In vivo interaction of NKD2 and TGFα and identification of endogenous NKD2. (A) NKD2 coimmunoprecipitates with TGFα in stably transfected MDCK cells. (Left) MDCK cells were cotransfected with EGFP-tagged hNKD2ΔC430 and human TGFα cDNA. TGFα was immunoprecipitated with a sheep polyclonal anti-human TGFα antibody (Tα), and then Western blots were probed with rabbit anti-EGFP antibody. A single 74-kDa band was observed (lane 1), which is consistent with the predicted size of EGFP-tagged NKD2. Lane 2 is a negative control with no primary antibody. (Right) MDCK cells were stably cotransfected with NKD2 cDNA and HA (ectodomain)/Flag (C terminus) dual-tagged proTGFα cDNA (15). Supernatant fractions were immunoprecipitated with rabbit anti-EGFP or Flag antibody, as shown, and blotted with anti-Flag mAb. (B and C) MDCK and COS-7 cells were stably transfected with EGFP-tagged NKD2 and dual-tagged proTGFα. Ectodomain of proTGFα was probed with anti-HA mAb, and Alexa 594-labeled goat anti-mouse antibody was used as a secondary antibody. In a single scanned image of one representative MDCK cell (B), NKD2 (green fluorescence) seems to envelop TGFα ectodomain staining (red fluorescence) with one example shown in higher power in the lower left corner. In a single scanned image of one COS-7 cell, TGFα, but not NKD2, is detected in the Golgi. There is punctate colocalization of TGFα and NKD2 that is most prominent at the cell periphery (higher power is shown in the left upper corner). (D) Reciprocal coimmunoprecipitations between endogenous NKD2 and TGFα in HaCat cells stably transfected with dual-tagged TGFα construct. Endogenous NKD2 was recognized by using R44, a rabbit polyclonal antibody raised to an N-terminal peptide of human NKD2 (see Materials and Methods). (Right) Preimmune serum was used as negative control in the first lane. (E) An ≈55-kDa band was detected by Western blotting with R44 (0.5 μg/ml) of lysates prepared from two human colorectal cancer cell lines (Caco-2 and HCA-7), a human skin keratinocyte cell line HaCat, and EGFP-tagged NKD2-expressing MDCK cells. R44 recognizes EGFP-tagged NKD2 and endogenous NKD2. (F) NKD2 is detected in mouse GI tract and skin. Lysates were prepared and endogenous NKD2 was detected by immunoblotting with R44 (0.5 μg/ml; see Materials and Methods).

G2A-NKD2 selectively affects basolateral localization of TGFα. (A) In polarized MDCK cells that had been stably transfected with inducible doxycycline-off G2A-NKD2 and dual-tagged TGFα constructs, there was no ectodomain staining for TGFα with HA antibody at the lateral membrane in the presence or absence (data not shown) of TACE/ADAM17 inhibitor WAY022 (5 μg/ml for 2 h before fixation). After 48 h of 1 μg/ml doxycycline treatment to inhibit G2A-NKD2 expression, ectodomain staining for TGFα was observed at the lateral plasma membrane. (B) Ectodomain staining for TGFα was found at the lateral plasma membrane in stably transfected wild-type and vector control (data not shown) polarized MDCK cells in the presence of TACE/ADAM17 inhibitor. (C) HA fluorescence was observed at the cell surface of G2A-NKD2-expressing MDCK cells transiently transfected with an AR-HA cDNA construct (see Materials and Methods). Na⁺, K⁺ ATPase α1 (D) and E-cadherin (E) immunofluorescence was observed at the lateral surface of wild-type (data not shown) and G2A-NKD2-expressing polarized MDCK cells. (F) Texas red transferrin was added to the basolateral medium of wild-type NKD2-expressing MDCK cells. No colocalization of NKD2 and endocytosed transferrin was observed from 5–30 min after administration of labeled transferrin.

Characterizing NKD2 and its interaction with TGFα. (A) Motif comparison among NKD family members. MS, myristoylation site; NHR, Naked homology regions; TTB, TGFα tail binding; H, histidine. Purple shading indicates a conserved EF-hand motif shared among Naked Cuticle, NKD1, and NKD2. Pink indicates a homologous region shared by NKD1 and NKD2. The putative region that Dvl binds to NKD1 is indicated. (B) Mapping of reciprocal interacting domains between NKD2 and TGFα cytoplasmic tail (TCT) by yeast two-hybrid analysis.

Functional assessment of NKD2. (A) MDCK cells stably cotransfected with NKD2 and TGFα were pulse-labeled with [³⁵S]methionine for 20 min and chased with unlabeled medium for the times indicated, and TGFα was immunoprecipitated with anti-Flag antibody. NKD2 accelerates appearance of TGFα 36-kDa and 16-kDa bands. (B) Myristoylation of NKD2. Stably transfected EGFP-tagged, doxycycline-inducible wild-type and G2A mutant NKD2 MDCK cells were grown on plastic without (–) or with (+) doxycycline. Cells were pulsed for 2 h with [³H]myristic acid; lysates were prepared, immunoprecipitated with anti-EGFP Ab, and resolved on a 10% SDS gel (see Materials and Methods). The last lane represents a Western blot with anti-EGFP Ab to cells expressing epitope-tagged wild-type NKD2. (C and D) Cellular localization of NKD2 is affected by myristoylation. Epitope-tagged wild-type and G2A-NKD2-expressing MDCK cells on Transwell filters were induced and examined by confocal microscopy. Wild-type NKD2 staining was observed at the lateral membrane below the tight junction marker Z01, but G2A mutant staining accumulated in the cytoplasm at the base of the cells asymmetrically. (E) Clusters of vesicles were observed at the base of the cytoplasm of polarized G2A-NKD2-expressing MDCK cells by electron microscopy. (Left) Arrow indicates area that is magnified in Right. Bars in Left and Right represent 500 nm and 100 nm, respectively. (F) Immunoelectron microscopic staining of NKD2 in wild-type NKD2-expressing MDCK cells. R44 primary antibody and horse-radish peroxidase-labeled goat anti-rabbit complex were used sequentially before embedding (see Materials and Methods). DAB dense precipitation was found coating different-sized vesicles (70–350 nm) evenly and unevenly. The cores of stained vesicles were lucent (20%, medium to large vesicles, open arrow), cloudy (60%, medium vesicles), and dense (20%, mediumto small vesicles, black arrow).