PDZD2 (PDZ-domain-containing 2; also known as PAPIN AIPC and PIN1) can be a ubiquitously indicated multi-PDZ-domain proteins. PDZD2) which has two PDZ domains. Manifestation of PDZD2 was recognized in several cells. Furthermore sPDZD2 secretion can be suppressed from the mutation of the sequence that presents similarity to caspase reputation motifs or by treatment having a caspase inhibitor. In conclusion PDZD2 may be the 1st reported multi-PDZ proteins that is prepared by proteolytic cleavage to create a secreted peptide including two PDZ domains. CGS 21680 HCl Intro PDZ (for PSD95 Discs-large and ZO-1) domains are conserved protein-protein discussion modules made up of ～80 to 100 proteins (reviewed lately in Harris & Lim 2001 Sheng & Sala 2001 They understand particular carboxy-terminal sequences and generally exist in protein as tandem repeats. PDZ domains are believed to operate by performing as molecular scaffolds to facilitate the set up of macromolecular complexes. PDZ domains are known greatest for their features in the clustering of plasma-membrane-associated protein and in intracellular signalling. Nevertheless the finding of interleukin-16 (IL-16) shows that PDZ-containing protein may also mediate extracellular signalling. IL-16 was initially CGS 21680 HCl defined as a T-lymphocyte chemotactic activity in mitogen-stimulated peripheral bloodstream mononuclear cells (evaluated in Cruikshank et al. 2000 Secreted IL-16 can be produced in many cell types from its multi-PDZ precursor pro-IL-16 by caspase-3-mediated proteolysis (Zhang et al. 1998 Sciaky et al. 2000 Sharma et al. 2000 PIN1/PAPIN/AIPC is a expressed multi-PDZ proteins of unknown function ubiquitously. We 1st determined PIN1 (for PDZ site proteins 1 isolated through the rat insulinoma cell range INS-1; Thomas et al. 1999 like a proteins that interacts with the essential helix-loop-helix transcription element E12. Individually full-length CGS 21680 HCl rat and human being PIN1 complementary DNAs had been cloned and called PAPIN (for plakophilin-related Armadillo-repeat-protein interacting PDZ proteins; Deguchi et al. 2000 and AIPC (for triggered in prostate tumor; Chaib et al. 2001 respectively based on the protein’s binding and manifestation properties. Lately the gene encoding the human being homologue of the proteins was mapped to 5p13.2 and named PDZD2 from the Human being Genome Nomenclature Committee (http://www.gene.ucl.ac.uk/nomenclature). PDZD2 can be used while the real name for PIN1/PAPIN/AIPC in this specific article in order to avoid misunderstandings. In this record we utilized V5- and green fluorescent proteins (GFP)-tagged constructs showing that PDZD2 can be localized towards the endoplasmic reticulum (ER) and the nucleus. Importantly we demonstrated by western blot analysis mass spectrometry and site-directed mutagensis that PDZD2 like pro-IL-16 is proteolytically cleaved at its C terminus to generate a secreted peptide sPDZD2 (for secreted PDZD2) that contains two PDZ domains. These findings give a new insight into the cellular role of PDZD2. RESULTS Analysis of the subcellular localization of PDZD2 Previous immunohistochemical analysis of PDZD2 expression gave conflicting results. PDZD2 was detected at cell-cell contacts in lung sections and in normal rat kidney (NRK) cells (Deguchi et al. 2000 whereas PDZD2 staining was reported to be mainly cytoplasmic in prostate sections (Chaib et NFATc al. 2001 To examine the subcellular localization of CGS 21680 HCl PDZD2 in more detail we tagged CGS 21680 HCl the full-length rat protein at the C-terminus with the viral V5 epitope (the PDZD2C-V5 construct Fig. 1A). After transient transfection into COS7 cells the CGS 21680 HCl subcellular distribution of V5-tagged PDZD2 was analysed by immunostaining. V5 immunoreactivity was detected at the perinuclear region in most of the transfected cells (Fig. 1Ba). This perinuclear region was defined as the ER by counterstaining with fluorescent concanavalin A (conA; a lectin) conjugates that bind to glycoproteins in the ER (Fig. 1Bb c). Furthermore a smaller sized percentage of transfected cells (～8%) demonstrated immunoreactivity that co-localized with nuclear DNA labelled by propidium iodide (Fig..