Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment

Summary Cells respond to mechanical stimuli with altered signaling networks. Here, we show that mechanical forces rapidly induce phosphorylation of CD97/ADGRE5 (pCD97) at its intracellular C-terminal PDZ-binding motif (PBM). Biochemically, this phosphorylation disrupts CD97 binding to PDZ domains of the scaffold protein DLG1. In shear-stressed cells, pCD97 appears not only in junctions, retracting fibers, and the attachment area but also in lost membrane patches, demonstrating (intra)cellular detachment at the CD97 PBM. This motif is critical for the CD97-dependent mechanoresponse. Cells expressing CD97 without the PBM are more deformable, and under shear stress, these cells lose cell contacts faster and show changes in the actin cytoskeleton when compared with cells expressing full-length CD97. Our data indicate CD97 linkage to the cytoskeleton. Consistently, CD97 knockout phenocopies CD97 without the PBM, and membranous CD97 is organized in an F-actin-dependent manner. In summary, CD97 shapes the cellular mechanoresponse through signaling modulation via its PBM

Overexpression of CD97 in Intestinal Epithelial Cells of Transgenic Mice Attenuates Colitis by Strengthening Adherens Junctions

The adhesion G-protein-coupled receptor CD97 is present in normal colonic enterocytes but overexpressed in colorectal carcinoma. To investigate the function of CD97 in colorectal carcinogenesis, transgenic Tg(villin-CD97) mice overexpressing CD97 in enterocytes were generated and subjected to azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis-associated tumorigenesis. Unexpectedly, we found a CD97 cDNA copy number-dependent reduction of DSS-induced colitis in Tg compared to wild-type (WT) mice that was confirmed by applying a simple DSS protocol. Ultrastructural analysis revealed that overexpression of CD97 strengthened lateral cell-cell contacts between enterocytes, which, in contrast, were weakened in CD97 knockout (Ko) mice. Transepithelial resistance was not altered in Tg and Ko mice, indicating that tight junctions were not affected. In Tg murine and normal human colonic enterocytes as well as in colorectal cell lines CD97 was localized preferentially in E-cadherin-based adherens junctions. CD97 overexpression upregulated membrane-bound but not cytoplasmic or nuclear β-catenin and reduced phospho-β-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3β (GSK-3β) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional β-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis

The Interaction of CD97/ADGRE5 With β-Catenin in Adherens Junctions Is Lost During Colorectal Carcinogenesis

The adhesion G-protein-coupled receptor CD97/ADGRE5 is present in adherens junctions of human normal intestinal cells and upregulated in colorectal carcinomas. Here, we examined whether CD97 directly interacts with junctional proteins in normal and malignant colorectal tissue. We identified an association of CD97 with β-catenin using a proximity ligation assay and confirmed the interaction between both endogenous proteins at the biochemical level by co-immunoprecipitation in human and mouse tissues and cell lines. Glutathione S-transferase-pulldown revealed that CD97 binds β-catenin through its seven-span transmembrane/intracellular domain(s). To study tumor-associated changes in the interaction of CD97 and β-catenin in situ, we quantified and correlated both proteins at the membrane, and in the cytoplasm and nuclei of colorectal carcinomas and their corresponding normal tissues (n = 111). In normal colon, membranous levels of CD97 and β-catenin correlated strongly (p < 0.0001). To some degree both molecules disappeared in carcinomas simultaneously from the membrane of tumor cells (p = 0.017). CD97 accumulated in the cytoplasm, whereas β-catenin emerged in the cytoplasm and nuclei. CD97 and β-catenin levels in the cytoplasm correlated well (p < 0.0001). Irrespective of their subcellular localization, interaction of CD97 with β-catenin in tumor cells was also restricted to the cell contacts. Accordingly, CD97 did not regulate β-catenin-dependent TCF-mediated transcriptional activity. In summary, while CD97 and β-catenin interact in adherens junctions, their interaction is lost and both molecules follow different functional paths inside tumor cells

The Interaction of CD97/ADGRE5 With β-Catenin in Adherens Junctions Is Lost During Colorectal Carcinogenesis

The adhesion G-protein-coupled receptor CD97/ADGRE5 is present in adherens junctions of human normal intestinal cells and upregulated in colorectal carcinomas. Here, we examined whether CD97 directly interacts with junctional proteins in normal and malignant colorectal tissue. We identified an association of CD97 with β-catenin using a proximity ligation assay and confirmed the interaction between both endogenous proteins at the biochemical level by co-immunoprecipitation in human and mouse tissues and cell lines. Glutathione S-transferase-pulldown revealed that CD97 binds β-catenin through its seven-span transmembrane/intracellular domain(s). To study tumor-associated changes in the interaction of CD97 and β-catenin in situ, we quantified and correlated both proteins at the membrane, and in the cytoplasm and nuclei of colorectal carcinomas and their corresponding normal tissues (n = 111). In normal colon, membranous levels of CD97 and β-catenin correlated strongly (p < 0.0001). To some degree both molecules disappeared in carcinomas simultaneously from the membrane of tumor cells (p = 0.017). CD97 accumulated in the cytoplasm, whereas β-catenin emerged in the cytoplasm and nuclei. CD97 and β-catenin levels in the cytoplasm correlated well (p < 0.0001). Irrespective of their subcellular localization, interaction of CD97 with β-catenin in tumor cells was also restricted to the cell contacts. Accordingly, CD97 did not regulate β-catenin-dependent TCF-mediated transcriptional activity. In summary, while CD97 and β-catenin interact in adherens junctions, their interaction is lost and both molecules follow different functional paths inside tumor cells

The Adhesion GPCR CD97/ADGRE5 inhibits apoptosis

The Adhesion G protein-coupled receptor (GPCR) CD97/ADGRE5 is induced, upregulated, and/or biochemically modified in various malignancies, compared to the corresponding normal tissues. As tumor cells are generally more resistant to apoptosis, we here studied the ability of CD97 to regulate tumor cell survival under apoptotic conditions. Stable overexpression of wild-type CD97 reduced serum starvation- and staurosporine-induced intrinsic and tumor necrosis factor (TNF)/cycloheximide-induced extrinsic apoptosis, indicated by an increase in cell viability, a lower percentage of cells within the subG0/G1 phase, expressing annexin V, or having condensed nuclei, and a reduction of DNA laddering. Protection from cell death by CD97 was accompanied by an inhibition of caspase activation and modulation of anti- and pro-apoptotic members of the BCL-2 superfamily. shRNA-mediated knockdown of CD97 and, in part, truncation of the seven-span transmembrane (TM7) region of CD97 increased caspase-mediated apoptosis. Protection from apoptosis required not only the TM7 region but also cleavage of the receptor at its GPCR proteolysis site (GPS), whereas alternative splicing of its extracellular domain had no effect. Together, our data indicate a role of CD97 in tumor cell surviva

Skeletal muscle expression of the adhesion-GPCR CD97: CD97 deletion induces an abnormal structure of the sarcoplasmatic reticulum but does not impair skeletal muscle function

CD97 is a widely expressed adhesion class G-protein-coupled receptor (aGPCR). Here, we investigated the presence of CD97 in normal and malignant human skeletal muscle as well as the ultrastructural and functional consequences of CD97 deficiency in mice. In normal human skeletal muscle, CD97 was expressed at the peripheral sarcolemma of all myofibers, as revealed by immunostaining of tissue sections and surface labeling of single myocytes using flow cytometry. In muscle cross-sections, an intracellular polygonal, honeycomb-like CD97-staining pattern, typical for molecules located in the T-tubule or sarcoplasmatic reticulum (SR), was additionally found. CD97 co-localized with SR Ca2+-ATPase (SERCA), a constituent of the longitudinal SR, but not with the receptors for dihydropyridine (DHPR) or ryanodine (RYR), located in the T-tubule and terminal SR, respectively. Intracellular expression of CD97 was higher in slow-twitch compared to most fast-twitch myofibers. In rhabdomyosarcomas, CD97 was strongly upregulated and in part more N-glycosylated compared to normal skeletal muscle. All tumors were strongly CD97-positive, independent of the underlying histological subtype, suggesting high sensitivity of CD97 for this tumor. Ultrastructural analysis of murine skeletal myofibers confirmed the location of CD97 in the SR. CD97 knock-out mice had a dilated SR, resulting in a partial increase in triad diameter yet not affecting the T-tubule, sarcomeric, and mitochondrial structure. Despite these obvious ultrastructural changes, intracellular Ca2+ release from single myofibers, force generation and fatigability of isolated soleus muscles, and wheel-running capacity of mice were not affected by the lack of CD97. We conclude that CD97 is located in the SR and at the peripheral sarcolemma of human and murine skeletal muscle, where its absence affects the structure of the SR without impairing skeletal muscle functio

CD97 Tg mice are protected from colitis in the AOM/DSS model.

<p>A Treatment protocol: Mice underwent three cycles of AOM/DSS treatment (n = 16/group). B Intestinal CD97 overexpression changed the number of tumors in the colon of Tg mice (*, p<0.05). C–F Disease activity index (DAI) and separate scores of this index during the first treatment cycle causing acute colitis in WT animals. Disease activity (C), fecal blood (D), diarrhea (E), and weight loss (F) were strongly reduced in Tg mice. Lowest disease activity was found in Tg2 mice.</p