TIP-1 has PDZ scaffold antagonist activity

Abstract

PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.

Figure 1.

Kir 2.3 C-terminus specifically interacts with the novel PDZ protein TIP-1. (A) Yeast were cotransformed with AD-TIP-1 and LexA DNA-binding domain fusions of wild-type (WT) Kir 2.3 C-terminus (top), a mutant Kir 2.3 C-terminus, lacking the last 3 amino acids (ΔPDZ; middle), or bicoid, an unrelated Drosophila protein (bottom). Transfected yeast were diluted and spotted onto selection/reporter plates, X-gal or -Leu. Transcriptional activation of the lacZ reporter produces blue yeast on X-gal plates; Activation of leu2 reporter allows yeast to grow on “leucine drop-out” plates (-Leu). (B) Schematic of the TIP-1 protein compared with mLin-7. Atypical of PDZ proteins, TIP-1 contains a single protein–protein interaction domain.

Figure 2.

TIP-1 expression. (A) Northern blots of poly(A⁺) RNA from human tissues hybridized with TIP-1 cDNA probes. Actin probe control is shown in the bottom panel. (B) Anti-TIP-1 antibody specifically recognized a 14-kDa band in immunoblots of rat kidney lysate. Excess antigen (+peptide) blocked antibody binding with the kidney 14-kDa protein. (C) Immunolocalization of TIP-1 (a, c, and e) corresponds to sites of Kir 2.3 expression (b, d, and f) in the kidney. Both proteins localize in distal convoluted tubule (a and b), connecting tubule (c and d), and collecting duct (e and f). Controls show that immunolabeling for TIP-1 is blocked by an excess of the immunizing peptide (g) and preimmune serum shows minimal nonspecific Kir 2.3 label (h). Scale bar, 17.5 μm.

Figure 3.

Kir 2.3 and TIP-1 interact in mammalian cells. COS cells transiently transfected with FLAG-TIP-1– and/or VSV-Kir 2.3–expressed proteins of the predicted size as detected with either anti-FLAG (A) or anti-VSV (B, lane 2) antibodies. (B) COS cells expressing VSV-Kir 2.3 alone or coexpressed with myc-TIP-1 were solubilized, and lysates were immunoprecipitated with anti-FLAG antibodies and immunoblotted with anti-VSV antibody. The FLAG antibody, but not an irrelevant control antibody (C), specifically coimmunoprecipitated Kir 2.3, verifying a bona fide interaction.

Figure 4.

Kir 2.3 and TIP-1 interact in a PDZ-dependent manner. (A) Interaction of AD-TIP-1 and LexA-Kir 2.3 wild-type (WT) or mutant Kir 2.3 C-termini was inferred from β-galactosidase activity in the yeast two-hybrid system. The GAL1 promoter induces the expression of the AD-tagged TIP-1 in the presence of galactose (■) but not in the presence of glucose (). Reporter activation is observed only in the presence of galactose. Mean ± SE; n = 3; * p ≤ 0.01; statistical significance as measured by ANOVA. (B) Three-dimensional structure of the TIP-1-binding pocket as modeled on the crystal structure of the related third PDZ domain of PSD-95 (PDB,1BFE; Doyle et al., 1996). Two residues, K20 and H90, predicted to interact with the PDZ ligand (blue) are shown in red. (C) GST pulldown assays using GST-Kir 2.3 C-terminus wild-type (WT) or a mutant GST-Kir 2.3 C-terminus lacking the PDZ interaction motif (ΔPDZ) and different purified recombinant His-tagged TIP-1 proteins (WT, wild-type TIP-1, and TIP-1-bearing K20A, H90A, or double K20A/H90A mutations). His-TIP-1 proteins specifically bound to the GST-Kir 2.3 proteins were detected by immunoblotting with anti-His antibodies (“pull-down”). In bottom panels, Ponceau S stain of GST-Kir 2.3 input and Coomassie stain of His-TIP-1 input are shown as loading controls. (D) The amount of each TIP-1 construct bound to GST-Kir 2.3 was assessed by densitometry, background subtracted, and analyzed relative to the WT TIP-1 band intensity. The mean ± SE relative densitometry of four pulldown experiments repeated in triplicate (p ≤ 0.01) is shown.

Figure 5.

TIP-1 competes with mLin-7 for binding to Kir 2.3. (A) Diagram illustrating the procedure for competitive immunoprecipitation experiments as described below and in Materials and Methods. (B) Kir 2.3-mLin-7 complexes were recovered on protein G-Sepharose beads with anti-VSV antibodies from COS cells expressing VSV-tagged Kir 2.3 and HA-tagged mLin-7. Three separate competition reactions were run in parallel. Lysate from COS cells transfected with pcDNA 3.1 was added (pc DNA lysate++) to one sample as a control. Another sample contained an equal volume of COS cell lysate containing TIP-1 (TIP-1-myc++). The other sample contained an equal volume of a 1:1 mix of the TIP-1-myc and pcDNA lysates (TIP-1-myc+/pcDNA+). After incubation at RT, HA-mLin-7, and TIP-1-myc bound to Kir 2.3 was recovered and detected by immunoblot. (C) Amount mLin-7 bound to Kir 2.3 relative to control (pC DNA++) was evaluated by densitometry of experiments shown in B. Mean ± SE; n = 3; p < 0.005.

Figure 6.

Kir 2.3 is mislocalized to a vesicular compartment in MDCK/TIP-1 cells. (A) Confluent monolayers of wild-type (WT, top panels) or MDCK cells stably transfected with myc-TIP-1 (MDCK myc-TIP-1, bottom panels), were transiently transfected with VSV-Kir 2.3 and stained with anti-VSV antibody. Images were chosen to document the full spectrum of phenotypes observed for each cell type. Scale bar, 10 μm. (B) Cells were scored for intracellular localization of Kir 2.3 (2, strong; 1, moderate; 0, none) by a blinded observer. Data are reported as the average Kir 2.3 intracellular localization score for cells from three separate infections (n = 100; p < 0.001). (C) MDCK cells stably expressing a mutant Kir 2.3 protein lacking the PDZ ligand (ΔPDZ) display a similar mislocalization phenotype as caused by TIP-1 expression. Cells are labeled with anti-VSV antibodies, detecting Kir 2.3. (D) MDCK WT and myc-TIP-1 cells were stained with anti-mLin-7 antibodies for endogenous mLin-7. Scale bar, 20 μm.

Figure 7.

TIP-1 is efficiently expressed in MDCK Kir 2.3-VSV cells by an adenoviral delivery system. Confluent, filter-grown MDCK cells stably expressing Kir 2.3-VSV were infected with wild-type (WT) or H90A TIP-1-myc adenoviruses. (A) After infection with TIP-1 adenoviruses, MDCK Kir2.3-VSV cells were decorated with biotin on the basolateral membrane and then costained with streptavidin (red) to mark the basolateral membrane and anti-myc antibodies (green) to visualize TIP-1. Scale bar, 10 μm. (B) Lysates from these cells were immunoblotted with anti-myc antibodies to detect WT and H90A TIP-1. (C) TIP-1 was immunoprecipitated with anti-myc antibodies and then immunoblotted with anti-VSV antibodies for Kir 2.3-VSV. (D) Transepithelial resistance (TER) of infected cells. Neither WT nor H90A viruses reduce the TER. Instead infection of either virus causes a small, reproducible increase in TER (n = 4; p ≤ 0.01). Mean ± SE.

Figure 8.

Adenoviral-mediated expression of TIP-1 in MDCK Kir2.3-VSV cells redistributes the channel to a vesicular compartment. (A) Shown are confluent monolayers of MDCK-Kir2.3 VSV cells grown on permeable supports after mock infection or infection with TIP-1-myc adenoviruses. Cells are colabeled with anti-VSV antibodies to detect Kir 2.3 (green) and with streptavidin to mark the basolateral membrane (red). All cells in the middle and right panels express TIP-1 except those identified with the asterisk (∗). (B) Z-plane images of cells were scored for intracellular localization of Kir 2.3 (2, strong; 1, moderate; 0, none) by a blinded observer. For both TIP-1 groups, only cells that were positively identified as expressing myc-TIP-1 by anti-myc labeling were scored. Data are reported as average Kir 2.3 intracellular localization score for cells from three separate infections. Expression of wild-type TIP-1 but not H90A TIP-1 resulted in a statistically significant increase in intracellular Kir 2.3. Mean ± SE; n = 24; p < 0.01. (C) Cells stained with anti-NaK/ATPase α1 antibodies. TIP-1 expression did not effect the localization of this endogenous basolateral marker. (D) Cells labeled with anti-VSV antibodies to detect Kir 2.3 (green) and anti-Rab11 antibodies (red) in mock and wild-type TIP-1–infected MDCK Kir 2.3-VSV cells. (E) Cells stained with anti-mLin-7c antibodies (green) and with streptavidin to mark the basolateral membrane (red). Scale bar, (A, C, D, and E) 10 μm.

Figure 9.

TIP-1 expression reduces amount of Kir 2.3 associated with the mLin-7/CASK complex. (A) Confluent MDCK Kir 2.3-VSV cells were infected with wild-type (WT) or H90A TIP-1-myc adenoviruses and compared with mock-infected cells (Ad/TIP-1-myc−). Lysates were immunoprecipitated with anti-mLin-7c antibodies (+) or no-antibody (−) and then immunoblotted with anti-VSV antibodies for Kir 2.3-VSV (above) or anti-CASK antibodies (below). (B) Mean ± SE densitometry (immunoprecipitated protein/input) from three separate infections; n = 3; p ≤ 0.05.