From the Polypeptide Hormone Laboratory, the
Department of Anatomy and Cell Biology, McGill University,
Montreal, Quebec H3A 2B2, Canada and the § Department de
Medecine et le Centre de Recherche du CHUL, Universite Laval, St. Foy,
Quebec G1V 4G2, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Incubating endosomes with ATP decreased binding
of 125I-insulin but not 125I-labeled
human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased -subunit tyrosine phosphorylation and
insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations
-subunit tyrosine phosphorylation and IRK activity were markedly
decreased. This was not observed with nonhydrolyzable analogs of ATP,
nor with plasma membrane IRK, nor with endosomal epidermal growth
factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by
bafilomycin A1, indicating a role for endosomal proton
pump(s). The inhibition of IRK was not due to serine/threonine
phosphorylation nor was it influenced by the inhibition of
phosphotyrosyl phosphatase using
bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the
-subunit with 1 mM ATP did not prevent the
inhibition of IRK activity on incubating with 5 mM ATP. To
evaluate conformational change we incubated endosomes with
dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis
under nonreducing conditions. Without DTT the predominant species of
IRK observed was
2
2. With DTT the
dimer predominated but on co-incubation with 5 mM ATP the
2
2 form predominated. Thus,
ATP-dependent endosomal acidification contributes to the
termination of transmembrane signaling by, among other processes,
effecting a deactivating conformational change of the IRK.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Following insulin binding to its receptor in intact cells the insulin receptor kinase (IRK)1 undergoes tyrosine autophosphorylation and kinase activation (1, 2). Equally rapidly there is internalization of activated insulin-IRK complexes into ENs (3, 4). IRK signaling appears largely to involve tyrosine phosphorylation of adaptor proteins, including insulin receptor substrate-1 (5), insulin receptor substrate-2 (6, 7), and SHC (8), which function as docking entities to entrain the insulin signaling sequence. The observation that the activated IRK is internalized to ENs is consistent with the occurrence of transmembrane signaling intracellularly (9, 10). Indeed studies in liver parenchyma have shown that the accumulation of activated IRKs exclusively in ENs is sufficient to promote insulin receptor substrate-1 tyrosine phosphorylation (11). In adipocytes it has also been shown that internal membranes are the principal sites where insulin receptor substrate-1 phosphorylation and phophatidylinositol 3-kinase activation occur (12).
Given the above, understanding the mechanisms controlling receptor function in ENs is important for understanding the regulation of insulin signaling. Studies have shown that the level of IRK tyrosine phosphorylation and hence activity is altered and ultimately reduced by an IRK-associated phosphotyrosine phosphatase in ENs (4, 13). Discovery of the endosomal acidic insulinase (14) has demonstrated a mechanism by which intraendosomal insulin concentration may be reduced, hence decreasing the proportion of IRKs occupied by ligand (15). In a cell-free system it was demonstrated that ATP-dependent endosomal acidification promoted both insulin dissociation from the IRK and subsequent degradation of free insulin by the endosomal acidic insulinase (15-17). In the present study we report that ATP-dependent endosomal acidification also leads to decreased IRK binding capacity and to marked deactivation of IRK activity toward exogenous substrates. Our data indicate that this latter phenomenon derives from an acidification dependent conformational change in the intraluminal aspect of the endosomal IRK with attendant deactivation of the cytosolic tyrosine kinase. These data thus identify another process leading to attenuation of the activated state of the IRK, and hence transmembrane signaling during the course of endocytosis.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals-- Female Sprague-Dawley rats (140-160-g body weight) were purchased from Charles River Ltd. (St. Constant, Quebec) and were fasted overnight prior to killing.
Reagents--
Porcine insulin (26.8 IU/mg) was a gift from Eli
Lilly Research Laboratories. Human growth hormone (hGH, 2.2 IU/mg was
from the NIH pituitary hormone and antisera program (Baltimore, MD). Carrier-free [125I]iodine and [-32P]ATP
(1000-3000 Ci/mmol) were purchased from NEN Life Science Products.
NaCl, MgSO4, trichloroacetic acid, and glycerol were from
Anachaemia Ltd. (Lachine, Quebec). Wheat germ agglutinin (WGA)-Sepharose 6-MB and protein A-Sepharose were from Amersham Pharmacia Biotech (Dorval, Quebec). Nucleosides tri-, di-, and monophosphates, AMP-PCP, AMP-PNP, and AMP-PSP were from Boehringer Mannheim. Chemicals for SDS-PAGE were from Bio-Rad. Kodak X-Omat AR
films were purchased from Picker International Canada (Montreal, Quebec). Immobilon was from Millipore Canada Ltd. (Mississauga, ON).
Bafilomycin A1, poly(Glu, Tyr) (4:1),
N-acetyl-D-glucosamine, and other chemicals were
from Sigma. The peroxovanadium compound bpV(phen) was synthesized and
purified as previously reported (18).
Antibodies--
Antibody to the juxtamembrane domain (residues
942-968) of the insulin receptor (960) and to phosphotyrosine were
prepared and purified as described previously (4). Affinity-purified goat anti-rabbit antibodies (whole molecule) were purchased from Sigma
and iodinated to a specific activity of 6 × 108
dpm/µg of IgG using a chloramine T procedure (19).
Subcellular Fractions, Binding, and Protein Determination-- Rats were anaesthetized with ether and were injected via jugular vein with a dose (per 100 g of body weight) of insulin (1.5 µg), or bpV(phen) (0.6 µmol), dissolved in 0.2 ml of phosphate-buffered saline (pH 7.4), 0.1% bovine serum albumin. Animals were killed by decapitation at 2 and 15 min postinjection of insulin and bpV(phen), respectively (13). Livers were rapidly excised, placed in ice-cold homogenizing buffer (50 mM HEPES (pH 7.4), 0.25 m sucrose, 1 mM phenylmethylsulfonyl fluoride, 1 mM MgCl2, 1 mM benzamidine) and minced before homogenization. Combined ENs and PM fractions were prepared as described previously (3) except that the buffer used throughout was that in which the livers were minced (see above). These subcellular fractions have been characterized in detail both morphologically and biochemically (3, 20-23). Hormone binding was assayed with 125I-insulin or 125I-labeled hGH prepared to a specific activity of 100-200 µCi/µg using the chloramine-T method as described previously (19). Protein content in the fractions was determined by a modification of Bradford's method using serum albumin as a standard (24).
Insulin Receptor Phosphotyrosine Content--
The
phosphotyrosine content of endosomal insulin receptors was determined
by subjecting WGA-purified preparations to SDS-PAGE and immunoblotting
with 960 and
PY as described previously (4).
Insulin Receptor Kinase Assays-- Insulin receptors from subcellular fractions were partially purified by chromatography on WGA-Sepharose 6MB columns, and receptor content and tyrosine kinase activity were measured as described previously (3, 4, 20). It has been previously shown that, after insulin administration, tyrosine kinase activity of WGA-purified endosomal preparations was at least 90% attributable to the IRK (4).
Insulin Receptor Autophosphorylation and Phosphoamino Acid
Analyses--
ENs were removed from the 0.6/1.0 m sucrose interface of
the gradient used in endosomal purification (20) and diluted with 0.25 M sucrose to a final protein concentration of 50 µg/ml. A 30-ml aliquot was centrifuged at 200,000 × gav for 40 min prior to resuspending in 500 µl
of cell-free system buffer (44 mM HEPES (pH 7.4), 0.55 M sucrose, 333 mM KCl, 11 mM NaCl,
11 mM MgCl2). Incubations were initiated by
adding 500 µl of 10 mM Tricine buffer containing either 2 or 10 mM ATP at a specific activity of 0.5 mCi of
32P/mmol. After incubating for 15 min at 37 °C the
reaction was stopped by adding an equal volume of ice-cold 20 mM HEPES, 0.25 M sucrose, 2 mM
phenylmethylsulfonyl fluoride, 2 mM benzamidine, 4 mM sodium orthovanadate, and 80 mM sodium
fluoride. ENs were solubilized by incubating for an additional 30 min
at 4 °C in a final concentration of 1% Triton X-100, 20 mM pepstatin, 20 mM leupeptin, and 10 mg/ml
aprotinin, after which IRKs were immunoprecipitated with 960, washed
in buffer, subjected to SDS-PAGE, transferred to Immobilon-P membranes,
and subjected to autoradiography and/or amino acid analyses as
described previously (25).
Insulin Receptor under Nonreducing Conditions-- Endosomal pellets were resuspended (500 µg of protein/ml) in a final concentration of 50 mM Tris (pH 6.9), 10% glycerol, and 20 mM N-ethylmaleimide. Samples were incubated at room temperature for 5 min and then solubilized without heating in 2.3% SDS, 0.05% bromphenol blue before subjecting to SDS-PAGE in the absence of reductants as described previously (26).
EGF Receptor Autophosphorylation in the Presence and Absence of ATP-- EGF (10 µg/100 g of body weight) was injected via the jugular vein into anesthetized rats. The animals were sacrificed by decapitation 15 min later, and ENs were prepared as noted above. Resuspended ENs were incubated for 15 min with 5 mM ATP after which autophosphorylation was initiated by adding [32P]ATP. The reaction was stopped after 15 min of incubation at 37 °C by adding Laemmli sample buffer after which aliquots (100 µg of protein) were subjected to SDS-PAGE, alkali digestion, and radioautography as described previously (13).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Following in vivo administration, 125I-insulin concentrates in ENs attained maximum levels at 2 min postinjection (19, 23, 27). When subsequently incubated in vitro, intraendosomal 125I-insulin undergoes dissociation from its receptor and degradation in a temperature and ATP-dependent manner (15). Previous work showed that this insulin degradation was effected by a relatively specific endosomal acidic insulinase (14, 15).
Effect of ATP on Insulin Binding and IRK Activity
In the present study we continued to evaluate the processes
involved in reducing the association of intraendosomal insulin with its
receptor. We examined the effect of incubating intact ENs with ATP on
subsequently measured insulin and hGH binding in both solubilized and
WGA-purified receptors. Table I
illustrates that, at increasing ATP concentrations (0.1, 1, 3, 5, and
10 mM), there was a progressive decrease of insulin but not
hGH binding. The decrease of insulin binding was not produced by ADP,
AMP, sodium pyrophosphate, adenosine, or nonhydrolyzable ATP analogs (Table II). The loss of binding activity
was temperature-dependent, as no changes were observed when
ENs were incubated on ice in the presence of 10 mM ATP
(Table II). These data strongly suggest that the
ATP-dependent effect was specific for insulin and
necessitated hydrolysis of the -phosphate of ATP.
|
|
Preincubating ENs with ATP also influenced IRK activity and
phosphotyrosine content (Fig. 1). In the
absence of ATP, IRK activity reflected the effect of preinjected
insulin (3, 4, 18). At 0.1 and 1.0 mM ATP there was an
increase, whereas at 5 mM ATP there was a marked decrease
in IRK activity even to a level below that observed in the absence of
ATP. At 10 mM ATP, IRK activity was virtually abolished.
IRK phosphotyrosine content was markedly increased on incubating ENs
with 1 mM ATP (Fig. 1B), whereas at 5 and 10 mM ATP this was significantly reduced and virtually
abolished, respectively, without affecting -subunit levels.
|
To verify the importance of the high energy bond of ATP in producing the above changes, we incubated ENs with 0 and 1 mM ATP in the presence (5 mM) or absence of the nonhydrolyzable ATP analog, AMP-PCP. No effect of 5 mM AMP-PCP either in the presence or absence of 1 mM ATP was found (Fig. 2). The effect of the analog to reduce IRK phosphotyrosine concentration to a modest extent may reflect some inhibition of IRK autophosphorylation.
|
To determine whether ATP-dependent attenuation of IRK activation is specific to the endosomal compartment, we preincubated PM with ATP and observed an augmentation of WGA-purified IRK activity (Fig. 3A). At 10 mM ATP the increase in PM IRK activity was greater than that observed at 1 mM in sharp contrast to what was found in ENs where preincubation with 10 mM ATP suppressed IRK activity completely.
|
Preincubation with ATP did not attenuate EGF receptor autophosphorylation. Thus incubating ENs from EGF-treated rats with 5 mM ATP did not significantly reduce 32P-labeling of the EGF receptor (Fig. 3B).
Role of Endosomal Acidification
Various studies have established that in ENs there is a progressive luminal acidification through the action of ATP-dependent proton pumps (28-31). To evaluate the role of ATP-dependent acidification on the IRK activation state we incubated ENs with bafilomycin A1, a potent inhibitor of endosomal ATPases (31), in the presence and absence of ATP. As noted in Fig. 4A coincubation with bafilomycin produced a marked attenuation in the ability of 5 and 10 mM ATP to effect a reduction in IRK activation. Furthermore in the presence of bafilomycin the level of IRK tyrosine phosphorylation at 5 mM ATP was comparable to that observed at 1 mM ATP (Fig. 4B).
|
Studies on Attenuation of IRK Activation
We subsequently sought to determine the mechanism by which ATP-dependent endosomal acidification results in diminution of the IRK activation state.
Serine/Threonine Phosphorylation--
Previous work demonstrated
that serine/threonine phosphorylation of the -subunit of the IRK
results in inhibition of IRK activity (32, 33). We thus determined if
ATP promotes the phosphorylation of IRK on serine and threonine
residues by incubating ENs with 1 or 5 mM
[
-32P]ATP (0.5 mCi/mmol). IRKs were subsequently
purified by immunoprecipitation and SDS-PAGE and subjected to
two-dimensional phosphoamino acid analyses of the
-subunit. The IRK
showed no detectable [32P]phosphoserine from either the 1 or 5 mM ATP incubations (Fig. 5).
|
Activation of Phosphotyrosyl Phosphatase(s)--
To assess whether
higher ATP concentrations might promote activation of endosomal
phosphotyrosyl phosphatases and effect a reduction in the
phosphotyrosine content of the IRK -subunit, we blocked
phosphotyrosyl phosphatase activity using bpV(phen), a potent
phosphotyrosyl phosphatase inhibitor (18). Coincubating ENs with
bpV(phen) did not prevent the marked suppression of IRK activity seen
in the presence of 5 mM ATP (Fig.
6). Furthermore, the augmented IRK
activity in ENs isolated from rats pretreated with bpV(phen) was
also suppressed on incubating these ENs with 5 mM ATP. Nor
did bpV(phen) influence the reduction observed in
-subunit tyrosine
phosphorylation seen in the presence of 5 mM ATP (Fig.
6B). We conclude that phosphotyrosyl phosphatase activity plays no role in effecting a reduction of endosomal IRK function at
higher ATP concentrations.
|
Dissociation of IRK Activity and its Autophosphorylation
State--
To determine the relationship between IRK activity and
-subunit tyrosine phosphorylation we preincubated ENs with 1 or 5 mM ATP for 15 min followed by a second incubation with 5 mM ATP. As observed in Fig. 7
preincubation with 1 mM ATP followed by a second incubation
with 5 mM ATP resulted in marked reduction of IRK activity
in the presence of a substantial retention of its phosphotyrosine
content. Sequential incubations in 1 mM ATP had no
deleterious effect on either IRK activity or phosphotyrosine content.
Thus endosomal acidification results in the inactivation of an
autophosphorylated IRK.
|
Evidence for an Acidification-dependent Conformational Change of the Endosomal IRK
The above observation suggested that intraluminal events were
responsible for deactivation of the endosomal IRK. To determine whether
there might be a conformational change in the IRK consequent to
ATP-dependent acidification we incubated ENs with ATP in
the presence or absence of 10 mM DTT to assess the ease
with which type I disulfide bonds might be reduced in the
heterotetrameric molecule (2
2). After
incubating in the presence or absence of 5 mM ATP the
heterotetramer was readily identified (Fig.
8, lanes 1 to 3).
When the incubation contained 10 mM DTT, the heterotetramer was readily reduced to the heterodimer (
) in the absence but not
the presence of 5 mM ATP (lane 4 versus 5). Thus, consequent to
ATP-dependent acidification of endosomes, there is a change in
-subunit conformation which renders the type I disulfide bonds relatively resistant to reduction by DTT.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The endosomal apparatus consists of a series of distinct
tubulovesicular components involved in the uptake and sorting of ligand-receptor complexes (34, 35). The ENs employed in these studies
have been previously characterized and shown to contain Golgi elements
but to be substantially free of plasma membrane and other subcellular
constituents (19, 20, 36-38). The addition of ATP to these ENs
augments both dissociation and degradation of internalized insulin (15)
due to stimulation of an ATP-dependent proton pump. The
resulting acidification (pH 5.5) of the intraendosomal milieu activates
a relatively specific insulin protease (i.e. endosomal
acidic insulinase) (14-17, 27, 39, 40). The coupling of dissociation
at acid pH with insulin degradation facilitates removal of internalized
receptor-bound insulin. However, given the small volume of an endocytic
vesicle ( 10
17 liter) (41), the extent of dissociation
of insulin from its receptor might be expected to be limited even at pH
5.5-6.0.
We thus considered that other mechanism(s) for abrogating IRK
activation in ENs might exist. Indeed incubating ENs with ATP resulted
in a loss of insulin receptor binding capacity. This effect was
specific for the IRK since no decrease in binding was observed for hGH
(Table I). The loss of insulin binding was not observed at 4 °C, and
the presence of the -phosphate of ATP was necessary since no
inhibition occurred with adenosine, AMP, ADP, and sodium pyrophosphate.
The presence of a high energy bond was necessary as nonhydrolyzable ATP
analogs were unable to produce the effect (Table II). The reduction of
insulin binding activity represents another mechanism for sustaining
the dissociation-degradation sequence for insulin in ENs.
An ATP-dependent process is implicated in deactivation of
the IRK within ENs. Although IRK activity and -subunit
phosphotyrosine content increased in parallel at 0.1 and 1 mM ATP, they were both reduced at higher ATP concentrations
(Fig. 1). This was not due to proteolysis of the
-subunit since none
was lost in these experiments. The effect requires the high energy bond
of ATP, and was unique to the endosomal compartment since PM IRK
activity was not suppressed at high ATP concentrations. ENs have a
slightly acidic interior maintained by an ATP-dependent
proton pump (15, 30, 31). The observation that
ATP-dependent inhibition of IRK activity was reversed
by bafilomycin (Fig. 4) strongly supports the idea that proton pump
acidification of ENs is critical to this process. It is noteworthy that
the levels of ATP promoting IRK inhibition approximate estimated
intracellular concentrations (42-44).
We explored the mechanism by which ATP-dependent endosomal acidification effects inactivation of the IRK. Since serine/threonine phosphorylation of the IRK has been show to reduce IRK activation (28, 29, 45), we examined the phosphoamino acid content of the endosomal IRK incubated in the presence of 1 versus 5 mM ATP. Two-dimensional phosphoamino acid analyses showed that serine/threonine phosphorylation of IRK was not augmented at higher ATP concentrations. The marked reduction in phosphotyrosine content of the IRK at 5 mM ATP was not a consequence of augmented phosphotyrosyl phosphatase activity, since bpV(phen) did not antagonize the inhibitory effect of 5 mM ATP on either IRK phosphotyrosine content or activity.
The ATP inhibitory effect was shown to be independent of the
phosphorylation state of the -subunit (Fig. 7), suggesting that an
intrinsic defect in IRK function secondary to a conformational change
might explain our observations. Indeed the ability of DTT to reduce
tetrameric IRK molecules was significantly decreased subsequent to the
incubation of ENs with 5 mM ATP (Fig. 8). The reduced
susceptibility of the type I disulfide bond between the
- and
-subunits to DTT implies the occurrence of a
pH-dependent modification of the IRK. We suggest that a
conformational change of the IRK, effected by the
ATP-dependent intraluminal drop in pH, was transmitted to
its cytosolic domain producing decreased IRK activity. This is
consistent with crystallographic studies (46, 47) suggesting that,
whereas activation of IRK occurs through a trans-autophosphorylation
reaction, deactivation occurs through a cis-intramolecular mechanism
(cis-inhibition) (46). The presumed change in the intraluminal portion
of the IRK may be responsible for the observed decrease in insulin
binding (cf. Table I).
This study indicates that the regulation of endosomal IRK activity is multifaceted and that the deactivation involves several discrete components. Previous work has shown that insulin signaling occurs from the endosomal system (9-11). The present study supports the view that there is a temporal window of signaling delimited in part by progressive acidification of ENs due to the activity of ATP-dependent proton pumping. Endosomal acidification contributes to IRK inactivation by: 1) promoting insulin dissociation from the IRK, 2) activating endosomal acidic insulinase, 3) decreasing the binding capacity of IRK, and 4) altering the conformation of the IRK thus reducing intrinsic activity.
Other studies have documented the importance of endosomal acidification
in regulating a range of biological processes. Vesicular stomatitis and
rabies viruses enter cells through receptor-mediated endocytosis but
are rendered competent to enter cytosol after accessing the low pH of
ENs. In this environment the viral envelope undergoes a conformational
transition permitting fusion of viral membrane with endosomal membranes
(48, 49). This transition involves the exposure of a hydrophobic
segment within the glycoprotein whose ability to interact with
membranes effects fusion and extrusion of the viral core through the
wall of ENs (49). Low pH-driven conformational changes in ENs have been
described for the diphtheria toxin and constitute a prerequisite for
the subsequent reduction of the diphtheria toxin interchain disulfide
bond, the rate-limiting step in translocation of toxin into cytosol
(50). Recent data suggest that a key determinant regulating
dephosphorylation and resensitization of the -adrenergic receptor is
the association of internalized receptor and phosphatase in a step
involving pH-sensitive conformational change(s) in receptor and/or
phosphatase (51).
The regulation of intraendosomal pH may play a role in modulating insulin sensitivity in vivo (52-54) since, in type II diabetic patients, the acidotropic agent chloroquine improved glucose metabolism (55-61). Because chloroquine inhibits intraendosomal insulin degradation it may be inferred that the endosomal accumulation of intact insulin is responsible for the improved insulin sensitivity. The present work raises the possibility that the metabolic effects of chloroquine are due to an influence on IRK conformation and function. Indeed it may be that pH-dependent disturbances in IRK function contribute to the pathogenesis of type II diabetes mellitus.
![]() |
ACKNOWLEDGEMENT |
---|
We express our appreciation to Sheryl Jackson for help with typing and editing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Medical Research Council of Canada, the National Cancer Institute of Canada, the Fonds de Recherche du Quebec, and the Cleghorn Fund at McGill University.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Chercheur Boursier, Junior 2 of the Fonds de Recherche en Sante du Quebec.
** To whom correspondence should be addressed: Polypeptide Hormone Laboratory, McGill University, Strathcona Anatomy and Dentistry Bdg., 3640 University St., Rm. W315, Montreal, Quebec Canada. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail: mc85{at}musica.mcgill.ca.
The abbreviations used are:
IRK, insulin
receptor kinase; EN, endosome; WGA, wheat germ agglutinin; PM, plasma
membrane; bpV(phen), bisperoxo(1,10-phenanthroline)oxovanadate anion; PY, phosphotyrosine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycinePAGE, polyacrylamide gel electrophoresishGH, human growth hormoneDTT, dithiothreitolEGF, epidermal growth factorAMP-PNP, adenosine
5'-(,
-imino)triphosphateAMP-PCP, adenosine
5'-(
,
-methylene)/triphosphate AMP-PSP, adenosine
5'-(3-thiophosphate)GAR, goat anti-rabbit antibody.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|