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

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.

The Interaction of CD97/ADGRE5 With ß-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 ß-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 ß-catenin through its seven-span transmembrane/intracellular domain(s). To study tumor-associated changes in the interaction of CD97 and ß-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 ß-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 ß-catenin emerged in the cytoplasm and nuclei. CD97 and ß-catenin levels in the cytoplasm correlated well (p < 0.0001). Irrespective of their subcellular localization, interaction of CD97 with ß-catenin in tumor cells was also restricted to the cell contacts. Accordingly, CD97 did not regulate ß-catenin-dependent TCF-mediated transcriptional activity. In summary, while CD97 and ß-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 survival.

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 function.

Mice overexpressing CD97 in intestinal epithelial cells provide a unique model for mammalian postnatal intestinal cylindrical growth.

Postnatal enlargement of the mammalian intestine comprises cylindrical and luminal growth, associated with crypt fission and crypt/villus hyperplasia, respectively, which subsequently predominate before and after weaning. The bipartite adhesion G protein-coupled receptor CD97 shows an expression gradient along the crypt-villus axis in the normal human intestine. We here report that transgenic mice overexpressing CD97 in intestinal epithelial cells develop an upper megaintestine. Intestinal enlargement involves an increase in length and diameter but does not affect microscopic morphology, as typical for cylindrical growth. The megaintestine is acquired after birth and before weaning, independent of the genotype of the mother, excluding altered availability of milk constituents as driving factor. CD97 overexpression does not regulate intestinal growth factors, stem cell markers, and Wnt signaling, which contribute to epithelial differentiation and renewal, nor does it affect suckling-to-weaning transition. Consistent with augmented cylindrical growth, suckling but not adult transgenic mice show enlarged crypts and thus more crypt fissions caused by a transient increase of the crypt transit-amplifying zone. Intestinal enlargement by CD97 requires its seven-span transmembrane/cytoplasmic C-terminal fragment but not the N-terminal fragment binding partner CD55. In summary, ectopic expression of CD97 in intestinal epithelial cells provides a unique model for intestinal cylindrical growth occurring in breast-fed infants.

Thy-1 (CD90) is an interacting partner for CD97 on activated endothelial cells.

Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97(+) myeloid cells colocalized with Thy-1(+) EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1(+)-activated human dermal EC, Thy-1(+) CHO cells, and immobilized Thy-1 protein. Binding of the CD97(+) CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1(+)-activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1-blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

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 beta-catenin and reduced phospho-beta-catenin, labeled for degradation. This was associated with inactivation of glycogen synthase kinase-3beta (GSK-3beta) and activation of Akt. In summary, CD97 increases the structural integrity of enterocytic adherens junctions by increasing and stabilizing junctional beta-catenin, thereby regulating intestinal epithelial strength and attenuating experimental colitis.

Individual cell-based models of tumor-environment interactions: Multiple effects of CD97 on tumor invasion.

The presence of scattered tumor cells at the invading front of several carcinomas has clinical significance. These cells differ in their protein expression from cells in central tumor regions as recently shown for the EGF-TM7 receptor CD97. To understand the impact of such heterogeneity on tumor invasion, we investigated tumor cells with modified CD97 expression in vitro and in vivo. Applying an individual cell-based computer model approach, we linked specific cell properties of these cells to tumor invasion characteristics. CD97 overexpression promoted tumor growth in scid mice, stimulated single cell motility, increased proteolytic activity of matrix metalloproteinases, and secretion of chemokines in vitro in an isoform-specific manner. We demonstrated by computer simulation studies that these effects of CD97 can increase the invasion capacity of tumors. Furthermore, they can cause the appearance of scattered tumor cells at the invasion front. We identified local tumor environment interactions as triggers of these multiple capabilities. Experimentally, our simulation results are supported by the finding that CD97 expression in tumor cells is regulated by their environment. Our combined experimental-theoretical analysis provides novel insight to how variations of individual cell properties can be linked to individual patterns of tumor cell invasion.

Diversity of CD97 in smooth muscle cells.

CD97, an epidermal growth factor (EGF)-TM7 receptor, is not restricted to hematopoetic and carcinoma cells but is also found on smooth muscle cells (SMC). We have examined its location and biochemical structure in various normal and tumorigenic SMC-containing tissues. SMC of the urinary bladder, lung bronchi and bronchioles, myometrium, and gastrointestinal tract were immunohistologically stained by using monoclonal antibodies (mabs) to the CD97 stalk region (CD97(stalk)). Mabs directed against an N-glycosylation-dependent epitope within the EGF-domains (CD97(EGF)) did not bind to normal SMC. Vascular SMC, which was also CD97(EGF)-negative, showed further CD97 heterogeneity. Only a few, if any, SMC from the aorta or elastic arteries of the systemic circulation were positive for CD97 mRNA and therefore also for CD97(stalk). CD97(stalk)-positive SMC were slightly more numerous in muscular and peripheral arteries. In contrast, most venous SMC expressed CD97(stalk). A comparison with other SMC molecules revealed a similar but not identical staining pattern for CD97(stalk) and desmin. Further CD97 heterogeneity was observed during SMC transformation. All leiomyomas (n=5) and nine out of 21 leiomyosarcomas were positive for both CD97(stalk) and CD97(EGF). As expected, CD97(EGF)-positive SMC tumors expressed partly N-glycosylated CD97. Seven out of 21 leiomyosarcomas were completely devoid of CD97. Thus, CD97 showed variable expression in vascular and biochemical modification in tumorigenic SMC, suggesting that the function of the molecule is specific for the SMC subtype.