(A–D) Schematic diagram of human OTUD4 domains and strategy to engineer a series of OTUD4 deletion mutants. Mapping of the molecular domain on OTUD4 involving in the interaction with CD73. The interactions between CD73 and OTUD4 fragments were examined by coimmunoprecipitation experiments in HEK-293T cells. Amino acids stretching from 380–410 on OTUD4 (D) were identified as the region that mediates the interaction between OTUD4 and CD73. (E) Schematic diagram of human CD73 domains and strategy to engineer a series of CD73 deletion fragments. (F) The interactions between OTUD4 and CD73 fragments were determined. Amino acids stretching from 275–311 on CD73 were identified as the region that mediates the interaction between OTUD4 and CD73. (G) Structural model for the interaction of CD73 with OTUD4. Residues R380–N410 (red) of OTUD4 (green) interact with the residues V275–D311 (yellow) of CD73 (cyan). Salt bridges are formed between E296 and E293 of CD73 and K409 and K403 of OTUD4, respectively. The interface is further stabilized by hydrophobic interactions between V300 and V301 of CD73 (yellow) and F404 (red) of OTUD4, and contacts between H304 (yellow) of CD73 and S397 (red) of OTUD4. (H) Validation of interaction domains between OTUD4 with CD73 by coimmunoprecipitation of ectopic V5-tagged CD73^WT and V5-tagged CD73^V300P/I301Q mutant in HEK-293T cells. The ubiquitylation status of CD73^WT and CD73^V300P/I301Q mutant was also determined. (I) MDA-MB468-CD73^WT and MDA-MB468-CD73^V300P/I301Q cells were treated with CHX and MG-132 and CD73 protein turnover were determined. (J) The adenosine levels were determined in MDA-MB468-CD73^WT and MDA-MB468-CD73^V300P/I301Q. (K) MDA-MB468, MDA-MB468-CD73^WT and MDA-MB468-CD73^V300P/I301Q were cocultured with human PBMCs and percentage of IFN-γ ⁺ in CD8⁺ T cell population quantified using flow cytometry. Data (mean ± SEM) are representative of at least 3 independent experiments. **P < 0.01 and ***P < 0.001, by unpaired t test, 1-way ANOVA with Tukey’s multiple comparisons test.