable GPR133-H543R or an empty vector were analyzed with SDS-PAGE and western blot for GPR133 levels using an anti-GPR133 C-terminal antibody. (C) Volcano plot of fold enrichment with the GPR133 bait and statistical significance of 2,138 total detected proteins. The 569 candidate proteins with subcellular annotation at the plasma membrane or in the extracellular space were filtered for enrichment with the GPR133 bait versus control vector samples across all cell lines, resulting in 87 consistently enriched proteins. (D) Volcano plot depicting these 87 proteins, 23 of which were significantly enriched (p < 0.05; blue dots). (E) Overview heatmap of the 38 most (p < 0.1) significantly enriched GPR133-binding protein candidates with subcellular localizations at the plasma membrane or extracellular space. The purple heatmap depicts detected protein intensities as a log₁₀ mean of three biological replicates per culture per condition. The subcellular localization column was derived using Ensembl database annotations (detailed in STAR Methods). The mean enrichment column depicts the ratio of log₁₀ mean intensities across all cell cultures and replicates from the GPR133 bait samples over the empty vector control samples. The p value column depicts the p value calculated from a mixed-effects model of enrichment with the GPR133 bait across all samples. The CRAPome column depicts the frequency of detection of proteins by mass spectrometry across 716 publicly available affinity purification datasets, and thereby identifies possibly non-specific interactors. See also Figure S1.

Figure 2.. Biochemical validation of candidate binding proteins detects PTK7 as the most robust GPR133 interactor

(A) Validation affinity co-purification assay testing six of the top interactors identified in the screen with a TwinStrep-tagged GPR133-H543R bait in HEK293T cells. All candidate interactors were C-terminally tagged with the Myc epitope. (Ai) Western blots of input whole-cell lysates stained against Myc tag of ligand candidate proteins and anti-GPR133 C terminus. (Aii) Western blots of eluates after Strep-Tactin 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 in the enrichment volcano plot as depicted in Figure 1D. (D) Normalized intensities of peptides corresponding to GPR133 and PTK7 as detected in the mass spectrometry analysis in Figure 1E, detailing all biological replicates. (E) Comparison of the GPR133-binding interaction of PTK7 vs. the previously reported ligand PLXDC2 using the Strep-Tactin 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 not co-purify at detectable amounts under the same experimental conditions. See also Figure S2.

Figure 3.. Biochemical characterization of the GPR133-PTK7 interaction

(A) Co-affinity purification of GPR133 variants with co-expressed PTK7. We generated a TwinStrep-tagged GPR133-H543R deletion mutant lacking the PTX domain (GPR133-DPTX-H543R, aa 277–875, contains endogenous signal peptide) and compared its PTK7 binding with that of full-length GPR133-H543R using Strep-Tactin purification. The interaction was not altered by deletion of the PTX domain. Inclusion or omission of DTSSP, an extracellular crosslinker, did not influence the binding. Representative western blots are shown. (B) Reverse co-purification experiment using PTK7 as bait. Both the full-length GPR133-H543R (aa 1–875) and a secreted TwinStrep-tagged GPR133 NTF (aa 1–563) lacking transmembrane domains co-purified with PTK7 using Ni-NTA beads. Pink, yellow, and red arrowheads denote the monomeric full-length GPR133-H543R, multimers of the full-length GPR133-H543R, or the secreted GPR133 NTF lacking transmembrane domains, respectively. Purple arrowheads denote non-specific antibody off-targets. Multimerization of GPR133 due to its transmembrane domains is reduced but not entirely prevented by the use of 1% ndodecyl b-D-maltoside. GPR133 multimerization is discussed in detail in Frenster et al. (C) Affinity co-purification of soluble secreted GPR133 NTF and PTK7 NTF. GPR133 NTF with N-terminal TwinStrep tag and secreted Myc-tagged PTK7 with no transmembrane domains were expressed alone, expressed together in adjacent cells, or co-expressed in the same cells. Secreted PTK7 co-purified with GPR133 in both mixed-culture and co-expression conditions (green arrows). Representative blots are depicted. See also Figure S2.

Figure 4.. PTK7 increases GPR133 signaling through a trans interaction

(A) Confocal microscopic image from “sandwich cultures” shows layered cells expressing either GPR133 (green) or PTK7 (red). Top view from a central confocal slice (center panel: x and y dimension) and orthogonal views (upper panel: z and x; right panel: z and y) depicted on the same scale. Scale bars, 10 μM. (B) HTRF assays in “sandwich cultures” demonstrate that PTK7 in neighboring cells increased cAMP levels in cells expressing wild-type (WT) cleaved GPR133, but not the uncleavable H543R mutant. A secreted PTK7 with no transmembrane domain (no TM, aa 1–703) did not influence GPR133 signaling. Two-way ANOVA: middle layer F_{2, 96} = 4.62, not significant; outer layer F_{2, 96} = 3.93, not significant; interaction of inner and outer layer F_4, 96 = 7.29, p < 0.0001. Tukey’s multiple comparisons: GPR133-WT-expressing cells with empty vector vs. PTK7 co-culture, p < 0.0001; GPR133-WT-expressing cells with full-length PTK7 vs. secreted PTK7 (no TM) co-culture, p < 0.0001. All other comparisons not significant; n = 4–22 independent experiments. (C) Pre-coating wells with PTK7 NTF (aa 1–703) significantly increased cAMP levels in WT GPR133-expressing, but not H543R uncleavable mutant-expressing cells. COL6 (native purified human collagen VI) had no effect on signaling. Two-way ANOVA: GPR133 variant expressed F_{2, 80} = 11.65, p < 0.0001; protein coating F_{2, 80} = 12.90, p < 0.0001; interaction of factors F_{4, 80} = 9.24, p < 0.0001. Tukey’s multiple comparisons: GPR133-WT-expressing cells on PTK7 NTF-coated vs. uncoated dishes, p < 0.0001; GPR133-WT-expressing cells on PTK7 NTF-coated vs. COL6-coated dishes, p < 0.0001). All other comparisons not significant; n = 5–15 independent experiments. (D) Combinatorial effect of PTK7 binding and p13 Stachel peptide treatment on GPR133 signaling. HEK293T cells expressing either WT GPR133 or empty vector control were seeded onto PTK7 NTF-coated (1.17 μg/cm²) or control-coated wells. Cells were then treated with synthetic p13 Stachel peptide (500 μM), inactive control peptide (500 μM), or solvent controls, and cAMP levels were measured by HTRF. The combination of PTK7 NTF binding and Stachel peptide elicited an additive response in GPR133 signaling compared with the individual treatments. Two-way ANOVA, GPR133 expression effect F_{1, 48} = 365.1, p < 0.0001; treatment effect F_{7, 48} = 13.2, p < 0.0001; interaction of GPR133 expression and treatment F_{7, 48} = 13.2, p < 0.0001. Tukey’s multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 4 independent experiments. Data are depicted as mean ± SEM. See also Figures S3 and S4.

Figure 5.. GPR133 and PTK7 expression profiles in GBM and healthy human tissues

(A) Epifluorescence microscopic images from two GBM surgical specimens show expression of both GPR133 (green) and PTK7 (red) within tumors. (B) Higher-magnification epifluorescence images demonstrate a complementary expression pattern in GBM specimens. (C) Confocal microscopic image with orthogonal views of a z stack indicates that cells tend to either predominantly express GPR133 or PTK7. (D) GTEx portal mRNA data from human tissues shows overlap in PTK7 and GPR133 expression in several tissues. (E) On a whole-tissue level, the expression of PTK7 mRNA showed a significant positive correlation with GPR133 mRNA (Pearson correlation, p < 0.0001). Each dot represents the whole-tissue average gene expression from the human GTEx tissues listed in (D). (F) Single-cell gene expression analysis of the seven available GTEx single-cell datasets—esophagus mucosa, esophagus muscularis, heart, lung, prostate, skeletal muscle, and skin—filtered for cells with RNA integrity number (RIN) >8 demonstrates that GPR133 only (5%) and PTK7 only (5.9%) expressing cells are more common than cells co-expressing both genes (0.3%) (130,840 cells included, single-cell expression normalized as log₁₀ of copy number per 10,000 reads). (G) Within the lung, which exhibits high expression of GPR133 and PTK7 on a whole-tissue level, individual cells preferentially express only GPR133 (6.1%) or only PTK7 (10.9%) rather than co-expressing both genes (1.1%), resulting in a negative correlation (Pearson correlation r = 0.83, p < 0.0001, n = 2,597 cells; cells expressing neither gene were excluded from correlation analysis to avoid zero inflation). (H) All seven available single-cell datasets from the GTEx portal reflect the same negative correlation between GPR133 and PTK7 expression in human tissues observed in (G), with GPR133 only (red) and PTK7 only (green) expressing cells being more common than cells expressing both genes (blue). Detailed single-cell analyses of these tissues are depicted in Figure S7. Scale bars in (A) and (B) represent 50 μM or 400 μM as annotated; scale bars in (C) represent 50 μM. See also Figures S5–S7.

Figure 6.. Knockdown of PTK7 phenocopies that of GPR133 in patient-derived GBM cells

(A) We tested four different shRNAs targeting endogenous PTK7 in patient-derived GBM cells and found significant knockdown for all by western blots (Ai) and densitometric analysis (Aii). A non-targeting scrambled shRNA (scr-sh) was used as control. (Bi) Similar to the effect of our previously described shRNA targeting GPR133 (Bayin et al.), shRNAs targeting PTK7 reduced tumorsphere formation in vitro (Tukey’s multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001; n = 4 independent experiments with six technical replicates per condition each). (Bii) Representative example images of tumorsphere formation assay. Data are depicted as mean ± SEM.

Figure 7.. Graphical summary of proposed PTK7-GPR133 interaction model

(A) The extracellular portion of PTK7 binds the GPR133 NTF in trans. This interaction does not depend on either protein’s transmembrane domain nor on GPR133’s pentraxin domain. (B) The binding may cause conformational changes and/or mechanical dissociation of the GPR133 NTF to increase receptor signaling activity. This interaction is allosteric and does not compete with orthosteric Stachel peptide agonism (in dashed parentheses). (C) Additional mechanical shear stress may facilitate NTF-CTF dissociation, causing the irreversible activation of GPR133 signaling.