A Secretory-type Protein, Containing a Pentraxin Domain, Interacts with an A-type K+ Channel

University of South Florida, Department of Otolaryngology, Head and Neck Surgery, Tampa, Florida 33612, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 05/2005; 280(15):15165-72. DOI: 10.1074/jbc.M500111200
Source: PubMed


A-type K(+) channels belonging to the Shal subfamily are found in various receptor and neuronal cells. Although their kinetics and cell surface expression are regulated by auxiliary subunits, little is known about the proteins that may interact with Kv4 during development. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus. Studies with chick cochlear tissues reveal that Kv4.2 coprecipitates PPTX and that both proteins are colocalized to the sensory and ganglion cells. A yeast two-hybrid assay demonstrated that the last 22 amino acids of the PPTX C terminus interact with the N terminus of Kv4.2. Chinese hamster ovary cells transfected with recombinant PPTX reveal secretory products in both non-truncated and truncated forms. Among the secreted variants are several blocked by Brefeldin A, suggesting export via a classical pathway. PPTX is soluble in the presence of sodium carbonate, suggesting localization to the cytosolic side of the plasmalemma. Immunohistochemical studies show that Kv4.2 and PPTX colocalize in the region of the plasmalemma of Chinese hamster ovary cells; however, both are locked in the endoplasmic reticulum of COS-7 cells, suggesting that PPTX does not act as a shuttle protein. Reverse transcription-PCR demonstrates that PPTX mRNA is found in tissues that include brain, eye, heart, and blood vessels.

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Available from: Bernd Sokolowski, Oct 08, 2015
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    • "Typically this tag is used at either the N-or C-terminal ends, as is the viral hemagglutinin coat protein or HA tag. However, both can be used as an epitope tag within the C-and N-terminal domains, since tagging at the very end of either terminal may interfere with a protein-protein interaction (Duzhyy et al., 2005). Moreover, if the protein is a signaling protein, a tag at the N-terminus will be cleaved-off the main body of the protein and thus, not resolvable on a gel. "
    Selected Works in Bioinformatics, 10/2011; , ISBN: 978-953-307-281-4
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    • "In the CNS, immunohistochemical analysis shows that Kcnd2 (Kv4.2) is expressed in rat striatal cholinergic interneurons [70] and is clustered on the postsynaptic membrane [71]. In chick cochleae, Kv4.2 is expressed post-synaptically by ganglion cells and at the basolateral aspects of the intermediate and short hair cells (analogs of mammalian outer hair cells), but is absent in tall hair cells (analogs of mammalian inner hair cells) [72], [73]. Thus, spatial expression patterns of Kv4.2 expression suggest that it modulates the electrical signals originating from efferent synaptic activity. "
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    PLoS ONE 02/2010; 5(2):e9058. DOI:10.1371/journal.pone.0009058 · 3.23 Impact Factor
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    • "Yeast two-hybrid screening. The full BK channel inserted into pBD-Gal4 Cam (Stratagene) at the EcoRI and SalI restriction sites was used as bait to screen a cDNA library made from embryonic days 14–19 Gallus gallus sensory epithelium from the basilar papilla (i.e., cochlea) as described previously [8]. Yeast AH109 cells (Clontech) were transformed sequentially, first with pBDGal-4 containing the bait construct and then with pADGal-4/cDNA library using the lithium acetate/single-stranded carrier DNA/polyethylene glycol method [9]. "
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