These data provide strong evidence that TIP-1 can prevent association of Kir 2.3 with mLin-7/CASK without altering the integrity of the basolateral membrane scaffold complex. Open in a separate window Figure 9. TIP-1 expression reduces amount of Kir 2.3 associated with the mLin-7/CASK complex. proteinCprotein conversation modules, which allows them to act as molecular scaffolds that recruit membrane proteins, cytoskeletal elements, trafficking machinery, and signal transduction molecules into multimeric complexes at distinct membrane domains (Brone and Eggermont, 2005 ; Campo 2005 ). Many play important functions in the generation and maintenance of epithelial and neuronal polarity. A prototypical example is usually provided by a group of PDZ proteins, Lin-7 (mLin-7/Veli/MALS), Lin-2 (CASK) and Lin10 (Mint-1/X11), that were first discovered in (Kaech 1998 ). In vulvar precursor cells, these molecules form a tripartite protein complex that interacts with the Let-23 receptor to coordinate its expression around the basolateral membrane (Simske 1996 ; Kaech 1998 ; Rongo 1998 ). Studies in and mammalian systems have revealed a probable mechanism. Lin-2 (CASK) interacts with cell structural elements, anchoring the complex to the membrane (Cohen 1998 ). Lin-10 offers a means of delivering the complex via its interactions with microtubule motors (Setou 2000 ) and Munc-18 docking machinery (Hata 1993 ; Borg Mouse monoclonal to ATM 1998 ; Butz 1998 ). The smaller Lin-7 protein acts as the bridge between receptor and scaffold complex, binding the receptor through a type I PDZ conversation and the scaffold via a L27 domain name conversation (Doerks 2000 ) with CASK (Kaech 1998 ). In PDZ protein complex have been identified in mammalian tissues (Borg 1998 ; Butz 1998 ; Straight 2001 b; Olsen 2002 ). Although renal epithelia do not Coelenterazine express a Lin-10 orthologue (Borg 1998 ), the mLin-7 and CASK complex is usually evolutionarily conserved and localizes exclusively at the basolateral membrane (Olsen 2005 ; Straight 2001 ). Recently, we exhibited that mLin-7 binds the inwardly rectifying potassium channel Kir 2.3 through a PDZ conversation and that the mLin-7/CASK complex plays an important role in coordinating polarized expression of the channel at the basolateral membrane by preventing the channel from traveling in the endocytic pathway (Olsen 2002 ). The mLin-7/CASK Coelenterazine complex has also been implicated in basolateral expression of the epithelial GABA transporter BGT-1 (Perego 1999 ), the EGF-like receptor ErbB-2/Her2 (Shelly 2003 ), and other potassium channels (Leonoudakis 2004b ), suggesting that conversation with the mLin-7/CASK complex represents a general Coelenterazine mechanism for the polarized expression of basolateral membrane proteins made up of a PDZ-binding motif. While investigating the mechanism for polarized expression of Kir 2.3, we discovered a novel PDZ-binding partner for the channel, Tax-interacting Protein-1 (TIP-1). A partial TIP-1 clone was originally identified through a yeast two-hybrid screen as one of many binding partners for the viral oncoprotein Tax (Rousset 1998 ). Here, we show that this full-length TIP-1 encodes a protein consisting of a single PDZ domain name and appears to be devoid of other proteinCprotein conversation modules. This represents a departure from the classic PDZ protein retention/clustering scheme because PDZ proteins rely on multiple proteinCprotein conversation motifs to effectively Coelenterazine scaffold membrane, cytoskeletal, and signaling proteins. Indeed, TIP-1 antagonizes the retention function of the mLin-7/CASK complex by competing for conversation with Kir 2.3. This molecular switch results in internalization of Kir 2.3 channels. The competitive PDZ conversation described here represents a novel mechanism for regulating the surface expression of Kir 2.3 channels and may provide a more general means for regulating other PDZ protein interactions. MATERIALS AND METHODS Yeast Two-Hybrid Interaction Trap Library Screen The yeast two-hybrid conversation trap system was Coelenterazine used according to established methods (Gyuris 1993 ; Golemis and Khazak, 1997 ) to capture proteins that interact with the PDZ-binding motif in Kir 2.3. A sequential, two-step library screen with two different baits was performed. In the first stage of the screen, a Lex A fusion protein of the Kir 2.3 C-terminal targeting domain name (amino acids 417-445) was.