spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 14 February 2006
doi: 10.1242/jcs.02799

Journal of Cell Science 119, 898-909 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
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 HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carteron, C.
Right arrow Articles by Cabedo, H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carteron, C.
Right arrow Articles by Cabedo, H.
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   Add to Twitter  
What's this?

Characterization of a neural-specific splicing form of the human neuregulin 3 gene involved in oligodendrocyte survival

Christelle Carteron1, Antonio Ferrer-Montiel2 and Hugo Cabedo1,3,*

1 Instituto de Neurociencias UMH-CSIC, Alicante, Spain
2 Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Alicante, Spain
3 Unidad de Investigación del Hospital de Sant Joan d'Alacant, Alicante, Spain


Figure 1
View larger version (47K):

[in a new window]
  		Fig. 1. Sequence comparison of hFBNRG3 and hNRG3. (A) Alignment of the hFBNRG3 and hNRG3 transcripts to the human genome by BLAST search. Exon 1 is spliced out in hFBNRG3, whereas exons 2 and 3 are spliced out in the hNRG3 transcript. Exons 4 to 11 are shared by both transcripts. (B) Predicted aa sequences for both transcripts are compared. EGF-like domains and transmembrane segments are indicated, and also the N-terminal hydrophobic segment for hFBNRG3. Both polypeptides diverge completely in the N-terminal domain and are preserved from the EGF-like to C-end. Perfectly conserved aa are underlined with asterisks.

 

Figure 2
View larger version (33K):

[in a new window]
  		Fig. 2. hFBNRG3 is expressed in the human foetal brain. RT-PCR was used to detect mRNA expression in the human foetal tissues. An amplicon of the expected size was obtained from human foetal brain but not from other foetal tissues (top). The molecular identity of the amplicon was verified after its cloning and sequencing. Quality of cDNA synthesis was checked by amplifying the housekeeping gene ß-actin (bottom).

 

Figure 3
View larger version (62K):

[in a new window]
  		Fig. 3. hFBNRG3 is a surface-exposed plasma membrane protein. (A) Confocal microscopy images show plasma membrane location of the hFBNRG3 polypeptide (COS-7 cells were transfected with hFBNRG3-myc, incubated with MG-132 and processed for immunocytochemistry as described in Materials and Methods). (B) hFBNRG3 is also inserted to the plasma membrane of the neuronal-model cell line PC12. (C) CFP-hFBNRG3 polypeptide localizes on the plasma membrane of COS-7 cells. (D) CFP-SMDF, a plasma membrane neuregulin, was used as control. (E) The soluble CFP showed a wide distribution pattern. Arrows indicate plasma membranes. Bars, 10 µm. (F) hFBNRG3 is considerably associated with the insoluble fraction of crude plasma membrane preparations (P) but not with the soluble fraction (S). SMDF was used as a control. (G) Biotinylation increased the recovery of neuregulin in the streptavidin precipitates, showing that hFBNRG3 is a surface exposed polypeptide. Lysate of nonbiotinylated cells was used as control.

 

Figure 4
View larger version (36K):

[in a new window]
  		Fig. 4. The ubiquitin/proteasome system regulates the protein stability of hFBNRG3. (A) Proteasome inhibition allows the detection of hFBNRG3 protein expression in anti-myc western blots of transiently transfected COS-7 cells. Nontreated cells (lane 5) showed no immunoreactivity at these conditions. Treatment with three different proteasome inhibitors Lactacystin, Z-Leu-Leu-Phe-CHO (Z-L-L-F-al) and MG-132 (lanes 6, 7 and 8, respectively) resulted in the appearance of immunoreactivity. The transfection of the inversely oriented cDNA produced no immunoreactivity (lane 1), even in the presence of the proteasome inhibitors (lanes 2, 3 and 4). Similar results were obtained in the neuron-like PC12 cells (lanes 9 to 12). Although a faint band was detected in nontreated PC 12 cells (lane 12) it was shown to be unspecific because it was also present in cells transfected with inversely oriented cDNA. (B) Time course of protein stabilization after proteasome inhibition with 2 µM MG-132 in COS-7 cells. As shown, immunoreactivity was already detected after 4 hours of proteasome inhibition. (C) Impairment of the ubiquitylation machinery by incubating the cells at 42°C increased the protein stability in the mammalian conditional mutant cell line ts20. An unspecific band of ~65 kDa was detected at 42°C even in cells that had been transfected with inversely oriented cDNA.

 

Figure 5
View larger version (25K):

[in a new window]
  		Fig. 5. Constructs used in this study. The ORF from hFBNRG3 was cloned in-frame with a myc epitope at the C-terminus of the protein (a) or an HA epitope in the N-terminal domain (b). Amino acids 3 to 55 were deleted in the myc-tagged construct (c). The ORF of hFBNRG3 was cloned in-frame after CFP (d). Extracellular and transmembrane (e) intracellular (f) or N-terminal domains (g) were deleted from the CFP-hFBNRG3 construct. A construct bearing the N-terminal domain and part of the EGF-like domain attached to CFP was also produced (h). Synaptotagmin I with a myc-epitope at the C-terminus (i) and a chimeric protein containing the 1-92 N-terminal aa of hFBNRG3 fused to synaptotagmin I myc (j) were also used.

 

Figure 6
View larger version (49K):

[in a new window]
  		Fig. 6. The N-terminal domain bears a cleavable signal sequence and confers instability to hFBNRG3. (A) Attachment of the HA epitope to the N-terminal domain of hFBNRG3-myc produced no immunoreactivity in anti-HA immunoblots (lane 4) even in the proteasome inhibited cells (lane 3), whereas an HA-tagged control protein was clearly detected (lane 2). As expected, HA immunoreactivity was not detected in non-HA-tagged hFBNRG3 (lanes 5 and 6) and nontransfected cells (lane 1). The band of ~90 kDa present in the immunoblots is unspecific as demonstrated by its presence in the nontransfected cells. (B) HA-hFBNRG3-myc was expressed at levels comparable with those of hFBNRG3-myc as demonstrated by the anti-myc immunoblot of aliquots of the same cell extracts. (C) Protein stability is increased when the peptide harbouring the signal sequence is deleted (lane 3). The attachment of the peptide to the transmembrane protein synaptotagmin I downregulates its expression levels in transfected cells (lane 5). (D) N-terminal domain bearing CFP chimeras (lanes 1, 3 and 5) were hardly detectable in anti-GFP immunoblots, whereas peptide-deleted chimeras (lanes 2 and 4) showed a considerable increase in protein levels. COS-7 cells were transfected with the indicated vectors and cell extracts were immunoblotted with anti-myc, anti-HA or anti-GFP antibody.

 

Figure 7
View larger version (29K):

[in a new window]
  		Fig. 7. hFBNRG3 is exported to the plasma membrane and released to the extracellular medium. (A) hFBNRG3 is post-translationally modified with N-acetyl-galactosamine but not with N-acetyl-glucosamine as demonstrated by the absence of affinity for the WGA lectin (lanes 2 and 3) and its binding to SBA lectin (lanes 4 and 5). Nonspecific binding was ruled out by elution with galactose (lane 6 and 7). (B) Dose-response curve of erbB4 phosphorylation. COS-7 cells transiently transfected with pcDNA3-erbB4 were trypsinized and re-seeded to assure identical levels of erbB4 receptor expression per well; after serum deprivation, cells were incubated with the indicated amount of recombinant NRG3 (inset) for 5 minutes, harvested with sample buffer and western blotted with anti-phosphotyrosine antibody. Increases in phosphotyrosine levels were normalized after re-probing the membranes with anti-PKC-{alpha}. The results of three different experiments are given as the mean + s.e.m. (top). (C) The same approach was used to detect neuregulin activity in conditioned medium (CM) of hFBNRG3 transfected cells. As shown, erbB4 phosphorylating activity was found in the medium of MG-132-treated, transiently transfected cells (lane 6). Although in some experiments neuregulin activity in the medium of nontreated cells was detected, differences were not statistically significant (lane 4 and bar labelled CM). Basal phosphorylation of erbB4 was estimated from conditioned medium of cells transfected with the inversely oriented hFBNRG3 cDNA without (lane 3) or with (lane 5) MG-132 and also in GST-treated cells (lane 7), and subtracted from calculations. Positive phosphorylation control was performed by adding recombinant GST-NRG3-EGF-like protein (lane 8). No protein bands were detected in nontransfected COS-7 cells even when GST or recombinant neuregulin was added (lanes 1 or 2, respectively). Data are given as mean ± s.e.m (n=3, *P≤0.05); Student's t-test was used.

 

Figure 8
View larger version (30K):

[in a new window]
  		Fig. 8. NRG3 is a survival factor for oligodendrocytes. (A) Serum-starved oligodendrocyte precursor cultures were immunostained with anti-NG2 antibody; a.1 and a.3 overlays; a.2 and a.4 Hoechst dye staining of nuclei. Apoptotic (arrows) and non-apoptotic (arrowheads) cells from 750 to 1500 NG2-positive cells per experiment were counted. Bars, 10 µm. (B) The results of three different experiments are shown. GST-NRG3 (150 nM) decreased apoptosis to similar levels found in non-serum-starved cultures. GST-purified protein was used as control. Data are given as mean ± s.e.m.; the Student's t-test was used (*P≤0,015). (C) Recombinant neuregulin 3 increases the phosphoryaltion of Akt in a dose-dependent way in the rat C6 glioma cell line. Recombinant neuregulin 1 (GST-SMDF) was used as a control. The densitometric quantification of a typical experiment is shown. (D) Time course of Akt phosphorylation in oligodendrocyte precursor cultures. (E) Inhibition of PI 3-kinase pathway with 20 µM LY294002 abrogates the anti-apoptotic effect of recombinant NRG3. Data are given as mean ± s.e.m.; the Student's t-test was used, *P≤0.015.

 

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   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006