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

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.

PPTX, a pentraxin domain‐containing protein, interacts with the T1 domain of Kv4

“…After 3 days, a single colony from each plate was isolated and grown O/N in SD-TRP medium at 308C. The yeast containing the bait plasmid was transformed with the pAD-Gal4 plasmid containing PPTX (Duzhyy et al, 2005) as described previously. An aliquot of 100 ll transformed cells was plated on SD-HLAT, SD-HLT plates, and SD-LT. Media were made from BIO 101 reagents (QBiogene Systems, Irvine, CA).…”

“…In the present study, we demonstrate that specific amino acids in the T1 domain of K v 4.2 bind PPTX. In addition, this interaction increases the expression of K v 4.2 in vitro, although the change is not observed at the biophysical level (Duzhyy et al, 2005).…”

“…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 1 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.…”

“…In the chick, this channel is arachidonic acid sensitive, inasmuch as both current and voltage responses are altered in hair cells by this molecule. Previously, we performed a yeast two-hybrid (Y2H) screening of a cDNA library made from embryonic chick cochlear epithelium, using part of the K v 4.2 N-terminus (aa 2-179) as bait (Duzhyy et al, 2005), to determine how this channel might be regulated during development. This screening revealed an interaction with a pentraxin-containing protein, referred to as PPTX.…”

Exploiting Protein Interaction Networks to Unravel Complex Biological Questions

“…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.…”