|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Characterization of VAMP-2 in the lung: implication in lung surfactant secretion
Pengcheng Wang*†, Marcia D. Howard*, Honghao Zhang*, Narendranath Reddy Chintagari*, Anna Bell*, Nili Jin*, Amarjit Mishra* and Lin Liu*1
*Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, USA, and †Department of Microbiology and Immunology, Medical College of Jinan University, Guangdong Province, Guangzhou 510632, People's Republic of China
Lung surfactant is crucial for reducing the surface tension of alveolar space, thus preventing the alveoli from collapse. Lung surfactant is synthesized in alveolar epithelial type II cells and stored in lamellar bodies before being released via the fusion of lamellar bodies with the apical plasma membrane. SNAREs (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors) play an essential role in membrane fusion. We have previously demonstrated the requirement of t-SNARE (target SNARE) proteins, syntaxin 2 and SNAP-23 (N-ethylmaleimide-sensitive factor-attachment protein 23), in regulated surfactant secretion. Here, we characterized the distribution of VAMPs (vesicle-associated membrane proteins) in rat lung and alveolar type II cells. VAMP-2, -3 and -8 are shown in type II cells at both mRNA and protein levels. VAMP-2 and -8 were enriched in LB (lamellar body) fraction. Immunochemistry studies indicated that VAMP-2 was co-localized with the LB marker protein, LB-180. Functionally, the cytoplasmic domain of VAMP-2, but not VAMP-8 inhibited surfactant secretion in type II cells. We suggest that VAMP-2 is the v-SNARE (vesicle SNARE) involved in regulated surfactant secretion.
Key words: exocytosis, lung surfactant, membrane fusion, SNARE, VAMP
Abbreviations: CMV, cytomegalovirus, EGFP, enhanced green fluorescent protein, ER, endoplasmic reticulum, FBS, fetal bovine serum, GLUT, glucose transporter, HRP, horseradish peroxidase, LB, lamellar body, PFA, paraformaldehyde, RT–PCR, reverse transcription–PCR, SNAP-23, N-ethylmaleimide-sensitive factor-attachment protein 23, SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor, VAMP, vesicle-associated membrane protein
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Lung surfactant forms a monolayer at the air–liquid interface in alveoli to reduce the surface tension, thus preventing the alveoli from collapse. Surfactant is composed of phospholipids and surfactant proteins with most of the components being synthesized in the ER (endoplasmic reticulum) in alveolar epithelial type II cells, and stored in the specified organelles of the lamellar bodies. The secretion of surfactant is a highly regulated process, including the translocation, docking and fusion of lamellar bodies to the apical plasma membrane, and eventually the release of the contents into the alveolar lumen. Our previous studies have demonstrated the involvement of annexin A2, SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor), and some other regulatory factors in the secretion of lung surfactant (Abonyo et al., 2003, 2004; Gou et al., 2004). However, the precise underlying mechanism remains unclear.
SNAREs are a protein family which exist ubiquitously in eukaryotic cells and play a crucial role in membrane targeting, docking and fusion (Rothman, 1994; Chen and Scheller, 2001; Jahn et al., 2003; Brunger, 2005; Jackson and Chapman, 2006). SNARE proteins contain the characteristic coiled-coil domains termed SNARE motifs, which can form a trans-SNARE complex in two adjacent membranes to pull the membranes into close apposition, thus leading to membrane fusion (Fasshauer, 2003). There are two classes of SNARE proteins according to their localizations in the cell. The v-SNARE (vesicle SNARE), a VAMP (vesicle-associated membrane protein), is located on the membrane of secretory vesicles; the t-SNARE (target SNAREs), such as syntaxin and SNAP-23 (N-ethylmaleimide-sensitive factor-attachment protein 23)/SNAP-25, are located on the plasma membrane. VAMP-2 has been extensively studied in neurons, and plays a critical role in Ca2+-triggered fusion of synaptic vesicles with the presynaptic membrane. VAMP-2 is also involved in regulating secretion in other cells, such as adipocytes and pancreatic β-cells. The t-SNARE proteins, syntaxin 2 and SNAP-23, are required in lung surfactant secretion (Abonyo et al., 2004). We have identified the v-SNARE required in this process. We have demonstrated the presence of VAMP-2, -3, and -8 in alveolar type II cells at the mRNA and protein levels. VAMP-2 is localized on lamellar bodies. The cytoplasmic domain of VAMP-2 reduced surfactant secretion. VAMP-2 may therefore be the v-SNARE involved in the regulation of lung surfactant secretion.
2. Materials and methods
2.1. Reagents and chemicals
FBS (fetal bovine serum), trypsin-EDTA and DMEM (Dulbecco's modified Eagle's medium) were from Invitrogen. ECL® (enhanced chemiluminescence) reagent was from Amersham Pharmacia Biotech. Rabbit anti-VAMP-2 antibody was from Stressgen Bioreagents. Rabbit anti-VAMP-3 antibody was from Affinity Bioreagents. Rabbit anti-VAMP-8 antibody was from Abcam. Mouse anti-LB-180 antibody was from Covance. Goat anti-SP-C antibody was from Santa Cruz Biotechnology. Goat anti-rabbit HRP (horseradish peroxidase)-conjugated IgG was from Bio-Rad Laboratories. Rat anti-mouse HRP-conjugated IgG was from Jackson Immunoresearch Laboratories. Alexa Fluor® 488 goat anti-mouse, Alexa Fluor® 488 chicken anti-rabbit, Alexa Fluor® 546 donkey anti-goat and Alexa Fluor® 568 goat anti-rabbit antibodies were from Molecular Probe. 18S rRNA primers were from Ambion.
2.2. Isolation of alveolar type II cells
Alveolar type II cells were isolated from 180 to 200 g Sprague–Dawley rats, according to the method of Dobbs et al. (1986) (see also Liu et al., 1996). All of the animal procedures in this study were approved by the Institutional Animal Care and Use Committee of Oklahoma State University.
2.3. RT–PCR (reverse transcription–PCR)
Total RNAs were extracted from rat lung homogenate or freshly isolated type II cells with TRI reagent. Then 1 μg of total RNA was reverse transcribed to cDNA by using MMLV (Moloney-murine-leukaemia virus) reverse transcriptase and random hexamer primers, followed by PCR amplification with gene specific primers. The primer sequences are listed in Table 1. Then 18S rRNA was amplified by using classic 18S rRNA primer pairs. The conditions for PCR amplification were as follows: 95°C for 2 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by 72°C for 8 min. The PCR products were electrophoretically separated on 1% agarose gel.
Table 1 PCR primers for VAMP gene amplification
2.4. Isolation of lamellar body
Lamellar bodies were isolated from rat lung by upward flotation on a discontinuous sucrose gradient, as described by Chander et al. (1983) and Chattopadhyay et al. (2003). A perfused rat lung was briefly homogenized in 1 M sucrose and then loaded at the bottom of a sucrose gradient (0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 M). After centrifugation at 80000 g for 3 h, the LB fraction was collected at the 0.4 and 0.5 M interface, and diluted to 0.24 M with cold water. LBs were spun at 20000 g and resuspended in 0.24 M sucrose containing 10 mM Tris and 50 mM Hepes (pH 7.0). The protein concentration of LBs was determined by Bio-Rad protein assay.
2.5. Preparation of the plasma membrane
Plasma membranes from rat lung tissue were prepared as described by Chattopadhyay et al. (2003). A Sprague–Dawley rat lung was perfused with saline and homogenized in buffer B (10 mM Na-Pi, pH 7.4, 30 mM NaCl, 1 mM MgCl2, 5 μM PMSF and 0.32 M sucrose). Following a discontinuous sucrose gradient (0.5, 0.7, 0.9 and 1.2 M) centrifugation at 95000 g for 60 min, the plasma membrane fraction was collected at the 0.9 and 1.2 M interface and diluted to 0.32 M sucrose with cold buffer A (buffer B without sucrose). The plasma membrane was spun down at 120000 g and resuspended in buffer B.
2.6. Western blotting
Protein samples were fractionated on SDS/12% PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 5% (w/v) non-fat dried skimmed milk powder in TTBS (tris-buffered-saline with Tween 20: 20 mM Tris/HCl, pH 7.6, 150 mM NaCl and 0.1% Tween 20). The membrane was incubated with appropriate primary antibodies (VAMP-2, 1:100; VAMP-3, 1:500; and VAMP-8, 1:500) at 4°C overnight, and with secondary antibody (1:2500) at room temperature for 1 h. Finally, the signal was developed with ECL® reagents.
Freshly isolated alveolar type II cells were cultured on coverslips overnight and fixed with 4% (w/v) PFA (paraformaldehyde) for 20 min at room temperature. Immunocytochemistry was done as described earlier (Chintagari et al., 2006). In brief, cells were permeabilized with 1% Triton X-100 and blocked with 10% FBS in 50 mM PBS. The slides were incubated overnight at 4°C with anti-LB-180 antibody (1:1000 dilution) and anti-VAMP-2 or VAMP-8 antibodies (1:100 dilution). They were subsequently washed and incubated with Alexa Fluor® 488 goat anti-mouse and Alexa Fluor® 568 goat anti-rabbit antibodies at 1:250 dilutions for 1 h at room temperature. Finally, the slides were washed, mounted and examined with a fluorescence microscope (Nikon Inc).
Immunohistochemistry was performed as described earlier (Chintagari et al., 2006). The rat lungs were perfused with PBS and lavaged with normal saline. The lungs were fixed with 4% PFA and then the paraffin-embedded lungs were sectioned (2 μm) and placed on glass slides (Fisher Scientific). The slides were de-paraffinized with xylene and rehydrated with graded alcohol and PBS. Inactivation of endogenous peroxidase by incubating the slides with 4% H2O2, and antigen retrieval was done by boiling the slides in citrate buffer (10 mM disodium citrate, pH 6.0 and 0.05% Tween-20) for 20 min. The sections were permeabilized with 1% Triton X-100 and blocked with 10% donkey serum in 50 mM PBS. They were incubated overnight at 4°C with anti-SP-C antibody (1:100 dilution) and anti-VAMP-2 (1:100 dilution). Subsequently, they were washed and incubated with Alexa Fluor® 488 chicken anti-rabbit and Alexa Fluor® 546 donkey anti-goat antibodies at 1:250 dilutions for 1 h at room temperature. Finally, the slides were washed, mounted and examined with a fluorescence microscope.
For fetal lung tissue, ABC staining was used due to the interference of red blood cells with immunofluorescence. The lung tissue sections were dewaxed and rehydrated. The slides were treated with 3% H2O2. Antigen retrieval was done by boiling the slides in citric buffer (pH 6.0) for 20 min. The slides were permeabilized in 0.3% Triton X-100 for 10 min and blocked in 10% normal horse serum. The tissue sections were incubated with anti-VAMP-2 (1:100), followed by secondary antibodies (1:100). The slides were incubated in ABC reagents for 30 min. Signals were detected using DAB (diaminobenzidine) substrate. The slides were counter stained with haematoxylin & eosin.
2.9. Construction of adenoviral vectors
We constructed adenoviruses for the overexpression of the cytoplasmic domains of VAMPs because of the difficulty in transfecting primary type II cells. The cytoplasmic domain (amino acids 1–94) of VAMP-2 or the cytoplasmic domain (amino acids 1–75) of VAMP-8 plasmids were provided by Dr Richard H Scheller (Stanford University), and were cloned into the pENTR/CMV (cytomegalovirus)-EGFP (enhanced green fluorescent protein) vector between EGFP and the SV40 polyA terminal sequence (Bhaskaran et al., 2007). The insert was switched to the adenoviral, pAd/PL-DEST vector (Invitrogen) through LR recombination. The vector was linearized by Pac I digestion and transfected into HEK-293A cells (human embryonic kidney cells) to generate adenoviruses. The virus titre was determined by infecting HEK-293A cells with a serial dilution of virus stock and counting virus-infected cells through GFP (green fluorescent protein).
2.10. Surfactant secretion assay
Lung surfactant secretion was determined by monitoring the release of [3H]-labelled PC form type II cells (Liu et al., 1996). Freshly isolated type II cells (1×106) were infected overnight with adenoviruses at a MOI (multiplicity of infection) of 100 in the presence of 0.6 μCi [3H]choline. The cells were washed 6 times and stimulated with 100 μM ATP, 100 nM PMA and 10 μM terbutaline for 2 h. At the end of incubation, the lipids in media and cells were extracted with chloroform–methanol. The secretion was calculated as 100×(counts in medium/counts in medium and cells).
3.1. VAMP genes are expressed in lung and alveolar type II cells
We utilized the RT–PCR method to examine the expression pattern of VAMP genes in alveolar type II cells (there are seven members in VAMP family). Specific primer pairs were designed, and gene fragments of different VAMP isoforms were amplified from rat lung and highly purified alveolar type II cells. Brain tissue was used as a reference. Most of the VAMP isoforms were expressed in type II cells. VAMP-2 mRNA was found both in lungs and type II cells (Figure 1). High expression of VAMP-3 and VAMP-8 was also seen which appear to be enriched in type II cells in comparison with lungs.
3.2. VAMPs are expressed in LB fraction
The distribution of VAMPs in alveolar type II cells was investigated by Western blotting for the different fractions of type II cells. In addition to the band expressed in brain (18 kDa), there was another band for VAMP-2 with a lower molecular mass found in lung tissue, type II cells, LB fraction and the plasma membrane, which was dramatically enriched in the LB fraction (Figure 2). VAMP-8 was detected in both the LB and the plasma membrane factions, whereas VAMP-3 was mainly present in the plasma membrane fraction and was not detected in the LB fraction.
3.3. Localization of VAMP-2 in lung tissue and in alveolar type II cells
To examine the cellular localization of VAMP-2 in the lung, adult perfused lung tissue by the dual-immunolabelling technique, using anti-SP-C (a type II cell marker) and anti-VAMP-2 antibodies. The signal of VAMP-2 staining was overlapped with that of SP-C in the lung tissue, indicating that VAMP-2 is localized in type II cells (Figure 3A). The location of VAMP-2 in the fetal lungs was also determined. VAMP-2 was localized in airway epithelial cells at gestational day 16 (Figure 3B).
To check the localization of VAMP-2 protein in alveolar type II cells, we used dual-immunostaining of VAMP-2 and LB-180, the latter a LB marker protein, in isolated alveolar type II cells. VAMP-2 staining partially overlapped with LB-180. However, VAMP-8 signal did not overlap with LB-180 (Figure 4).
3.4. Cytoplasmic domain of VAMP-2 inhibits surfactant secretion
The role of VAMP-2 in surfactant secretion was explored by overexpressing the cytoplasmic domain of VAMP-2. The VAMP-2 cytoplasmic domain functions as a dominant-negative mutant since it does not contain a membrane domain, thus preventing the trans-SNARE complex formation. We constructed an adenoviral vector (V-2) that contains the CMV promoter and the cytoplasmic domain (amino acids 1–94) of VAMP-2. Two controls were included, CMV promoter-driven EGFP [CV (control virus)] and CMV promoter-driven cytoplasmic domain (amino acids 1–75) of VAMP-8 (V-8). VAMP-2 cytoplasmic domain inhibited surfactant secretion, while VAMP-8 cytoplasmic domain did not inhibit (Figure 5), suggesting a role of VAMP-2 in surfactant secretion.
SNARE proteins play a central role in eukaryotic membrane trafficking events, the underlying mechanism being conserved among different species. We have previously demonstrated that the t-SNAREs, syntaxin 2 and SNAP-23 are present in alveolar type II cells, and that they are required in surfactant secretion. We have now characterized VAMPs in lung tissues and type II cells, and studied their functional role in lung surfactant secretion.
From the RT–PCR results, various isoforms of VAMP genes are expressed in type II cells. VAMP-1 is highly homologous to VAMP-2, has a different cellular distribution pattern and is involved in calcium-dependent synaptic vesicle exocytosis (Jacobsson et al., 1998; Sherry et al., 2003; Raptis et al., 2005). VAMP-1 is expressed in non-neuronal tissues (Rossetto et al., 1996), but its function is unclear. VAMP-3 is ubiquitously expressed and preferentially associated with early/recycling endosomes. VAMP-3 plays an important role in platelet alpha granule secretion by using tetanus toxin (Flaumenhaft et al., 1999), antibody (Feng et al., 2002) and the cytoplasmic domain (Polgar et al., 2002). However, the reduction of VAMP-3 in transgenic mice had no effects on platelet function, indicating that it is not essential for platelet-releasing reaction in mice (Schraw et al., 2003). VAMP-7 is resistant to tetanus neurotoxin cleavage (Galli et al., 1998). It is associated with the late endosome, and is involved in endocytosis and intracellular trafficking between the ER and Golgi (Braun et al., 2004; Siddiqi et al., 2006). VAMP-8 is involved in the fusion between early and late endosomes of the endocytic pathway (Wong et al., 1998; Antonin et al., 2000). VAMP-8 is required in the regulated exocytosis of pancreatic acinar cells and platelets (Wang et al., 2004; Ren et al., 2007). VAMP-2 was initially identified as a v-SNARE of synaptic vesicles in neurons, thus playing an important role in synaptic vesicle exocytosis (Lin and Scheller, 2000). VAMP-2 is also involved in regulated transporting events in non-neuronal systems, such as trafficking of GLUT4 (glucose transporter 4) to the plasma membrane (Slot et al., 1997; Foster et al., 1998; Foster and Klip, 2000; Bryant et al., 2002; Watson et al., 2004). The VAMP isoforms abundantly expressed in alveolar type II cells were VAMP-2, VAMP-3 and VAMP-8.
From protein expression patterns of VAMP-2, -3 and -8 in lung and type II cells seen by Western blotting, we found that along a band with the same size as that in brain (18 kDa), a band with a lower mass for VAMP-2 was consistently detected by a polyclonal anti-VAMP-2 antibody. This is probably the degradation product of VAMP-2 or another VAMP-2 isoform. VAMP-2 was also detectable in the plasma membrane fraction. Immunostaining showed that VAMP-2 was partially co-localized with LB marker, LB-180, indicating that VAMP-2 is localized on lamellar bodies. Based on this association, it is reasonable to consider the role of VAMP-2 as a v-SNARE in the fusion of lamellar bodies with the plasma membrane. VAMP-3 was barely detected in lamellar bodies, but was present in type II cells. It is enriched in the lung plasma membrane fraction. Interestingly, a strong band for VAMP-8 was observed on lamellar bodies by Western blot analysis. However, immunostaining only exhibited a faint staining of VAMP-8 on lamellar bodies. This is probably due to the different exposure of the epitopes recognized by the antibody. The presence of more than one VAMP isoform and their distinctive distribution patterns suggest that different VAMP isoforms are involved in different processes in type II cells, rather than simply functional redundancy.
The VAMP-2 cytoplasmic domain does not contain its membrane domain, although it can form a SNARE complex (Hao et al., 1997; Poirier et al., 1998). Once it is expressed in the cells, the VAMP-2 cytoplasmic domain incorporates into the SNARE complex to compete with endogenous VAMP-2 effectively, preventing the formation of a functional trans-SNARE complex. Thus, the VAMP-2 cytoplasmic domain behaves as a dominant-negative mutant or as a competitive inhibitor. The overexpression of VAMP-2 cytoplasmic domain in type II cells inhibited surfactant secretion. This is consistent with a previous study demonstrating that the expression of VAMP-2 cytoplasmic domain decreased insulin-stimulated GLUT4 translocation (a process similar to regulated exocytosis) in adipocytes (Olson et al., 1997). VAMP-8 cytoplasmic domain had no effects on surfactant secretion. Along with the LB location of VAMP-2, these results indicate that VAMP-2 actively participates in surfactant secretion.
Pengcheng Wang, Marcia Howard, Honghao Zhang, Narendranath Chintagari, Anna Bell, Nili Jin and Amarjit Michra carried out the experiments. Pengcheng Wang drafted the paper. Lin Liu conceived the study, participated in its design and co-ordination, and helped to draft the paper.
We thank Ms Tazia Cook for her editorial assistance before submission.
This work was supported by the
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Received 9 March 2011/5 April 2012; accepted 10 May 2012
Published as Cell Biology International Immediate Publication 10 May 2012, doi:10.1042/CBI20110146
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)