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

ABSTRACT 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.

Download full-text


Available from: Bernd Sokolowski, Aug 05, 2015
  • Source
    • "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
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Owing to the multifaceted functions of the large conductance Ca(2+)-activated K(+) channel (BK), identification of protein-protein interactions is essential in determining BK regulation. A yeast two-hybrid screening of a cochlear cDNA library revealed a BK-ApoA1 interaction. Patch clamp recordings of excised membrane patches from transfected HEK293 cells showed that ApoA1 inhibits the BK alpha-subunit by significantly increasing activation and deactivation times, and shifting half-activation voltage to more positive potentials. Reciprocal coimmunoprecipitations verified the BK-ApoA1 interaction using excised sensory epithelium and ganglia. Additionally, immunocolocalization studies revealed BK and ApoA1 expression in both receptor cells and auditory neurons. These data suggest new avenues of investigation, given the importance of apolipoproteins in neurological diseases.
    Biochemical and Biophysical Research Communications 08/2009; 387(4):671-5. DOI:10.1016/j.bbrc.2009.07.074 · 2.28 Impact Factor
  • Source
    • "Mutation of either of these aa inhibits tertiary folding and, thus, tetramerization of K v 1.3. These experiments, in addition to the results reported here, suggest that PPTX may be important in channel trafficking or assembly, an interaction that is likely to begin as PPTX increases K v 4.2 channel density at the plasmalemma, as reported previously (Duzhyy et al., 2005). In that previous study, expression in COS cells suggested that the K v 4.2/PPTX complex was locked in the ER, a phenomenon that may occur in CHO cells under the present culture parameters. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Voltage-gated K(+) (K(v)) channels reside as tetramers in the membrane. The events that coordinate folding, trafficking, and tetramerization are mediated by an array of associated proteins and phospholipids whose identification is vital to understanding the dynamic nature of channel expression and activity. An interaction between an A-type K(+) channel, K(v)4.2, and a protein containing a pentraxin domain (PPTX) was demonstrated in the cochlea (Duzhyy et al. [ 2005] J. Biol. Chem. 280:15165-15172). Here, we present results based on fold recognition and homology modeling that revealed the tetramerization (T1) domain of K(v)4.2 as a potential docking site for interacting proteins such as PPTX. By using this model, putative sites were experimentally tested with the yeast two-hybrid system to assay interactions between PPTX and the T1 domain of K(v)4.2 wild type (wt) and mutants (mut). Results showed that amino acid residues 86 and 118 in the T1 domain are essential for interaction, because replacing these negatively charged with neutrally charged amino acids inhibits interactions. Cotransfections of Chinese hamster ovary cells with PPTX and K(v)4.2wt further revealed that PPTX increases K(v)4.2 wt expression in vitro when analyzing total lysates, whereas interactions with K(v)4.2 microt resulted in a decrease. These studies suggest that portions of the T1 domain can act as docking sites for proteins such as PPTX, further underscoring the significance of this domain.
    Journal of Neuroscience Research 06/2009; 87(8):1841-7. DOI:10.1002/jnr.22016 · 2.73 Impact Factor
Show more