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-dystroglycan
,1
* Department of Immunology and Key Laboratory of Molecular Medicine of the Ministry of Education, Shanghai Medical College of Fudan University, Shanghai, China; and
Immunology Division of E-Institutes of Shanghai Universities, Shanghai, China
1Correspondence: Department of Immunology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai, 200032, China. E-mail: sdxiongfd{at}126.com
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ABSTRACT |
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-dystroglycan receptors for agrin, only -dystroglycan could be detected in lymphocytes. Confocal examination showed that -dystroglycan colocalized with agrin in forming the immunological synapse. Down-regulation of -dystroglycan expression inhibited lymphocyte activation even in the presence of agrin. However, agrin involved in down-regulation of -dystroglycan receptors did not increase the inhibitory effect on lymphocytes activation. The anti--dystroglycan antibody also induced lymphocytes activation. Taken together, these findings strongly indicate that agrin and -dystroglycan mediate lymphocyte activation. Furthermore, agrin-involved lymphocyte activation is mediated by -dystroglycan.—Zhang, J., Wang, Y., Chu, Y., Su, L., Gong, Y., Zhang, R., Xiong, S. Agrin is involved in lymphocytes activation that is mediated by -dystroglycan.
Key Words: agrin • -dystroglycan • Immunological synapse • lymphocyte activation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. The interaction of aggregated T cell receptors (TCRs) with the major histocompatibility complex (MHC) loaded with antigen peptides at the immunological synapse results in gene transcription and subsequent T cell proliferation. The signaling thresholds at the immunological synapse also tune the T cell proliferation regulated by costimulatory molecules (6
7
8
9
10
11
12
13)
. While much is known about the molecular and cellular interactions that are important for a successful outcome (14)
, the triggering mechanism is unknown that elicits the TCR, MHC costimulatory molecules to aggregate to form an immunological synapse so as to activate T cells.
The heparin sulfate proteoglycan agrin, a key organizer of postsynaptic differentiation at the neuromuscular junctions (NMJ), is an essential extracellular matrix glycoprotein for synapse formation between motoneurons and muscle (15
16
17)
. It is thought to orchestrate the first phase of neuromuscular (NMJ) formation. During early development, agrin is synthesized by motoneurons, transported to the axon terminal, and secreted into the forming basal membrane. The deposition of agrin initiates the accumulation of synaptic proteins, for example the nicotinic acetylcholine receptor (AChR), at the site of contact between motoneuron and muscle. Since NMJ and lymphocyte synaptic structure and functions are similar to one another, it is reasonable to presume that agrin probably triggers immunological synapse formation and the activation of lymphocytes. In fact, Rupp et al. (18)
reported that agrin potentially functions at the immunological synapse. They demonstrated that agrin induces aggregation of lipid rafts that contain complexes of CD3 and T cell receptors, CD4 or CD8, and Lck, and that agrin at picomolar concentrations lowers the threshold within minutes for antigen-induced lymphocyte proliferation. More recently, Shaw/Allen (19)
and Bezakova/Ruegg, (20)
further discussed the potential functions of agrin in the immune system, which strongly indicates the involvement of agrin in the activation of lymphocytes.
In NMJ, several lines of evidence suggest that MuSK and -dystroglycan are candidate functional receptors of agrin. The tyrosine kinase MuSK is a signaling receptor required for agrin-induced postsynaptic differentiation. MuSK-deficient mice lack any postsynaptic specializations at the nerve-muscle contact and die perinatally (21)
. Cultured myotubes devoid of MuSK do not aggregate AChR in response to agrin. Furthermore, in this system MuSK expression restoration elicits agrin-induced AChR aggregation (22)
. -Dystroglycan is a highly glycosylated extracellular protein with a molecular mass of 120–190 kDa, and noncovalently anchored to a 43 kDa transmembrane ß-dystroglycan (23
24
25)
. -Dystroglycan links to the extracellular matrix via its several ligands such as agrin, laminin-1, laminin-2, etc. They in turn anchor themselves to the membrane fragment ß-dystroglycan. -Dystroglycan linked to the actin cytoskeleton via ß-dystroglycan, and dystrophin or utrophin (26
, 27)
. Monoclonal antibody IIH6C4 specific against -dystroglycan significantly inhibits agrin-induced AChR aggregation on C2 myotubes, and antisense -dystroglycan oligonucleotide inhibit agrin activity and dystroglycan–/– myotubes form only immature AChR clusters in response to agrin stimulation. Increasing data support the concept that -dystroglycan mediates downstream signaling leading to AChR aggregation (28
29
30
31
32
33)
. Therefore, both MuSK and -dystroglycan are crucial and significant for agrin-triggered synapse formation and signaling at the NMJ. However, the expression and function of MuSK and -dystroglycan have not been investigated in lymphocytes. To this end, in the present study the roles of agrin were evaluated in lymphocyte activation and immunological synapse formation. Furthermore, in lymphocytes candidate cognate agrin receptors were evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell preparation
Splenocytes suspensions were prepared from immunized mice or controls. T cells and B cells were purified from splenocytes by using negative selection magnetic beads (Miltenyi Biotec, Auburn, CA, USA). The efficiency of purification was determined by flow cytometry to be greater than 95% for both CD3+ and B220+. Bone marrow-deriveddentritic cells (DCs) were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng/mL) and/or tumor necrosis factor (TNF) (200 U/mL) in vitro as described (34)
. Cell purity at the end of an incubation period in all sets was assessed based on morphological criteria. The cells obtained from intraperitoneal lavage were cultured overnight and the nonadherent cells were decanted. The adherent cells were used as macrophages for further analysis.
RT-PCR
Total RNA extracts from splenocytes or other isolated primary cells were obtained using the guanidinium isothiocyanate acid phenol chloroform procedure with small modifications (35)
. In all samples, residual DNA was eliminated with DNase I (MBI, Hanover, MD, USA). Reverse transcription was performed using Moloney murine leukemia virus (MMTV) reverse transcriptase (MBI). Specific gene primers (Table 1
) were used for subsequent DNA amplification. Samples were analyzed on a 1.5% agarose/TBE gel. In some experiments, the cDNA products was quantitatively analyzed for the expression of agrin and -dystroglycan by real-time PCR using LightCycler Instrument (Roche Molecular Biochemical, Nutley, NJ, USA) with the DNA binding dye SYBR Green I. The amounts of mRNA were by normalizing them to GAPDH gene expression levels.
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Western blot
Splenocytes were stimulated with ConA (10 µg/mL) for 48 h and harvested for membrane protein extraction as described (18)
. The medium was collected and condensed by lyophilizing and concentrating them to 15 mg/mL of total protein. These membrane components and the medium were resuspended with sample buffer (50 mmol/L Tris-HCl, 100 mmol/L DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol), separated by 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with primary antibody against agrin or -dystroglycan (Chemicon International, Inc., El Segundo, CA, USA) followed by HRP-conjugated goat anti-mouse antibody (Chemicon International, Inc.), then visualized using chemiluminescence Western blot detection reagent (Pierce Biotechnology, Inc., Rockford, IL, USA).
Antisense plasmids construction and transfection
The amplified agrin or -dystroglycan DNA fragments were cloned into pUCm-T vector (Shennengbocai, Shanghai, China) in clockwise direction according to the manufacturer’s instruction and designated as pUCmT-AG and pUCmT-DG, respectively. Fragments digested by EcoRV and BamHI restriction enzymes from the plasmids were then reverse subcloned into pcDNA3 for the construction of antisense plasmids. PCR and DNA sequencing were used to validate the identity of the constructs. The antisense plasmid specific for agrin or -dystroglycan was termed as pcDNA-As-AG and pcDNA-As-DG, respectively. The splenocytes derived from the immunized mice or controls were transfected with pcDNA3, pcDNA-As-AG, pcDNA-As-DG, or pEGFP-DG (provided as a gift by Steve J. Wind) using lipofectamine2000 reagent (Life Technologies, Grand Island, NY, USA) as described by the manufacturer. Twenty-four to 48 h after transfection the cells were restimulated with ConA (10 µg/mL) or BSA (100 µg/mL). The inhibitory efficiency of the antisense approaches on the expression of agrin or -dystroglycan was assessed by FACS analysis.
Flow cytometry
Antibodies against -dystroglycan (6C1, IgG, CHEMICON International, Inc.), FITC-labeled rabbit anti-mouse IgG (Rockland, Gilbertsville, PA, USA), CD4-PerCP (RM4-5) (BD PharMingen, San Diego, CA, USA), CD8a-PE (53-6.7) (BD PharMingen), B220-PE (BD PharMingen) or appropriately labeled isotype control antibodies (BD PharMingen) were used for surface labeling. Antibody against agrin (MAB5204, Chemicon International, Inc.) was used for intracellular staining as described (36)
. All the cells were analyzed by flow cytometry (FACScalibur; Becton Dickinson, Franklin Lakes, NJ, USA) with Cellquest software. The secondary antibody or isotype antibodies were used as negative controls to set markers.
Proliferation assay
In each well of 96-well round-bottom microtiter plate (Nunc. Roskilde, Denmark), splenocytes (5x106 cells/mL) from immunized mice or controls were cultured in RPMI 1640 medium with either ConA (10 µg/mL, 5 µg/mL) or BSA (100 µg/mL). In some experiments, splenocytes were stimulated with or without ConA in the presence of antibody against -dystroglycan (IIH6C4, 10 µg/mL) or antibody against agrin (MAB5204, 10 µg/mL). All the cells were incubated at 37°C in a 5% CO2 humidified atmosphere for 56 h. [3H]-Thymidine (0.5 µCi) was added to each well and the cells were incubated for another 16 h. Cells were then harvested onto glass fiber paper using a semi-automatic cell harvester. Thymidine incorporation was determined by a liquid scintillation counter (Rihuan SN-695).
Slide preparation and confocal observation
Splenocytes (5x106cells/mL) activated with ConA (10 µg/mL) were incubated at 37°C for 2 h, then spread onto prewarmed poly (L-lysine) -coated slides (sigma). DCs and purified naive T cells (1:5) were mixed and centrifuged at 500 x g for 2 min in conical tubes. After centrifugation, the cells were incubated at 37°C for 30 min, resuspended and plated on poly-L-lysine-coated slides. Macrophages adhered to slides were preincubated with BSA (100 µg/mL) overnight, washed with PBS, and mixed with T cells purified from the splenocytes of BSA-immunized mice (1:5). All these slides were incubated for another 15 min at 37°C to promote cell attachment, fixed in freshly prepared 4% formaldehyde/PBS for 30 min, then washed three times in PBS. For staining with antibodies, all slides were first placed in blocking solution (1% BSA/0.1NaN3 in PBS) and stored at 4°C for at least 24 h. The slides were then stained with appropriate primary antibodies (Abs) against agrin or -dystroglycan diluted in blocking solution for 2 h at 4°C in a humidified chamber, then coupled with appropriate secondary Abs like Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), FITC-conjugated rabbit anti-mouse IgG (Rockland), rhodamine-labeled goat anti-mouse IgM (µ) (Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD, USA) for 1 h followed by three washes in PBS. After overnight drying at room temperature, slides were stored at 4°C and protected from light. Single- and two-color confocal analyses were performed using two-photon confocal microscopy (Leica).
Statistics
Statistical significance was assessed using a 1-way ANOVA. The LSD test was used for comparison between each group. P value of < 0.05 was considered significant, *P < 0.05, **P < 0.01.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A) and in isolated T cells and B cells, immature DCs (imDCs), mature DCs (mDCs) as well as macrophages (Fig. 1B
). The obtained size of the amplified cDNA was shown to be the same as that anticipated. The sequence of the amplified PCR products matched the reported sequences (Gene bank accession No.: NM_021604). With intracellular staining with agrin-specific antibody, FACS analysis showed the expression of agrin in splenocytes (Fig. 1C
) and in the isolated T cells, B cells, imDCs, mDCs as well as macrophages (Fig. 1D
). Western blot revealed that 
200-, 45 kDa, and an additional small band of proteolytic agrin were detected in the condensed supernatant of activated lymphocytes, but not the membrane protein extraction (Fig. 1E
), indicating that agrin is expressed in a secretory form in activated lymphocytes.
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Expression of agrin is closely associated with activation of lymphocytes
To address whether agrin expression was associated with lymphocyte activation, the kinetics of agrin expression in lymphocytes was observed. The expression of agrin increased 12 h after ConA stimulation, reached its peak at 48 h and dropped markedly at 96 h poststimulation, which matched the proliferation pattern of the ConA activated lymphocytes (Fig. 2
A, B). At 96 h poststimulation, the proliferation rate of lymphocyte evidently decreased, which was observed generally in the similar assays (Fig. 2B
). To further elucidate the function of agrin in the activation of lymphocytes, they were transfected with the antisense plasmids pcDNA-As-AG. The expression of agrin was found to be dramatically decreased after transfection (Fig. 2C
). Twenty-four hours after the transfection, the lymphocytes were restimulated with either ConA or corresponding antigen BSA. The activation was significantly inhibited in both ConA-activated (Fig. 2D
) and BSA-restimulated lymphocytes (Fig. 2E
) compared with that of the nontransfected cells, suggesting that agrin is indeed involved in the activation of lymphocytes, not only in antigen nonspecific but also in antigen-specific activation.
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Verification of agrin’s putative receptors on lymphocytes
Agrin exerts its functions through binding to its putative receptors including MuSK and -dystroglycan in the NMJ. However, it is still uncertain whether the same receptors are recruited in the immune synapse. In the present study, the expression of -dystroglycan and MuSK in splenocytes was examined. To our surprise, only -dystroglycan but not MuSK was detectable in repeated experiments (Fig. 3
A). Even after increasing mRNA template copies, MuSK expression was still not detected (data not shown), indicating a difference in roles for agrin receptors between NMJ and splenocytes. To determine the cell types that express -dystroglycan, T cells and B cells (isolated from splenocytes), imDC and mDC (derived from bone morrow) and macrophages (harvested from intraperitoneal lavage) were tested for the expression of -dystroglycan. The expression of -dystroglycan was detected in all abovementioned cell types by RT-PCR (Fig. 3B
). DNA sequencing verified the sequence homology of the -dystroglycan detected in the cells in this study with that reported (Gene bank accession No.: X86073). To localize the expression of -dystroglycan, FACS and Western blot analysis were performed. -Dystroglycan was detected on the surface of the lymphocytes (Fig. 3C, D
). Western blot analysis of the membrane extracts confirmed this finding (Fig. 3E
). The components complexed to -dystroglycan were examined since the presence of these components was essential and imperative for the function of -dystroglycan. Figure 3F
showed that dystrophin-glycoprotein complex components including syntrophin, utrophin, dystrophin, dystrobrevin, sarcospan, sarcoglycan , sarcoglycan ß, sarcoglycan 
, sarcoglycan 
and sarcoglycan 
besides -dystroglycan were found in splenocytes, implicating that -dystroglycan expressed in the splenocytes may possess the function.
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Agrin-involved lymphocyte activation is mediated by -dystroglycan
To convincingly demonstrate the function of -dystroglycan in the activation of lymphocytes, the expression of -dystroglycan was increased 12 h after ConA stimulation and reached its peak at 24 h and persisted in a high level at 48 and 96 h (Fig. 4
B). While -dystroglycan in the lymphocytes was down-regulated by transfection with pcDNA-As-DG, an antisense plasmid that decreases -dystroglycan expression (Fig. 4C
), both nonspecific (ConA-activated, Fig. 4D
) and antigen-specific (BSA-activated, Fig. 4E
) lymphocyte activation (P<0.05) were significantly inhibited, suggesting that -dystroglycan expression levels intrinsically affect lymphocyte activation status. Double down-regulation of -dystroglycan and agrin did not increase the inhibitory effect on lymphocyte activation compared with that of single down-regulation of -dystroglycan or agrin (Fig. 4F
). No difference was found comparing the transfection efficiency of agrin and -dystroglycanin cotransfection system to single-transfection (data not shown). In activated lymphocytes with cotransfection anti-sense plasmids, no significant difference of declined expression of agrin and/or -dystroglycan appeared between single-transfection and cotransfection system (data not shown). The conceivable explanation of no addictive inhibition of cotransfecting antisense -dystroglycan and agrin could be that agrin and -dystroglycan are involved in the same pathway that induces the activation of lymphocytes. By using neutralizing antibodies against agrin and -dystroglycan, nonsymmetrical effects on lymphocyte activation were observed: the antibody against -dystroglycan enhanced the activation of lymphocytes whereas the antibody against agrin did not elevate it, but rather slightly decreased lymphocyte activation (Fig. 4G
). We speculated that the antibody against -dystroglycan here served as analogous to agrin, which activated lymphocytes. Confocal microscopic evaluation showed that -dystroglycan and agrin were colocalized on the surface of the lymphocytes (Fig. 4H, I
), previously implying that agrin exerts its function through binding itself to -dystroglycan. Taken together, these findings strongly suggest that agrin and -dystroglycan are indeed involved in the activation of lymphocytes and that agrin-involved lymphocyte activation is mediated by -dystroglycan.
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Agrin and -dystroglycan are co-capped in immunological synapse
The formation of immunological synapse is considered to be a pivotal event in lymphocyte activation. To determine the role of agrin and -dystroglycan in the formation of immunological synapse, we found that agrin and CD3 are diffusely distributed on the surface of resting T cells, but co-capped on the surface of activated T cells (Fig. 5
A). Similarly, -dystroglycan co-capped with CD3 too after T cell activation (Fig. 5B
). When naïve T cells were cocultured with DCs, co-capping of agrin and -dystroglycan was observed (Fig. 5C
). Similar results were obtained after coculturing of the antigen-treated macrophages and the antigen-specific T cells (Fig. 5D
), indicating that agrin and -dystroglycan may participate in the formation of immunological synapse.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The neuromuscular junction (NMJ) is the most well-studied synapse and is widely regarded as structurally and functionally less complicated than neuronal synapses in the brain. Over the past two decades the skeletal neuromuscular synapse has received much attention as a model system by those interested in understanding how cells control the postsynaptic density of transmitter receptors. In the embryo, acetylcholine receptors (AChRs) are scattered diffusely across the surface membrane of the muscle cell, but during development they immediately cluster together at extremely high densities in membrane domains (37)
. Based on studies of the mechanisms of aggregation of AChR on the surface of muscle cells, agrin has been definitively identified as a crucial organizer of postsynaptic differentiation at the NMJ (15
16
17)
.
During synapse formation at the NMJ, a small patch on the muscle fiber surface differentiates into a complex postsynaptic apparatus. An early step in this process is the redistribution of AChRs within the myotube membrane, leading to their aggregation and colocalization with numerous other proteins normally found in the postsynaptic cytoskeleton and synaptic cleft. Motor axons frequently contact unspecialized portions of the myotube surface, and subneural high-density AChR aggregates form only after making contact with one another. Several agents are capable of stimulating the formation of AChR clusters on cultured myotubes. The best characterized is agrin, a proteoglycan initially isolated by McMahan and colleagues from Torpedo electric organ (38)
. Addition of agrin to cultured muscle cells triggers formation of AChR aggregates in the myotube membrane; typically agrin-treated mytobues bears >10-fold more aggregates per unit surface area than untreated myotubes. Like nerve-induced clusters, agrin-induced AChR clusters form by redistribution of diffusely distributed AChRs and are accompanied by extracellular matrix, membrane, and cytoskeletal components of the postsynaptic apparatus (39)
. Gautam and colleagues (16)
have shown that postsynaptic differentiation is profoundly impaired in agrin-deficient mice: nerve-associated AChR aggregates are fewer in number, smaller in size, and lower in receptor density on homozygous mutant myotubes than those on myotubes of littermate control. These studies strongly suggest that agrin is a crucial organizer of postsynaptic differentiation.
The concept of synapse has recently been adopted to describe the specialized connection that forms between APCs and T cells in the immune system (40)
. Growing evidence suggests that the similarity between immunological and neuronal synapses may be in more than just name (41)
, for example, the aggregation of surface molecules is a fundamental mechanism by which both lymphocytes and neurons regulate transmembrane signaling. The aggregation of pre- and postsynaptic molecules in the nervous system is essential for synapse formation and ensures that there is zonal and rapid exchange of information between neurons. Similarly, in the immune system, the ligand-induced clustering of membrane lipid microdomains is an essential event in the focal transduction of intercellular signals that regulate lymphocyte activation, growth, and differentiation (42
43
44)
. Since Dustin and colleagues (5)
first reported that the formation of immunological synapse is the machine for T cell activation, the components involved in the formation of immunological synapse were broadly explored. T cell activation is initiated by the interaction of T cell receptors (TCRs) with peptides that are displayed on the surface of antigen-presenting cells (APCs) (45)
. Costimulatory signals induce the recruitment of TCRs to lipid rafts and the clustering of rafts into a formed functional module polarized toward the APC (10)
. Rafts serve as platforms for signal transduction and, in clustering, form the TCR-APC contact junction (46)
. Furthermore, cytokines (47)
, chemokines (48)
, cytoskeletons (49
, 50)
, and signaling transducers (51)
were also verified to contribute to immunological synapse formation. In the past decade, immunologists have expended much effort to discover the key organizer in the formation of immunological synapse. In agrin-treated T cells, the lipid rafts was redistributed into clusters and dense caps, and extensive research showed that these raft clusters also contain other signaling molecules such as CD28, CD3 and MHC I, as well as coclustering with the Lck tyrosine kinase. Incubation with exogenous agrin markedly lowered the OVA threshold for lymphocyte activation and decreased the magnitude of the response to saturation amounts of OVA (18)
. In our studies, agrin expression was also found in various immunocytes. Along with the lymphocyte activation, the expression of agrin was up-regulated, whereas agrin was down-regulated, which was associated with significant declines in lymphocyte activation. These results further confirmed that agrin expression levels are closely associated with lymphocyte activation status. Taken together with previous studies, there is clear evidence suggesting that agrin plays an important role in lymphocyte activation; however, being a secretory protein, it is unclear how agrin exerts its function in the immune system.
-Dystroglycan and MuSK are considered as cognate receptors for agrin in NMJ, which provided us with important insight on how to probe for their involvement in the immune system. In the present study, our results show that -dystroglycan is present in immunocytes based on the results obtained with RT-PCR, FACS and confocal microscopy. On the other hand, MuSK, another crucial receptor of agrin in NMJ, was not detectable in immunocytes. Additional work suggests that agrin and -dystroglycan participate in the formation of immunological synapse and subsequently induce the activation of lymphocytes, and that agrin-involved activation of lymphocytes is mediated by -dystroglycan.
The formation of immunological synapse is a necessary step for activation of lymphocytes. Although our findings indicate that agrin and -dystroglycan co-capped in immunological synapse and played roles in lymphocyte activation, it is still unclear which molecules are induced to aggregate together. Furthermore, the signaling pathways linked to receptor activation by these mediators are unknown. We will undertake in the future additional to delineate the signaling cascades activated after agrin and -dystroglycan interaction.
It is well known that the T cell plays a pivotal role in adaptive immunology. Overactive immune responses always result in autoimmune diseases and graft rejection. On the contrary, T cell deficiency always results in tumor formation and microorganism invasiveness. Obviously, the activity of lymphocytes is very tightly linked with the status of immune defense. Therefore, interfering in the expression of agrin and -dystroglycan might contribute to the prevention and treatment of diseases. Taken together, our results might provide additional understanding for how recognition and interaction occurs between immunocytes. Furthermore, the results of comparative studies on NMJ and immunological synapse phenomenology will aid us in deciphering the mechanisms underlying synapse formation in the immune system.
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ACKNOWLEDGMENTS |
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Received for publication January 24, 2005. Accepted for publication September 26, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-Dystroglycan functions in acetylcholine receptor aggregation but is not a coreceptor for agrin-MuSK signaling. J. Neurosci. 18,6340-6348, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell 77,675-686[CrossRef][Medline]This article has been cited by other articles:
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  Y. Gong, R. Zhang, J. Zhang, L. Xu, F. Zhang, W. Xu, Y. Wang, Y. Chu, and S. Xiong {alpha}-Dystroglycan is involved in positive selection of thymocytes by participating in immunological synapse formation FASEB J, May 1, 2008; 22(5): 1426 - 1439. [Abstract] [Full Text] [PDF]
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 E. C. Jury, J. Eldridge, D. A. Isenberg, and P. S. Kabouridis Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients J. Immunol., December 1, 2007; 179(11): 7975 - 7983. [Abstract] [Full Text] [PDF]
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), respectively. RT-PCRs were performed to analyze the mRNA expression for agrin and GAPDH. Fragments with expected size were amplified from all above cells. C, D) FACS was conducted to analyze the expression of agrin with intracellular staining. Histograms show staining with antibody specific against agrin (black) and overlaid with the FITC-labeled rabbit anti-mouse IgG. E) Extracted membrane components (lane1, 2) and condensed medium (lane 3, 4) from Con A-activated splenocytes were blot with antibody against agrin (IgG1, No: MAB5204, Lot No: 23010562; CHEMICON International, Inc.).
