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Department of Immunology (K.T., N.K., Y.U., T.I., H.Y.)
Graduate School of Pharmaceutical Sciences Osaka University
Osaka 565-0871, Japan
Department of Cancer Chemotherapy
(T.F.) Institute for Cancer Research Kagoshima University,
Faculty of Medicine Kagoshima 890-8520, Japan
Dana-Farber
Cancer Institute and Department of Biological Chemistry and Molecular
Pharmacology (H.S.) Harvard Medical School Boston,
Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In contrast, many receptor-like PTPases have two tandemly repeated cytoplasmic PTPase domains. The membrane-proximal and membrane-distal PTPase domains are called D1 (domain 1) and D2 (domain 2), respectively. Previous studies on the structure-function relationships of the receptor-like PTPases indicated that: 1) D1 has PTPase activity by itself; 2) the conserved cysteine residue in the signature motif of D1 is essential for the PTPase activity; 3) the three-dimensional structures of D1 and D2 are very similar; and 4) D2 has no or very little intrinsic catalytic activity (8, 9, 10, 11). These results suggested that although D2 is structurally very similar to D1, its function may be to regulate the catalytic activity or specificity of D1.
Extracellular domains of receptor-like PTPases are composed of various
combinations of functional motifs such as the immunoglobulin-like (Ig)
repeat, the fibronectin type III (FnIII) repeat, and the carbonic
anhydrase-like domain, suggesting that the extracellular domain of
receptor-like PTPases may modulate PTPase activity by binding to
specific ligands. Specific ligands are, however, known for only a
handful of receptor-like PTPases: PTPß binds contactin and other
extracellular matrix molecules (12, 13, 14); PTP
, PTP
, and
PTPµ are homotypic receptors (15, 16, 17); and laminin-nidogen complex
binds to the fifth FnIII domain of human leukocyte common
antigen-related (LAR) molecule (18). Although effects of ligand binding
on PTPase activity are not known, it has been proposed that
homodimerization of receptor-like PTPases CD45 and PTP
may inhibit
their PTPase activities (19, 20). According to this model, PTPase
activity might be regulated by modulation (either promotion or
inhibition) of receptor dimerization induced by ligand binding.
However, the crystal structures of LAR and PTPµ do not lend
support to this model (11, 21).
Receptor-like PTPase LAR consists of non-covalently-bound subunits designated the E (extracellular) subunit and the P (phosphatase) subunit, generated by proteolytic cleavage of a single precursor protein between the eighth FnIII domain and a transmembrane segment (22, 23). The 150-kDa E subunit is composed of three Ig domains and eight FnIII domains, whereas the 85-kDa P subunit has a short extracellular domain, a transmembrane domain, and two tandemly repeated PTPase domains (D1 and D2) in the cytoplasm. The E and P subunits of LAR are connected by a noncovalent bond. There are several lines of circumstantial evidence that implicate LAR PTPase in insulin receptor signal transduction: 1) LAR is widely detected on a variety of insulin-sensitive tissues such as liver, muscle, and adipocytes (24, 25, 26); 2) LAR-deficient mice exhibit significantly lower levels of plasma glucose as well as a reduced rate of hepatic glucose production in the fasting state, and a significant resistance to insulin-stimulated glucose disposal and suppression of hepatic glucose output (27); 3) increased LAR PTPase activity toward the insulin receptor is detected in subcutaneous adipose tissue of obese subjects (28); 4) in in vitro experiments, the recombinant LAR PTPase catalytic domain dephosphorylates a regulatory phosphorylation site (Tyr-1150) in the insulin receptor ß-subunit (25); 5) in a rat hepatoma cell line in which the expression of the LAR protein is suppressed by antisense RNA, tyrosine phosphorylation of the insulin receptor is significantly enhanced after insulin stimulation (29); and 6) LAR binds to and dephosphorylates tyrosine-phosphorylated insulin receptor in Chinese hamster ovary cells that express human insulin receptor and LAR molecules (CHO-hIR/LAR) as well as in a rat hepatoma cell line (30, 31). These results suggest a negative regulatory role of LAR in insulin signaling, and the dysregulation of LAR PTPase activity might be a pathogenic factor in insulin-resistant diabetes.
The purpose of the current study is to clarify the functions of the two PTPase domains of LAR in the regulation of insulin receptor tyrosine dephosphorylation in vivo, using LAR mutants in which the catalytic cysteine residue in D1 or D2 has been changed to serine, or in which either D1 or D2 has been deleted. Using this approach, we showed that the LAR D1 is responsible for insulin receptor dephosphorylation, whereas the LAR D2 is mainly responsible for the recognition of the phosphorylated insulin receptor. Moreover, the association of extracellular domains of insulin receptor and LAR was supported by using a LAR deletion mutant without both PTPase domains. We found that LAR not only dephosphorylates insulin receptor but also serves as a substrate of insulin receptor tyrosine kinase. Thus, the extracellular domain and two PTPase domains of the receptor type PTPase LAR have distinct functional roles in insulin receptor signaling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Initially, COS-7 cells were cotransfected with IR, together with the
full-length LAR wild type (LAR WT), a LAR mutant that harbors the
Cys-1522 to Ser substitution at the catalytic center of PTPase D1 (this
mutation will be abbreviated as LAR D1CS), or the empty vector. The
Cys-1522 to Ser mutation completely abolishes the in vitro
LAR PTPase activity using artificial substrates. NP40 lysates were
prepared from the transfected cells at various times after the
stimulation with 100 nM insulin, and the
tyrosine-phosphorylated proteins were detected by immunoblotting with
antiphosphotyrosine monoclonal antibody (mAb) 4G10 (Fig. 1
). A 95-kDa protein was prominently
tyrosine phosphorylated 1 min after the insulin stimulation, and the
tyrosine phosphorylation levels remained almost constant for 15 min
after the insulin stimulation. When the same blot was reprobed with
anti-IR ß-subunit antiserum, a 95-kDa protein band was detected (data
not shown), indicating that the 95-kDa tyrosine-phosphorylated band is
the insulin receptor.
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In the LAR D1CS transfectants, several additional proteins were also
tyrosine phosphorylated prominently, even in the absence of insulin
stimulation. There are two possible explanations for the increased
tyrosine phosphorylation of these proteins: 1) the IR tyrosine kinase
remains in the active state, due to the inability of LAR D1CS to
dephosphorylate, and continues to tyrosine phosphorylate other
proteins; or 2) these phosphorylated proteins are also substrates of
LAR. To determine which of these two explanations was correct, COS-7
cells were cotransfected with LAR D1CS and IR and stimulated with
insulin for 1 min, and NP40 cell lysates were prepared. LAR in the
lysates was immunoprecipitated with anti-LAR E subunit mAbs (a mixture
of 11.1A, 75.3A, and 71.2E), and the precipitates were immunoblotted
with the antiphosphotyrosine mAb 4G10. Cys-to-Ser mutants of several
PTPases are known to tightly bind their specific
tyrosine-phosphorylated substrates without dephosphorylating them
(32, 33, 34, 35). By analogy, it might be expected that LAR D1CS would
tightly bind the tyrosine-phosphorylated form of its physiological
substrates. As shown in Fig. 2B, only
three bands of phosphorylated proteins, at 200 kDa, 95 kDa, and 85 kDa,
were detected in LAR D1CS immunoprecipitates. The 95-kDa protein is IR,
because it was detected with anti-IR antiserum (Fig. 2A). The other two
bands are the LAR precursor and the LAR P subunit (see below). These
results favor the first explanation.
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) are
about the same size as the LAR precursor and P subunit, respectively,
we examined whether LAR is tyrosine phosphorylated. To this end, we
generated a mAb (YU1) that recognizes the LAR P subunit by immunizing
mice with a glutathione-S-transferase (GST)-LAR cytoplasmic
domain fusion protein. The same filter as shown in Fig. 2B was reprobed
with the anti-LAR P subunit mAb YU1. As shown in Fig. 2C, 200-kDa and
85-kDa protein bands, which correspond precisely to the
tyrosine-phosphorylated proteins, were detected by YU1, indicating that
the 200-kDa and 85-kDa tyrosine-phosphorylated proteins are the LAR
precursor protein and the LAR P subunit, respectively.
LAR PTPase D2 Is Important for Recognition of the Insulin
Receptor
To examine whether the LAR D1 and D2 play distinct roles in the
LAR-IR interaction, we used three LAR mutants; namely LAR D1CS, LAR
D2CS (Cys-1813 to Ser mutation in D2), and LAR D1D2CS (a mutant with
both Cys-1522 to Ser in D1 and Cys-1813 to Ser in D2). These LAR
mutants, or LAR WT, were individually transfected together with IR.
Cell lysates were prepared 1 min after insulin stimulation, and LAR was
immunoprecipitated with anti-LAR E subunit mAbs. Immunoblots with the
antiphosphotyrosine mAb 4G10, the anti-LAR P subunit mAb YU1, and the
anti-IR antiserum are shown in Fig. 3, and the relative intensities of bands detected with the
antiphosphotyrosine mAb 4G10 were analyzed by densitometry (Fig. 4). Tyrosine-phosphorylated insulin
receptor was efficiently coimmunoprecipitated with LAR D1CS, moderately
coimmunoprecipitated with LAR WT and LAR D1D2CS, but only negligibly
coimmunoprecipitated with LAR D2CS (Fig. 3A). Interestingly, the
immunoblotting with anti-IR antiserum indicated that IR was present in
the LAR WT and LAR D1CS immunoprecipitates at about the same level, but
there was very little in the LAR D2CS or LAR D1D2CS immunoprecipitates
(Fig. 3B), even though the levels of immunoprecipitated LAR (Fig. 3C)
and the expression levels of insulin receptor (Fig. 3E) in these
transfected cells were very similar. These results indicate that the
cysteine residue in LAR D2 is important for the binding to the insulin
receptor and that such D2-mediated binding is needed for efficient
tyrosine dephosphorylation of the IR ß-subunit by the LAR D1 domain.
Consistent with this interpretation, the total tyrosine phosphorylation
level of IR in cell lysates of the LAR WT transfectant was
significantly lower than those of LAR D1CS, D2CS, and D1D2CS (Figs. 1D and 4B), indicating that both D1 and D2 are important for IR
dephosphorylation. Interestingly, the tyrosine phosphorylation levels
of the LAR P subunit were significantly increased in the LAR D1CS and
LAR D1D2CS transfectants compared with the LAR WT or LAR D2CS
transfectant (Figs. 3A and 4A), suggesting that the phosphorylated
tyrosine residue(s) of LAR P subunit is/are dephosphorylated mainly by
the catalytic activity of LAR PTPase D1.
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D1 and
LAR D2 (mutants lacking D1 and D2, respectively) and LAR PTPase
(a mutant lacking both D1 and D2). These LAR deletion mutants, or LAR
CS, were individually transfected into COS-7 cells together with IR as
described above. LAR was immunoprecipitated with anti-LAR E subunit
mAbs from the cell lysates prepared 1 min after insulin stimulation.
Figure 5A shows the immunoblot with the
antiphosphotyrosine mAb PY20 conjugated with horseradish peroxidase.
Tyrosine-phosphorylated IR was most prominently coimmunoprecipitated
with LAR D1CS, was moderately so with LAR PTPase and LAR D1, and
was only negligibly so with LAR D2. When the same blot was reprobed
with anti-LAR E subunit mAbs, almost the same intensities of 150-kDa
bands were detected in transfectants of LAR D1CS, D2, and PTPase,
but the intensity in LAR D1 was much weaker (Fig. 5B). However, the
obvious association of LAR D1 with tyrosine-phosphorylated IR was
detected in spite of its weak expression, indicating that LAR D2 plays
an important role in the recognition of tyrosine-phosphorylated IR.
Interestingly, the binding of tyrosine-phosphorylated IR to LAR
PTPase, which has no PTPase domains, was also detectable, suggesting
the possibility of association between the extracellular domains of
both LAR and IR. When the same blot was reprobed with anti-LAR P
subunit mAb YU1, 85-kDa, 53-kDa, 51-kDa, and 22-kDa bands were detected
in transfectants of LAR DCS, D2, D1, and PTPase, respectively
(Fig. 5C). These bands, with the exception of the 53-kDa band of LAR
D2, completely corresponded to the bands detected with an
antiphosphotyrosine mAb in Fig. 5A, indicating that tyrosine residue(s)
existing between the transmembrane domain and the PTPase domain 1 of
LAR were tyrosine phosphorylated by IR. An in vitro PTPase
assay indicated that LAR D2 immunoprecipitated from COS-7
transfectants with anti-LAR E subunit mAbs had obvious PTPase activity,
but LAR D1 or LAR PTPase immunoprecipitates had no activity (data
not shown). Therefore, we supposed that since LAR D2 could bind to
tyrosine-phosphorylated IR and promptly dephosphorylated the IR and
tyrosine-phosphorylated LAR, only a weak band of
tyrosine-phosphorylated IR was detected.
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, the LAR P subunit as well as the
coprecipitated IR ß-subunit were tyrosine phosphorylated in cells
transfected with the IR WT but not in those transfected with the IR MT.
In these experiments, expression levels of both IR and LAR were
comparable between the two transfected cells (data not shown). These
results thus indicate that the LAR P subunit is a substrate of the
insulin receptor tyrosine kinase. Moreover, when the same blot was
reprobed with the anti-IR antiserum, IR was detected in both IR WT and
IR MT transfectants (Fig. 6B), indicating that part of LAR-IR
association is independent of the IR phosphorylation state.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this study, our criteria for a physiological substrate of LAR were as follows: 1) a LAR substrate is dephosphorylated more efficiently by LAR PTPase than other tyrosine-phosphorylated proteins, and 2) a tyrosine-phosphorylated substrate of LAR has a high affinity to LAR PTPase D1. We demonstrated preferential dephosphorylation of tyrosine-phosphorylated insulin receptor by LAR PTPase and a specific association of LAR D1CS mutant with the tyrosine-phosphorylated insulin receptor. Recently, Ahmad et al. (30, 31) also reported a physical association of rat LAR and the human insulin receptor overexpressed in CHO cells. The association seems to increase in accordance with the tyrosine phosphorylation of insulin receptor. These results indicate that the insulin receptor is a likely physiological substrate of LAR. Based on these findings, our present study has further clarified distinct functions of the two PTPase domains of LAR for the insulin receptor by various kinds of LAR mutants in vivo.
In cotransfection experiments, we found that LAR WT efficiently
dephosphorylated the insulin receptor activated by insulin stimulation.
On the other hand, the tyrosine-phosphorylated insulin receptor was
coprecipitated with LAR D1CS, a substrate trapping mutant of LAR,
indicating the tight association of these two molecules. These results
support that LAR PTPase D1 is involved in a catalytic process, and that
Cys-1522 located at the catalytic center of the PTPase D1 is critical
for its catalytic activity in vitro (9) as well as in
vivo. In the same way, it might be expected that if LAR PTPase D2
has a catalytic effect on the phosphorylated insulin receptor, both LAR
D2CS and LAR D1D2CS mutants could efficiently trap the
tyrosine-phosphorylated insulin receptor as well. However, neither LAR
D2CS nor LAR D1D2CS showed a marked binding to the phosphorylated
insulin receptor as was seen in LAR D1CS, suggesting that LAR PTPase D2
does not catalyze the dephosphorylation of the insulin receptor.
Interestingly, the association of the insulin receptor to LAR
constructs that have no mutation in the domain 2, such as LAR WT and
LAR D1CS, is significantly weakened by the introduction of Cys to
Ser-substituted mutation in LAR D2. In contrast, LAR D1 that has
only a wild-type PTPase domain 2 in the cytoplasm showed obvious
binding to the phosphorylated insulin receptor. These results indicate
that LAR PTPase D2 is important for the recognition of a substrate, the
insulin receptor, and that Cys-1813 in the domain is essential for
recognition in vivo.
Ahmad and Goldstein (31) have reported that LAR is associated with the
insulin receptor and that the association is enhanced by insulin
stimulation. We also confirmed the physical association between LAR and
the insulin receptor in transfected COS-7 cells. However, LAR PTPase
was found to be associated with the insulin receptor in spite of the
complete absence of LAR PTPase domains. These results indicated that
not only the PTPase domains of LAR but also its extracellular domain
are involved in the association with the insulin receptor.
LAR undergoes proteolytic processing when cells are grown to a high density or in response to increases in cytoplasmic Ca levels and protein kinase C activity (23). Aicher et al. (36) have reported that the processing of LAR is detected 5 min and 40 min after calcium ionophore stimulation in A431 and Hela cells, respectively. In our transfection assay of COS-7 cells, we could hardly detect the shed LAR in the culture media at 1 min, and we could detect it only slightly at 5 min after insulin stimulation (data not shown). Therefore, the processing of the LAR extracellular domain would not affect the interpretation of the results from immunoprecipitation assays with anti-LAR E subunit mAbs in this study.
LAR was tyrosine phosphorylated by tyrosine kinase activity of insulin
receptor in response to insulin stimulation and rapidly
autodephosphorylated in vivo. Some tyrosine phosphatases,
such as PTP1C (37), PTP (38), and PTPase 1B (39), are also known to
be tyrosine phosphorylated by the insulin receptor tyrosine kinase. The
phosphorylation of PTP1C increases its PTPase activity, and that of
PTPase 1B is necessary for its interaction with insulin receptor. The
tyrosine phosphorylation of PTP leads to a reduction of the insulin
receptor signaling (the activation of an endogenous kinases, such as
Src, and the binding of Grb2) (40). The tyrosine phosphorylation sites
in LAR, the functional significance of the phosphorylation, and the
understanding of the change, if there is any, in the catalytic activity
caused by the phosphorylation are unclear. Nonetheless, the
phosphorylation and autodephosphorylation of LAR tyrosine residues are
likely to play important functions in the regulatory mechanism of
insulin-receptor signal transduction.
Based on the results of this study, we propose a new model of the tyrosine dephosphorylation mechanism of insulin receptor by LAR PTPase. LAR weakly associates with the insulin receptor at the steady state via the LAR extracellular domain. After insulin binding, insulin receptor tyrosine kinase induces autophosphorylation, leading to increased tyrosine kinase activity. The activated insulin receptor tyrosine kinase phosphorylates its substrates, including LAR. The tyrosine phosphorylation of LAR might induce the conformational change of the PTPases D1 and/or D2, resulting in a tighter association via LAR PTPase domain 2, and then dephosphorylating phosphotyrosine residues of the insulin receptor by the catalytic activity of the PTPase D1. A phosphorylated tyrosine residue(s) of LAR is also dephosphorylated by the catalytic activity of the PTPase D1 to return to the preactivation state.
Insulin receptor signaling is important for both cell growth and glucose metabolism. Molecular links between LAR and phosphorylated tyrosine residues of the insulin receptor are suggested. However, the elaborate regulatory mechanism by the two PTPase domains of LAR must still be clarified, perhaps using both the insulin receptor mutants with substitutions of tyrosine resides and various LAR mutants.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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296 expression
plasmid termed pSP65-SR2 (41). pSP65-SR2-LAR PTPase (amino
acids 1–1,343) and pSP65-SR2-LAR D2 (amino acids 1–1,590)
deletion mutants were constructed by removing appropriate restriction
fragments from pSP65-SR2-LAR WT. A cDNA sequence corresponding to
LAR PTPase domain 2 (amino acids 1,633–1,881) with appropriate
restriction sites for subcloning were generated by PCR and inserted
in-frame into the pSP65-SR2-LAR PTPase to construct LAR D1.
Their nucleotide sequences were confirmed by DNA sequencing. The
numbering of LAR amino acid residues is in accordance with Streuli
et al. (22). Human insulin receptor expression vectors,
pSR-human insulin receptor (pSR-IR) and pSR-IR K1030M, in
which Lys-1030 at the ATP binding site of the insulin receptor is
changed to Met, were kindly provided by Dr. Ebina.
Cell Culture and Transfections
COS-7 cells, obtained from the Human Science Research Resources
Bank (Osaka, Japan), were grown at 37 C in a 5%
CO2 atmosphere in RPMI 1640 medium (Nissui
Pharmaceutical, Tokyo, Japan) supplemented with 10%
heat-inactivated FCS (Life Technologies, Inc.,
Gaithersburg, MD) and 10 mg/ml kanamycin. Cells were cotransfected with
5 µg of pSP65-SR2-LAR and 1 µg of pSR-IR by the
diethylaminoethyl (DEAE)-dextran method (42). After incubation for
48 h, the transfected cells were starved for 16 h in
serum-free RPMI 1640 medium and then stimulated with 100 nM
insulin for the indicated times. After washing with ice-cold PBS
containing 1 mM sodium vanadate, 5 mM NaF, 5
mM sodium pyrophosphate, and 5 mM EDTA, the
cells were lysed on ice in 1 ml of ice-cold lysis buffer (1% NP40, 150
mM NaCl, 50 mM Tris, pH 7.4, 5 mM
EDTA, 10 mM iodoacetamide, 10 mM NaF, 10
mM sodium pyrophosphate, 0.4 mM sodium
vanadate, 0.1 mM phenylarsine oxide, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM benzamidine). The
cell lysates were centrifuged in a microcentrifuge to remove insoluble
materials before immunoprecipitation and immunoblotting.
Immunoprecipitation and Immunoblotting
Cell lysates were precleared with approximately 15 µg
isotype-matched control mAbs (Transduction Laboratories, Inc., Lexington, KY) and 20 µl Gamma-bind (Amersham Pharmacia Biotech, Arlington Heights, IL) for more than 2 h
at 4 C. For immunoprecipitation, cell lysates were incubated with 15
µg monoclonal anti-LAR mAbs (1:1 mixture of 11.1A and 75.3A) (23) for
1 h at 4 C, and then with 20 µl Gamma-bind for an additional
1 h at 4 C. After washing twice with 1 ml lysis buffer as
described above and once with 1 ml PBS with 10 mM NaF, 10
mM sodium pyrophosphate, 0.4 mM sodium
vanadate, and 0.1 mM phenylarsine oxide, immunoprecipitates
were resolved by SDS-PAGE. Proteins in immunoprecipitates and cell
lysates were transferred from a SDS-polyacrylamide gel to a
nitrocellulose membrane (Schleicher & Schuell, Inc.,
Keene, NH), blocked with 3% BSA in TBS-T (20 mM Tris-HCl
pH 8.0, 137 mM NaCl, and 0.1% Tween 20) and incubated with
primary mAbs at room temperature for 1 h, followed by washing
three times with TBS-T. To detect antibody binding, horseradish
peroxidase-conjugated antimouse IgG or horseradish
peroxidase-conjugated antirabbit IgG (Transduction Laboratories, Inc.) diluted in TBS-T was incubated at room temperature for
1 h. After washing in TBS-T three times, bound horseradish
peroxidase conjugates were visualized with enhanced chemiluminescent
reagent (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan). The antibodies used were antiphosphotyrosine mAb 4G10
(Upstate Biotechnology, Inc., Lake Placid, NY),
antiinsulin receptor ß-subunit (Transduction Laboratories, Inc.), anti-LAR E subunit mAbs (1:1:1 mixture of 75.3A, 11.1A,
and 71.2E) (23), and anti-LAR P subunit mAb (YU1). For the blot of
immunoprecipitates from lysates of COS-7 cells cotransfected with LAR
deletion mutants and IR, we used antiphosphotyrosine mAb PY20
conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Preparation of Antihuman LAR P Subunit mAb (YU1)
The anti-LAR P subunit mAb YU1 was generated by immunization
with a recombinant glutathione S-transferase fusion protein
with the LAR P subunit beyond transmembrane segment expressed in a
pGEX-2T vector (Amersham Pharmacia Biotech).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, and a grant from Fuso Pharmaceutical Industries, Ltd.
Received for publication January 19, 2000. Revision received September 25, 2000. Accepted for publication October 13, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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