JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M500111200 on February 11, 2005

J. Biol. Chem., Vol. 280, Issue 15, 15165-15172, April 15, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/15/15165    most recent
M500111200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duzhyy, D.
Right arrow Articles by Sokolowski, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duzhyy, D.
Right arrow Articles by Sokolowski, B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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

Dmytro Duzhyy, Margaret Harvey, and Bernd Sokolowski{ddagger}

From the University of South Florida Department of Otolaryngology, Head and Neck Surgery, Department of Physiology and Biophysics, Tampa, Florida 33612

Received for publication, January 4, 2005 , and in revised form, February 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ion channels define the excitatory characteristics of sensory and neuronal cells. The A-type transient K+ channel has a role in this regulation by governing the frequency and amplitude of action potentials (1–3). Several genes contribute to the diversity of this {alpha}-subunit, including members of the Shaker and Shal subfamilies (4). Additionally, auxiliary subunits increase diversity by dramatically changing the function and expression of the {alpha}-subunits. For example, Kv{beta} accessory proteins can confer properties of inactivation to Shaker {alpha}-subunits that are non-inactivating (5, 6) in addition to regulating their cell surface expression (7). Other accessory subunits such as KChIP, frequenin, DPPX, MiRP, and MinK can alter the biophysical properties of Shal-subfamily members, Kv4.2 and Kv4.3 (8–13). Thus, the association of these subunits is significant to both channel expression and the generation of normal electrical signals.

The pentraxins belong to a superfamily of proteins that include laminin-G and thrombospondins (14). Moreover, they are secretory proteins that can be divided into the classical "short" pentraxins and the more recently discovered "long" pentraxins (for review see Ref. 15). The short pentraxins (~25 kDa) were described first in the 1930s, resulting in a large body of literature that includes C-reactive protein and serum amyloid P. The genes that encode these proteins are turned on in response to trauma and infection and are regulated by interleukin-1 and -6, tumor necrosis factor, and glucocorticoids. Recently, members of the short pentraxins were found in Limulus, suggesting that both members were present in common ancestors of arthropods and chordates (16). The long pentraxins (~50 kDa) include members such as the neuronal pentraxins (NP1, NP2, NPR), apexin (a protein associated with sperm-egg fusion), and tumor necrosis factor-stimulated gene-14. An additional member is PTX3, which differs from tumor necrosis factor-stimulated gene-14 by one amino acid (aa)1 and which was cloned from human vascular endothelial cells (15).

Potassium channels in the auditory and vestibular system play a central role in processing stimuli at the level of the receptor or hair cell. Their acquisition during development regulates the onset of hearing sensitivity, as was demonstrated with the calcium-activated K+ channel (17, 18). However, channel expression can vary depending on hair cell type and location on the sensory epithelium. Recently, we identified an arachidonic acid-sensitive, A-type channel in the cochlea of chicken (19) that belongs to the Shal subfamily. This Kv4.2 channel is localized to hair cells that are innervated exclusively by large calyceal efferents (20), which hyperpolarize the receptor cell (21). This negative change in membrane potential recruits a greater number of A-type channels, which can thus shape the excitatory response to a depolarizing stimulus (19). The acquisition of this channel occurs early in development, but little is know about the mechanisms that regulate its expression. We performed a yeast two-hybrid screening of an embryonic cDNA library, to begin defining proteins that interact with the A-type channel during development. Here, we localize and describe the characteristics of one such polypeptide, a pentraxin-like protein that interacts with Kv4.2 and has secretory characteristics.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction and Yeast Two-hybrid Screening—A plasmid (pBD-N) containing the sequence for the N terminus (aa 2–179) of chick Kv4.2 (accession number AF075160 [GenBank] ) in fusion with the GAL4 binding domain (BD), was constructed by inserting a PCR-amplified DNA fragment into the vector pBD-GAL4 Cam (Stratagene, La Jolla, CA), using the primers 5'-CTAGAATTCGCAGCGGGCGTACCAGCTTGGTTA-3' and 3'-CTAGTCGACTTTAGTGTGGGTTTTCAAAGGCCCGCCACAT-5'. Using the pBD-N construct as "bait," protein-protein interactions with Kv4.2 were determined by using the yeast two-hybrid system to screen a cDNA library made from cochlear sensory epithelium of embryonic day 14–19 Gallus gallus. The library was constructed by cloning cDNA fragments into ZAPII by EcoRI and XhoI restriction sites (Stratagene). Yeast AH109 cells (Clontech) were transformed sequentially with the bait construct and the cDNA library using the lithium acetate/single-stranded carrier DNA/polyethylene glycol method (22). Cells were plated on SD-His-Leu-Ade-Trp (SD-HLAT) medium (BIO 101 Systems, Qbiogene, Irvine CA), and positive colonies were selected and analyzed. Once cDNA fragments coding for PPTX were obtained, the 5'-end was determined by PCR, using the pAD-plasmid library as a template and the primers 5'-CGCGTTTGGAATCACTACAGGGA-3' and 3'-TCGTCGCCTTCATCCAGCACGGAA-5', which have annealing sites within the vector and the partially known PPTX cDNA sequence.

To determine the importance of the N terminus in the PPTX-Kv4.2 interaction, deletion variants of the N terminus of PPTX (pAD-N1, pAD-N2, and pAD-N3) were used in a yeast two-hybrid assay, using the N and C termini of Kv4.2 as bait. The three PPTX variants of the N terminus were obtained from screenings of the cDNA library and contained fragments that had deletions of aa 1–14, 1–139, and 131–154 of PPTX, respectively. To verify the importance of the PPTX C terminus, deletions of this end (pAD-C1, pAD-C2, and pAD-C3) were obtained by using pAD-N1 and deleting aa 214–433, 321–433, and 412–433 using restriction endonucleases BglII, BamHI-XbaI, and SmaI-XbaI, respectively.

Expression constructs for full-length PPTX and mutated recombinants were made by inserting the PPTX coding region into the vector pCMS-EGFP (Clontech). These constructs were used to characterize PPTX in CHO and COS-7 cells. To make insertions possible and to increase protein expression, PPTX cDNA was amplified with a forward primer coding for the XhoI site and a consensus Kozak sequence upstream of an ATG start codon and a reverse primer containing an XbaI site. The pAD-plasmid library or plasmid pAD-N1 served as a template. PCR was accomplished using high-fidelity Tgo DNA polymerase (Roche Applied Science). DNA fragments were gel-purified, digested with a mixture of XhoI and XbaI, and ligated with the vector. PCR was performed on a PTC-200 thermocycler (MJ Research, Waltham, MA) under the following conditions: 94 °C for 2 min, 35–38 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 2 min, and 1 cycle at 72 °C for 7 min.

The plasmid pCMS-PPTX, which expressed full-length PPTX, was constructed using PCR with the pAD-plasmid library as a template and the primers 5'-ACTACTCGAGCCACCATGCTGCCTGGAGTGCTTT-3' and 3'-ACTATCTAGATTAAGAAACATACTGAGCTCCTCCATA-5'. The plasmid pCMS-PPTXmut1-NF, containing the coding sequence for PPTX with a mutation of aa 6–20 that consisted of a deletion of aa 6–9 and a substitution (AQYVSDYKDDDDK), which included a FLAG tag (Sigma). The plasmid was constructed by first generating a PCR fragment using pADP-N1 as a template with primers 5'-CTACAAGGACGATGACGACAAGTCCGTGCTGGATGAAGGCGACGA-3' and 3'-ACTATCTAGATTAAGAAACATACTGAGCTCCTCCATA-5'. The fragment was gel-purified and amplified again with primers 5'-ACTACTCGAGCCACCATGCTGCCTGGAGTGCTTT-3' and 3'-ACTATCTAGATTAAGAAACATACTGAGCTCCTCCATA-5'. A PCR-generated fragment with two copies of hemagglutinin (HA) on the C terminus of Kv4.2 was inserted into pcDNA3.1(+) (Invitrogen) by exchanging a fragment, coding for EcoRI-XhoI on the C terminus. The fragment was generated with cKv4.2 as a template and primers 5'-CTACTCGAGCAACTTCAGCCGTATCTACCACCAG-3' and 3'-ACTACTCGAGTTAGGCATAATCCGGTACATCATAAGGGTAGGCATAATCCGGTACATCATAAGGGTATAAAGCGGATACTCTGACAATGTT-5'.

Transfections, Coimmunoprecipitations, and Western Blotting—CHO cell cultures were transfected with plasmid DNA (6 µg) using Lipofectamine (Invitrogen). For cotransfections, plasmids were used in a 1:1 ratio. After 5 h of incubation, transfection medium was replaced with fresh medium, and cells were incubated for 30–34 h. In experiments examining the mode of secretory transport, Brefeldin A, an inhibitor of a classical secretory pathway, was added to cell cultures at a concentration of 5–10 µg/ml. Cells were lysed by pipetting and then sonicating on ice in Tris buffer with 1% Triton X-100 and 0.2% SDS containing protease inhibitors. Lysates were cleared by centrifugation at 20,800 x g for 15 min at 4 °C. Cells were resuspended for fractionation studies in either 100 mM Na2CO3, pH 10, or STE buffer (150 mM NaCl, 25 mM Tris-HCl, 5 mM EDTA, pH 7.5) with protease inhibitors. Homogenates were triturated, spun at 1000 x g for 5 min, and pellets (P1 fractions) were resuspended in STE buffer. The supernatants (S1 fractions) were spun at 20,800 x g for 30 min at 4 °C. Supernatants (S2 fractions) were collected, and pellets (P2 fractions) were resuspended in STE buffer. All fractions were solubilized in equal volumes of Laemmli sample buffer with 5% {beta}-mercaptoethanol and boiled for 5 min.

Lysates were cleared for coimmunoprecipitation by centrifugation at 20,800 x g for 30 min and incubated at 4 °C for 6 h with antibody-bound rec-protein G-Sepharose 4B beads (Zymed Laboratories Inc.) (7.5 µg of anti-FLAG M2 mAb, Sigma, or 2 µg of anti-HA pAb, Abcam, Cambridge, MA). Beads were washed with lysis buffer, then MOPS buffer plus 0.2% Triton X-100. Samples were prepared for SDS-PAGE under denaturing, reducing conditions. Conditioned medium was spun for 20 min at 3000 x g and used for immunoprecipitation of secreted FLAG-tagged PPTX variants. Supernatant (1 ml) was combined with protease inhibitors, and beads that were preincubated with 5 µg of anti-FLAG antibody. Samples were incubated overnight at 4 °C and then prepared for SDS-PAGE analysis.

All samples were run in 10% polyacrylamide gels (Bio-Rad) under denaturing conditions. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) and blocked in Tris-buffered saline-Tween 20 containing 5% nonfat dry milk. Primary antibodies were diluted in blocking buffer and incubated at 4 °C overnight. Western blots were probed with anti-FLAG M2 mAb (1:1000), anti-HA pAb (1:4000), or anti-HA mAb (1:5000) (Sigma). Blots were developed using ECL and photographed using Hyperfilm ECL (Amersham Biosciences). Densitometry analysis was done using Spot Denso software on a ChemiImager 5500 (Alpha Innotech Corp., San Leandro, CA). Coimmunoprecipitation of PPTX and Kv4.2 using lysates of cochlear tissue were carried out as described previously (23).

Immunohistochemistry—Sensory and ganglion cells of the chick cochlea were prepared for immunostaining as described previously when using either immunofluorescence or horseradish peroxidase (19). The antibody used for these experiments consisted of an anti-PTX3 polyclonal (Alexis Biochemicals, San Diego, CA) raised against the entire length of human PTX3 (24). Comparisons of chick N- and C-terminal ends with human PTX3 reveals an identity of 41 and 67%, respectively. Western blot analysis showed that this antibody identified a polypeptide of similar weight to FLAG-tagged PPTX in CHO cells transfected with recombinant PPTX (data not shown). The anti-Kv4.2 monoclonal antibody was raised against aa residues 209–225 of rat Kv4.2 (AAB19939 [GenBank] . Comparison of the chick sequence (AAL56633 [GenBank] with that of rat shows that chick Kv4.2 has two additional amino acids between aa 458 and 459 of rat and a substitution of glutamine for proline at aa 464 of rat.

Immunohistochemical preparation of CHO and COS-7 cells transfected with recombinants was carried out as follows. Cells were cultured on coverslips in 10-cm2 culture dishes. CHO cells were transfected with pCMS-PPTXmut1-FL and pcDNA3.1(+):Kv4.2HA using Lipofectamine and incubated as above. COS cells were transfected with 1 µg each of pCMS-PPTXmut1-FL and pcDNA3.1(+):Kv4.2HA with 6 µl of FuGENE 6 (Roche Applied Science), incubated for 12 h in 2 ml of medium, and cultured in fresh medium for 24–36 h. Cells were washed, fixed in 4% paraformaldehyde, blocked in 10% fetal bovine serum, and incubated with primary Ab. Protein expression was detected with anti-FLAG M2 mAb (1:250) and/or anti-HA pAb (1:500), using goat anti-mouse Texas Red-X (1:150) or goat anti-rabbit Alexa Fluor 488 (1:300, Molecular Probes, Eugene, OR).

RT-PCR—Cochlear ducts, blood vessels, brain, heart, aorta, and eye were excised from an adult chicken and homogenized in Tri-Reagent (Molecular Research Center, Cincinnati, OH) to obtain total RNA. Cells were processed for RT-PCR as described previously (19). Primers used to generate PPTX products were 5'-ACTACTCGAGCCACCATGCTGCCTGGAGGAGTGCTTT-3' and 3'-CTAGAATTCGAGGTACAGCTGGATCTCATAGG-5'. Chick {beta}-actin served as a positive control with primers 5'-TGGATGATGATATTGCTGCG-3' and 3'-CTCCATATCATCCCAGTGG-5'. Reactions performed without reverse transcriptase served as a negative control. Total RNA in a quantity equivalent to one cochlea was used in a 20-µl reverse transcriptase reaction at 58 °C for 1 h. Reactions were performed for one cycle at 94 °C for 2 min, 28–40 cycles at 94 °C for 20 s, 60 °C for 20 s, 72 °C for 20 s, and 72 °C for 7 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Putative Structure—Yeast two-hybrid screening of ~1.5 x 106 pAD-library clones resulted in positive clones selected on SD-HLAT medium. The pAD-library plasmids were isolated from selected clones, amplified in Escherichia coli strain XL1, and retransformed back to yeast cells containing the vector, pBD-GAL4 Cam, or the plasmid pBD-N, that expressed the N-terminal end of Kv4.2 in fusion with a GAL4 binding domain. Plasmids supporting the growth of yeast cells on an SD-HLAT medium in the presence of the pBD-N plasmid, but not in the presence of the pBD-GAL4 Cam vector, were used as templates for cDNA sequencing. Sequences obtained from the 5'-end of these isolates revealed a protein with a pentraxin domain (PPTX). PCR was used to amplify the longest part of the PPTX cDNA region located next to the pAD-vector sequence, because there was a possibility that the N-terminal sequence was incomplete in these isolated clones. One of the primers in this PCR had an annealing site within the pAD-vector sequence, whereas another primer had an annealing site within a previously determined part of the PPTX cDNA. The sequence of the longest isolated PCR fragment revealed an additional 5'-end coding sequence for PPTX. Of 40 clones isolated, 38 were matched 100% to the PPTX sequence (Fig. 1), whereas two coded for a PPTX isoform with a deletion of aa 130–155.



View larger version (74K):
[in this window]
[in a new window]
  		FIG. 1.
Nucleotide and deduced aa sequence of PPTX (accession number AY672618 [GenBank] ). Putative transmembrane regions are underlined. Coiled coils are shown in bold italics, and the PTX "signature sequence" is in boldface. {uparrow} refers to putative N-linked glycosylation site at 159 and 330. The signal peptide is found within the first 21 aa.

 
Sequences obtained from RT-PCR products of cochlea, eye, brain, heart, and blood vessels of an adult chicken confirmed the expression of the longer RNA isoform in all tissues (Fig. 2). Primers were designed for RT-PCR by comparing the alignment data between PPTX and PTX3 (accession number AAH22176 [GenBank] and the intron-exon structure of PTX3, so that the amplified region would include parts of the potential first and third exons. This design would identify different splice variants of PPTX RNA; however, no such variants were determined by RT-PCR, which confirms the longer isoform as the major isoform.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 2.
RT-PCR products for PPTX (876 bp) are found in brain, heart, cochlea, eye, and blood vessels. PCR was performed with (+) and without (-) reverse transcriptase and {beta}-actin (247 bp) served as a positive control.

 
Computer analysis (Top Pred2) predicted two transmembrane regions in the primary structure of PPTX. One region, located at the very N-terminal end, is recognized as a signal peptide (SignalP 3.0), whereas the other divides PPTX into two unequal parts. The N terminus (residues 1–245) contains a coiled coil structure that has a 39% sequence identity to the mouse homolog of PTX3. Sequence identity between PPTX and PTX3 is distributed unevenly in the N terminus. Two blocks of the PPTX aa sequence (31–40 and 61–74) are preserved in PTX3, whereas the first 82 aa are 58% identical. The C terminus (residues 265–433) of PPTX contains a pentraxin domain that is 69% identical to mouse PTX3. In contrast, residues 83–265 of PPTX are only 42% identical to mouse PTX3. The uneven distribution of the homology score between three regions of PPTX has a similar pattern to the exon organization of PTX3. This result suggests that PPTX is a chicken PTX3 isoform with a sequence that deviated during evolution in the second exon. The PPTX sequence contains two putative N-glycosylation sites that may be responsible for the observed higher molecular mass (~55 kDa) than theoretically calculated (~47 kDa). Also, PPTX has nine Cys residues in its sequence of which some are probably responsible for PPTX multimer formation.

Colocalization of PPTX and Kv4.2 in the Cochlea—The Kv4.2-PPTX association in vitro led to an examination of expression in cochlear tissues. The results show that Kv4.2 and PPTX are colocalized to specific hair cells of the sensory epithelium as well as to the ganglion cells that innervate these receptor cells (Fig. 3, A–G). A cross-section of the sensory epithelium in Fig. 3A shows a gradation in hair cell length from tall, to intermediate, to short. PPTX (Fig. 3, A–D) colocalizes with Kv4.2 (Fig. 3, F and G) to the basal-lateral regions of intermediate and short hair cells but not tall hair cells. Previously, we showed that Kv4.2 is limited to the soma of the cochlear ganglion cells and the dendritic endings that innervate tall hair cells (19). Similarly, PPTX is found in the soma of cochlear ganglion cells (Fig. 3E). The interaction of PPTX and Kv4.2 was verified in the cochlea by coimmunoprecipitation. The products of coimmunoprecipitation reactions carried out with an anti-Kv4.2 antibody were analyzed for coprecipitation of PPTX. Fig. 3H shows that PPTX coprecipitates with Kv4.2 when using tissue lysates from the chick cochlea.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 3.
Colocalization and coprecipitation of PPTX and Kv4.2 in the cochlea. A, immunofluorescent staining with FITC of a cross-section of the chick cochlea shows the expression of PPTX in the basal-lateral ends of intermediate (arrows) and short hair cells (IHC, SHC) and a lack of reactivity in tall hair cells (THC). The primary antibody used was an anti-hPTX3 polyclonal. B–D, a series of photos at higher magnification revealing regions of immunostaining in hair cells when using a horseradish peroxidase-conjugated secondary antibody and diaminobenzamidine for visualization. Again, there is an absence of immunostaining in tall hair cells, whereas intermediate and short hair cells show immunoreactivity in basolateral regions (arrows). The round arrow identifies a large calyceal efferent nerve terminal juxtaposed against the short hair cell. E, immunoreactivity for PPTX is found also in cochlear ganglion cells. F and G, immunostaining with an anti-Kv4.2 antibody reveals Kv4.2 expression in the basolateral regions of intermediate and short hair cells (arrows). H, coimmunoprecipitation and immunoprecipitation of PPTX using cochlear tissue. The chick homologue of PTX3, PPTX, coprecipitates with Kv4.2 when using an anti-Kv4.2 antibody bound to protein G-Sepharose beads (left lane). Immunoprecipitating PPTX reveals the monomer as well as multimeric forms of PPTX (middle lane). Right lane is the bead control.

 
Verification of Interaction with Kv4.2 in Vitro—PPTX deletion variants were used in a yeast two-hybrid analysis to localize the region of PPTX that interacts with Kv4.2. This analysis revealed that deletions at the N terminus of PPTX did not impair yeast cell growth, whereas deletions of the C terminus prevented growth, including the last 22 aa residues (Fig. 4). To additionally test and characterize the Kv4.2-PPTX association, total lysates of CHO cells that expressed both recombinant PPTXmut1-NF and Kv4.2HA were used in coimmunoprecipitation experiments (Fig. 5). The justification for using PPTX-mut1 was based on experiments showing that PPTX undergoes truncation during expression in CHO cells (see below). Truncated variants accumulate intracellularly and are likely candidates for interaction with the N-terminal end of Kv4.2. Consequently, FLAG tags placed at the N terminus were cleaved from the protein together with the signal peptide. In comparison, FLAG tags at the C-terminal end produced no coprecipitates, suggesting that the tag interfered with PPTX-Kv4.2 interactions (data not shown). By introducing a mutation at the cleavage site, we were able to stabilize the protein and preserve the N-terminal FLAG tag necessary for antibody recognition. Moreover, this construct maintained the primary structure of wild type PPTX downstream from the cleavage site, including the C terminus, which was needed for the interaction with Kv4.2. Using this variant, anti-FLAG antibody bound to recprotein G-Sepharose beads coprecipitated Kv4.2HA when expressed with PPTXmut1-NF, but not when Kv4.2HA was expressed alone (Fig. 5A, right and middle lanes, respectively). The reciprocal experiments showed that anti-HA pAb coprecipitated PPTXmut1-NF when expressed with Kv4.2HA, but not when expressed alone (Fig. 5B, right and middle lanes, respectively).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4.
Analysis of PPTX interactions with Kv4.2. AH109 cells were cotransformed with a plasmid (pBD-N) that contained a portion of the N terminus of Kv4.2 (aa 2–179), in fusion with the GAL4 binding domain, and a plasmid (pAD) that contained a deletion variant in the N (N1–N3) or C terminus (C1–C3) of PPTX, in fusion with the GAL4-activating domain. A plasmid (pBD-C) that codes for the C terminus of Kv4.2 (aa residues 433–632) in fusion with the GAL4-BD served as a negative control in cotransformations with pAD-N1. The result shows that there is no association with the N terminus of Kv4.2 when portions of the C terminus of PPTX are deleted, including the last 22 aa.

 



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 5.
Coprecipitation using lysates from CHO cells transfected with FLAG-tagged PPTXmut1 and HA-tagged Kv4.2 recombinants. A, anti-FLAG mAb brings down Kv4.2 when coexpressed with PPTXmut1 (right lane), but not when Kv4.2 is expressed alone (middle lane). B, anti-HA pAb brings down PPTXmut1 when coexpressed with Kv4.2 (right lane), but not when PPTX-mut1 is expressed alone (middle lane). Left lanes in A and B are lysate controls. The antibody with which each blot was probed appears at the bottom of each figure.

 
PPTX and Kv4.2 colocalization in vitro was demonstrated by transfecting two different mammalian heterologous expression systems and using fluorescent immunostaining to visualize the expression in different regions of the cell. CHO cells expressing PPTX and Kv4.2 were used initially, as these revealed transient currents when using the whole-cell tight seal recording technique (data not shown). These cells showed PPTXmut1-NF as fluorescent red-colored clusters at the membrane (Fig. 6A), whereas expression of Kv4.2HA resulted in fluorescent green-colored clusters (Fig. 6B) when viewed with rhodamine and fluorescein isothiocyanate filters, respectively. Overlapping the two images demonstrated colocalization of the two proteins as visualized by yellow immunofluorescence (Fig. 6C). However, CHO cells showed a clustering of Kv4.2 in the absence of PPTX (data not shown). Because of the large surface area of COS cells, immunohistochemical colocalization was repeated in these cells to visualize the ability of PPTX to act as a shuttle protein. Previous studies suggested that Kv4.2 is locked in the endoplasmic reticulum (ER) of COS cells in the absence of a shuttle protein such as KChIP (25). Our results show that Kv4.2 was colocalized with PPTX in the region of the ER (Fig. 6, D–F), whereas both proteins were absent at the membrane. Similar outcomes were obtained when cells were cotransfected with plasmids expressing Kv4.2HA and full-length, non-tagged, wild type PPTX, where the latter was detected with PTX3 pAb (data not shown).



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 6.
Immunolocalization of PPTX and Kv4.2 in CHO and COS-7 cells. CHO cells cotransfected with PPTX-mut1-NF (A) and Kv4.2HA (B) show fluorescent red (Texas Red-X) or green (Alexa Fluor 488) clusters when viewed with rhodamine or fluorescein isothiocyanate filters, respectively. C, overlapping the two images in A and B shows colocalization at the cell membrane, as visualized by fluorescent yellow. COS-7 cells cotransfected with PPTX-mut1-NF (D) and Kv4.2HA (E) show fluorescent red and green in regions of the ER when viewed with the aforementioned filters, respectively. F, overlapping the two images in D and E shows colocalization in the region of the ER as seen by fluorescent yellow.

 
PPTX Is a Secretory Protein—Most of the known long pentraxins, including NP1, Narp, and PTX3 are secreted proteins, whereas others, such as NPR, are membrane-bound receptors. Computer analysis predicted a signal peptide with a cleavage site between aa 20 and 21 of the N terminus. To determine how this signal peptide affects the intracellular and extracellular distribution of PPTX, we transfected CHO cells with either PPTXmut1-NF, which contained aa substitutions that included a FLAG sequence at the cleavage site (Fig. 7A), or with full-length PPTX, which contained a FLAG-tagged C terminus (PPTX-CF). The results show that the 55-kDa polypeptide (PPTX-CF lysate), observed in Fig. 7B, lane 1 (far left), is the full-sized PPTX protein. This result was confirmed with an anti-hPTX3 pAb, which also recognized a 55-kDa polypeptide for both wild type, non-tagged PPTX and PPTX-CF (data not shown). In addition to the full-length precursor (55 kDa), lane 1 shows that PPTX undergoes truncation, leading to the intracellular accumulation of 45- and 50-kDa polypeptides and to a lesser extent a 30-kDa species (Fig. 7B). A fifth band (36 kDa) in lane 1 did not immunoprecipitate with anti-FLAG Ab and is considered a nonspecific protein (data not shown). In comparison, Fig. 7B, lane 2 shows that the signal peptide is necessary for truncation as only the 55-kDa precursor is present when there is a mutation of this peptide (PPTXmut1-NF).



View larger version (34K):
[in this window]
[in a new window]
  		FIG. 7.
Secreted and non-secreted products of PPTX variants in CHO cells. A, alignment of the N-terminal sequences of wild type PPTX with the mutation variant shows the identical (boldface) and non-identical (italics) aa and the signal peptide cleavage site (underlined). The mutated variant has 4 aa deleted and 13 aa substituted with a FLAG-tag (PPTXmut1-NF) in the region of the signal peptide. B, Western blots of protein samples obtained from total lysates or immunoprecipitated (IP) secreted products. CHO cells were transfected with either full-length recombinant PPTX containing an intact signal peptide with a FLAG-tagged C terminus (PPTX-CF) or PPTXmut1-NF. When the signal peptide is intact, CHO cells express a 55-kDa full-length precursor polypeptide and truncated variants of 30, 45, and 50 kDa (lane 1, far left). The truncated variants are not expressed in cells transfected with PPTXmut1-NF (lane 2). In comparison, the secreted products of CHO cells transfected with PPTX-CF include the precursor, two truncated variants (30 and 50 kDa), and a 60-kDa species (lane 3). The administration of Brefeldin A (Bref. A) blocks all variants except for some of the 50- and 55-kDa species, suggesting some secretion via a non-classical pathway (lane 4). In comparison, a mutation of the signal peptide produces a single secretory product of 55 kDa regardless of the absence (lane 5) or presence (lane 6) of Brefeldin A. Immunoprecipitation was accomplished using anti-FLAG pAb, whereas Western blots were probed with anti-FLAG mAb preadsorbed against rabbit serum. Negative controls consisted of using medium containing secreted products and protein-G beads without Ab (lanes 8, and 9). The 36-kDa band is a nonspecific product (lanes 1 and 2).

 
We then examined the secretory products of PPTX-CF and PPTXmut1-NF by immunoprecipitation, in the absence and presence of Brefeldin A. Brefeldin A blocks protein transport between the ER and Golgi, allowing a determination of the secretory pathways taken by the precursor and each of the truncated variants. In the absence of Brefeldin A, CHO cells transfected with PPTX-CF secreted a 60-kDa species and to a lesser degree, 30-, 50-, and 55-kDa polypeptides (Fig. 7B, lane 3). The 60-kDa polypeptide may be a dimer of the 30-kDa truncated variant, although additional analyses are necessary to verify this observation. In the presence of Brefeldin A, CHO cells transfected with PPTX-CF secreted lesser amounts of the 50- and 55-kDa polypeptides, whereas the 30- and 60-kDa species were blocked completely (Fig. 7B, lane 4). CHO cells transfected with mutant PPTX secreted the 55-kDa precursor but not the truncated variants, regardless of the absence or presence of Brefeldin A (Fig. 7B, lanes 5 and 6, respectively). These results suggest that secretion of the 30- and 60-kDa species proceeds through a classical ER-Golgi pathway, requiring a signal peptide for processing, whereas some portion of the 50- and 55-kDa polypeptides is secreted via a non-classical pathway. Control experiments show that immunoprecipitation without an antibody resulted in the absence of product (Fig. 7B, lanes 7 and 8).

The prediction of transmembrane regions in PPTX (aa 245–265) and the accumulation of truncated PPTX variants inside the cell compelled us to investigate whether PPTX resides as a peripheral or transmembrane protein by examining the lysates of PPTX-CF-transfected CHO cells. For this purpose, we used a sodium carbonate buffer, which solubilizes both peripheral membrane proteins and membrane-enclosed contents but not transmembrane proteins (26, 27). Equivalent volumes of fractions P1, P2, S2, and total lysates were analyzed by Western blotting using anti-FLAG (i.e. PPTX) or anti-HA (i.e. Kv4.2) mAb (Fig. 8A). HA-tagged Kv4.2 served as a positive control, because this channel protein has six transmembrane regions. Treatment with carbonate buffer increased the amount of material in the membrane fraction (P2) and cleared the fraction of proteins not tightly associated with the membrane. Thus, the signal for Kv4.2HA (~74 kDa) increased with carbonate treatment in the cleaned membrane fraction, P2, compared with the soluble fraction (Fig. 8A, upper lanes). The 85-kDa species does not immunoprecipitate with HA-tagged Kv4.2 and is considered a nonspecific product. In contrast, the different intracellular PPTX variants (45, 50, and 55 kDa) segregated primarily to the soluble fraction in the carbonate-treated condition (S2; Fig. 8A, lower lanes). The solubility of the 55-kDa precursor and its 45- and 50-kDa truncated variants were quantified using densitometry. With carbonate treatment, the 55-(98 ± 2%), 45-(94 ± 5%), and 50-kDa (86 ± 8%) variants segregated primarily with the soluble fraction, whereas a portion (2, 6, and 14%, respectively) remained bound to the membrane (Fig. 8B). In comparison, nearly 100% of Kv4.2 remained in the pellet. A summary diagram of the interaction and distribution of PPTX and its truncated variants are shown in Fig. 9.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 8.
Localization of PPTX in cellular fractions treated with Na+ carbonate. CHO cells, expressing either full-length FLAG-tagged PPTX or full-length HA-tagged Kv4.2, were lysed and treated with either 0.1 M Na2CO3 or STE buffer. Samples consisted of total lysates or cell fractions (P1, P2, or S2) in either buffer. All fractions were solubilized in the same volume of Laemmli sample buffer, and equal volumes of samples were loaded on the gel. Western blots were probed with anti-HA or anti-FLAG mAbs. A, the results show that the 55-kDa precursor segregates to the soluble portion in either solution, whereas the truncated variants segregate to the soluble portion when treated with carbonate. In comparison, Kv4.2 segregates to the membrane portion. The 85-kDa species is a nonspecific product found in the soluble portion in either treatment. B, plot showing integral density (ID) values measured for each band of the cleared carbonate membrane P2 (p) or soluble S2 (s) fractions normalized to the total amount (IDp + IDs) in the two fractions. In carbonate buffer, products (45, 50, 55 kDa) obtained from CHO cells expressing wild type PPTX segregate primarily with the soluble fraction (open bars). In contrast, products (74 kDa) obtained from CHO cells transfected with Kv4.2HA segregate with the membrane fraction (closed bars).

 



View larger version (67K):
[in this window]
[in a new window]
  		FIG. 9.
A drawing showing the intra- and extracellular distribution of different PPTX species. The full-length (55 kDa) variant of PPTX is a peripheral membrane protein, with an N-terminal signal peptide (SP) and a cleavage site between aa 20 and 21. The last 22 aa of the C-terminal end are necessary for PPTX to interact with the N terminus of Kv4.2. This ion channel contains six transmembrane domains and a voltage-activated pore between the fifth and sixth domains. PPTX undergoes truncation leading to the accumulation of 30-, 45-, and 50-kDa variants. The 30-, 50-, and 55-kDa species are secreted and found with a 60-kDa species that does not accumulate in the cytosol. This species may be a dimer of the 30-kDa variant, which is produced via a classical secretory pathway (solid arrow). In comparison, a portion of the 50- and 55-kDa species may follow this path (stippled arrow). Presently, only full-length PPTX appears to interact with Kv4.2 in native tissues.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Protein sequence analysis showed that PPTX belongs to the family of long pentraxins, which include PTX3 and the neuronal pentraxins NP1, Narp, and NPR (28–31). All members of this family have a pentraxin domain in their C terminus that is homologous to the short pentraxins, such as C-reactive protein and serum amyloid P (32, 33). The C terminus of the long pentraxins is more highly conserved among family members than the N terminus, which contains a coiled coil structure and a putative signal peptide. The latter region is necessary for the export of secreted proteins and serves as a membrane anchor in the case of extracellularly localized NPR (31). PPTX shares all the aforementioned structural features of the long pentraxins. In this study, we determined the specific characteristics of PPTX expression and function. Some of these characteristics, such as secretion, are similar to those displayed by other pentraxins. However, unlike these proteins, the PPTX-Kv4.2 interaction occurs intracellularly as demonstrated by the yeast two-hybrid assay and by models of Kv4.2, which show that the N terminus is located on the cytoplasmic side of the plasmalemma. Furthermore, our data suggest that interactions may occur with truncation variants of PPTX that accumulate in CHO cells. Presently, the Narp-AMPA receptor interaction, which occurs extracellularly, is the only known case of a pentraxin-ion channel association (34); however, PPTX is only 21% identical to Narp at the aa level.

The cochlea of the chick is composed of specific cell types that are defined by their length. The tall hair cells are thought to be analogous to the inner hair cells in mammals, whereas the intermediate and short hair cells are analogous to the outer hair cells and, like these receptors, receive most of the efferents originating in the central nervous system. This pattern of innervation is particularly defined in the short hair cells of the chick where large calyceal efferent endings synapse at the base of these cells (20). Thus, the colocalization of PPTX and Kv4.2 to intermediate and short hair cells underscores their potential as interacting partners, and this potential was realized by the coprecipitation of PPTX with Kv4.2.

The presence of a putative signal peptide sequence at the N-terminal end of PPTX led us to hypothesize that this protein belongs to a family of classically secreted proteins. These proteins use the ER-Golgi pathway and contain a signal peptide that is cleaved during secretion (35). In our experiments, PPTX was expressed as a 55-kDa protein with truncated variants of 30-, 45-, and 50-kDa. In comparison, secreted species had molecular masses of 30, 50, 55, and 60 kDa. A mutation introduced at the cleavage site of the signal peptide prevented the truncation of both secreted and non-secreted products indicating that truncation is dependent on the integrity of this region. This outcome, in addition to the partial inhibition of full-length PPTX secretion by Brefeldin A, suggests that this polypeptide is secreted partly via a classical pathway. However, Brefeldin A did not inhibit secretion of the 50-kDa polypeptide nor the PPTXmut1, suggesting that certain variants of PPTX may undergo non-classical export. These results are corroborated by the finding that a portion of the 50- and 55-kDa polypeptide segregates (14 and 2%, respectively) with the membrane fraction, suggesting that non-classical secretion may occur through interactions with the membrane by mechanisms that remain to be determined. Examples of proteins that follow such a path include hydrophilic acylated surface protein B (HASPB) and galectins (for review see Ref. 36). Galectins and pentraxins have similar functions and belong to a larger family, the lectins (for review see Ref. 37).

In addition to affecting truncation, mutations of the signal peptide affect the size of products that are secreted and that accumulate intracellularly. Mutation of aa 6–20 impaired the intracellular accumulation of 30, 45 and 50 kDa PPTX polypeptides, suggesting the modification of a signal sequence that defines the truncated forms. Thus, this region includes aa residues that contain signals necessary for modifications leading to both the accumulation of intracellular variants and the truncation of PPTX prior to secretion.

We examined the possible transmembrane localization of PPTX, because the computer model predicted two such regions in the primary structure. The results show that a major portion of PPTX remains in the soluble fraction when using a carbonate buffer. In contrast, the transmembrane protein, Kv4.2 clearly segregates to the membrane fraction. Thus, whereas PPTX may not interact with the cellular membrane directly, it may associate with the membrane through carbohydrate groups as reported for HASPB and galectin-3 (38, 39). Moreover, a recent finding shows that neuronal pentraxins, containing a chromo-domain, are localized in the cytosol and associate with the face of the inner plasma membrane (40). Various isoforms of this protein are found in the brain within distinct cell types, suggesting that the function of pentraxin-containing proteins is more complex than originally thought.

The yeast two-hybrid and coprecipitation studies show that PPTX interacts with Kv4.2. Localization studies confirm that PPTX and Kv4.2 cocluster at the plasmalemma surface of CHO cells. Presently, the only known case of a pentraxin-ion channel association is that of the Narp-AMPA receptor (34). However, we did not see qualitative differences in the ability of Kv4.2 to form clusters in CHO cells in the presence or absence of PPTX. A potential reason for this result may be that Kv4.2 clustering in CHO cells is regulated by other proteins or Kv4.2 is prone to aggregate naturally, particularly when overexpressed. Our colocalization experiments in COS cells suggest that PPTX does not release Kv4.2 from the ER. This characteristic is unlike that of KChIP, which releases Kv4 from the ER and acts as a shuttle protein (25). Moreover, our whole-cell tight seal recordings suggest that PPTX does not alter the biophysical properties of inactivation of Kv4.2 when coexpressed in CHO cells. Consequently, the role of PPTX in relation to Kv4.2 may be one that has not yet been identified for accessory subunits that interact with these voltage-gated channels.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY672618 [GenBank] .

* This work was supported by Grant DC43095 from the NIDCD, National Institutes of Health (to B. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: University of South Florida, Otology Laboratory MDC83, Dept. of Otolaryngology-Head and Neck Surgery, 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel.: 813-974-5988; Fax: 813-974-1483; E-mail: bsokolow{at}hsc.usf.edu.

1 The abbreviations used are: aa, amino acid; CHO, Chinese hamster ovary; HA, hemagglutinin; MOPS, 4-morpholinepropanesulfonic acid; RT-PCR, reverse transcription-PCR; mAb, monoclonal antibody; pAb, polyclonal antibody; ER, endoplasmic reticulum; AMPA, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Back


   ACKNOWLEDGMENTS
 
We thank Drs. S. Heller (Harvard University) for the cochlea cDNA library, J. S. Trimmer (University of California, Davis) for the anti-Kv4.2 monoclonal antibody, and C. Venkataramu for excising and amplifying the cDNA library.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Connor, J. A. & Stevens, C. F. (1971) J. Physiol. 213, 21-30[Abstract/Free Full Text]
  2. Neher, E. (1971) J. Gen. Physiol. 58, 36-53[Abstract/Free Full Text]
  3. Rogawski, M. A., Beinfeld, M. C., Hays, S. E., Hokfelt, T. & Skirboll, L. R. (1985) Ann. N. Y. Acad. Sci. 448, 403-412[CrossRef][Medline] [Order article via Infotrieve]
  4. Salkoff, L., Baker, K., Butler, A., Covarrubias, M., Pak, M. D. & Wei, A. (1992) Trends Neurosci. 15, 161-166[CrossRef][Medline] [Order article via Infotrieve]
  5. Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej, D. N., Dolly, J. O. & Pongs, O. (1994) Nature 369, 289-294[CrossRef][Medline] [Order article via Infotrieve]
  6. Heinemann, S. H., Rettig, J., Wunder, F. & Pongs, O. (1995) FEBS Lett. 27, 383-389
  7. Shi, G., Nakahira, K., Hammond, S., Rhodes, K. J., Schechter, L. E. & Trimmer, J. S. (1996) Neuron 16, 843-852[CrossRef][Medline] [Order article via Infotrieve]
  8. Kuryshev, Y. A., Gudz, T. I., Brown, A. M. & Wible, B. A. (2000) Am. J. Physiol. 278, C931-C941
  9. An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S. & Rhode, K. J. (2000) Nature 403, 553-556[CrossRef][Medline] [Order article via Infotrieve]
  10. Nakamura, T. Y., Poutney, D. J., Ozaita, A., Nandi, S., Ueda, S., Rudy, B. & Coetzee W. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12808-12813[Abstract/Free Full Text]
  11. Nadal, M. S., Ozaita, A., Amarillo, J., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A. & Rudy, B. (2003) Neuron 37, 449-461[CrossRef][Medline] [Order article via Infotrieve]
  12. Zhang, M., Jiang, M. & Tseng, G. N. (2001) Circ. Res. 88, 1012-1019[Abstract/Free Full Text]
  13. Deschenes, I. & Tomaselli, G. F. (2002) FEBS Lett. 528, 183-188[CrossRef][Medline] [Order article via Infotrieve]
  14. Beckmann, G., Hanke, J., Bork, P. & Reich, J. G. (1998) J. Mol. Biol. 275, 725-730[CrossRef][Medline] [Order article via Infotrieve]
  15. Goodman, A. R., Cardozo, T., Abagyan, R., Altmeyer, A., Wisniewski, H. G. & Vilcek, J. (1996) Cytokine Growth Factor Rev. 7, 191-202[CrossRef][Medline] [Order article via Infotrieve]
  16. Shrive, A. K., Metcalfe, A. M., Cartwright, J. R. & Greenhough, T. J. (1999) J. Mol. Biol. 290, 997-1008[CrossRef][Medline] [Order article via Infotrieve]
  17. Fuchs, P. A. & Sokolowski, B. H. (1990) Proc. R. Soc. Lond. B. Biol. Sci. 241, 122-126[Medline] [Order article via Infotrieve]
  18. Kros, C. J., Ruppersberg, J. P. & Rüsch, A. (1998) Nature 394, 281-284[CrossRef][Medline] [Order article via Infotrieve]
  19. Sokolowski, B. H. A., Sakai Y., Harvey M. C. & Duzhyy D. E. (2004) J. Neurosci., 24, 6265-6276[Abstract/Free Full Text]
  20. Fischer, F. P. (1992) Hear. Res. 61, 167-178[CrossRef][Medline] [Order article via Infotrieve]
  21. Fuchs, P. A. & Murrow, B. W. (1992) J. Neurosci. 12, 800-809[Abstract]
  22. Gietz, R. D. & Woods, R. A. (2002) Methods Enzymol. 350, 87-96[CrossRef][Medline] [Order article via Infotrieve]
  23. Sokolowski, B. H. A., Harvey, M. C., Venkataramu, C., Duzhyy, D. E. (2005) J. Neurosci. Res. 79, 459-467[CrossRef][Medline] [Order article via Infotrieve]
  24. Battazii, B., Vouret-Craviari V., Bastone, A., De Gioia, L., Matteucci, C., Peri, G., Spreafico, F., Pausa, M., D'Ettorre, C., Gianazza, E., Tagliabue, A., Salmona, M., Tedesco, F., Introna, M. & Mantovani, A. (1997) J. Biol. Chem. 272, 32817-32823[Abstract/Free Full Text]
  25. Shibata, R., Misonou, H., Campomanes, C. R., Anderson, A. E., Schrader, L. A., Doliveira, L. C., Carroll, K. I., Sweatt, J. D., Rhodes, K. J. & Trimmer, J. S. (2003) J. Biol. Chem. 278, 36445-36454[Abstract/Free Full Text]
  26. Fujiki, Y., Hubbard, A. L., Fowler, S. & Lazarow, P. B. (1982) J. Cell Biol. 93, 97-102[Abstract/Free Full Text]
  27. Howell, K. E. & Palade, G. E. (1982) J. Cell Biol. 92, 822-832[Abstract/Free Full Text]
  28. Breviario, F. S., d'Aniello, E. M., Golay, J., Peri, G., Bottazzi, B., Bairochll, A., Sacconell, S., Marzella, R., Predazzi, V., Rocchi, M., Della Valle, G., Dejana, E., Mantovanil, A. & Introna, M. (1992) J. Biol. Chem. 267, 22190-22197[Abstract/Free Full Text]
  29. Schlimgen, A. K., Helms, J. A., Vogel, H. & Perin, M. S. (1995) Neuron 14, 519-526[CrossRef][Medline] [Order article via Infotrieve]
  30. Tsui, C. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Barnes, C. & Worley, P. F. (1996) J. Neurosci. 16, 2463-2478[Abstract/Free Full Text]
  31. Dodds, D. C., Omeis, I. A., Cushman, S. J., Helms, J. A. & Perin, M. S. (1997) J. Biol. Chem. 272, 21488-21494[Abstract/Free Full Text]
  32. Oliveira, E. B., Gotschlich, C. & Liu, T. Y. (1979) J. Biol. Chem. 254, 489-502[Abstract/Free Full Text]
  33. Gewurz, H., Zhang, X. H. & Lint, T. F. (1995) Curr. Opin. Immunol. 7, 54-64[CrossRef][Medline] [Order article via Infotrieve]
  34. O'Brien, R. J., Xu, D., Petralia, R. S., Steward, O., Huganir, R. L. & Worley, P. (1999) Neuron 23, 309-323[CrossRef][Medline] [Order article via Infotrieve]
  35. Walter, P., Gilmore, R. & Blobel, G. (1984) Cell 38, 5-8[CrossRef][Medline] [Order article via Infotrieve]
  36. Nickel, W. (2003) Eur. J. Biochem. 270, 2109-2119[Medline] [Order article via Infotrieve]
  37. Kaltner, H. & Gabius, H. J. (2001) Adv. Exp. Med. Biol. 491, 79-94[Medline] [Order article via Infotrieve]
  38. Denny, P. W., Gokool, S., Russell, D. G., Field, M. C. & Smith, D. F. (2000) J. Biol. Chem. 275, 11017-11025[Abstract/Free Full Text]
  39. Mehul, B. & Hughes, R. C. (1997) J. Cell Sci. 110, 1169-1178[Abstract]
  40. Chen, B. & Bixby, J. L. (2005) J. Comp. Neurol. 481, 391-402[CrossRef][Medline] [Order article via Infotrieve]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/15/15165    most recent
M500111200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duzhyy, D.
Right arrow Articles by Sokolowski, B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duzhyy, D.
Right arrow Articles by Sokolowski, B.
Social Bookmarking
Add to CiteULike   Add to Complore&