From the Department of Cell and Molecular Biology,
Northwestern University Medical School, Chicago, Illinois 60611, the
§ Department of Ophthalmology, Pharmacology, and Chemistry,
University of Washington School of Medicine, Seattle, Washington
98195-6485, and the ¶ Ralph and Muriel Roberts Laboratory for
Vision Research, Sun Health Research Institute,
Sun City, Arizona 85372
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ABSTRACT |
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Desensitization is a ubiquitous response of
guanine nucleotide-binding protein-coupled receptors (GPCRs)
characterized by the waning of effector activity despite continued
presence of agonist. Binding of an arrestin to the activated, often
phosphorylated GPCR triggers desensitization. We reported for the
luteinizing hormone/choriogonadotropin receptor (LH/CG R) that
The luteinizing hormone/choriogonadotropin receptor (LH/CG
R)1 is a guanine
nucleotide-binding protein-coupled receptor (GPCR) localized on the
cell surface predominately of ovarian theca, granulosa and luteal cells
and testicular Leydig cells (1, 2). Agonist activation of the LH/CG R
leads to activation of adenylyl cyclase (AC) via Gs (3)
and, in some cells, to activation of phospholipase C (4-6). Like the
responsiveness of most GPCRs (7), agonist-stimulated AC activity wanes
in response to persistent stimulation of LH/CG R with saturating
agonist concentrations (8-10). Desensitization of LH/CG R-stimulated
AC activity occurs physiologically in preovulatory ovarian follicles in
response to the mid-cycle surge of luteinizing hormone ,which induces
ovulation and corpus luteum formation (8, 11-13). The initial 60 min
of follicular desensitization in which receptor-stimulated AC activity is reduced ~50% (14-17) without a concomitant reduction in LH/CG R
numbers (18-22) can be mimicked in cell-free follicular membrane preparations (23-30). Desensitization of LH/CG R-stimulated AC activity in follicular membranes requires GTP (Km = ~70 nM) (23, 25-27) and is reversed by the GDP analog
GDP LH/CG R desensitization in follicular membranes is, however, dependent
upon an endogenous arrestin, likely Materials--
Visual arrestin was purified from bovine retinas
(37); recombinant Desensitization and AC Assay--
A partially purified membrane
fraction enriched in AC activity was isolated from porcine ovarian
follicles (34) and stored at Binding of Statistics--
Results (means ± S.E.) were analyzed using
Student's t test (p < 0.05) (41).
Experiments were conducted to determine whether synthetic peptides
(Fig. 1) corresponding to the entire 2i
and 3i cytoplasmic loops or the N-terminal portion of the cytoplasmic
tail (4i) of the porcine LH/CG R (1) could competitively disrupt
agonist-dependent desensitization of AC activity in porcine
ovarian follicular membranes. To this end, membranes were first
preincubated (30 min at 4 °C) with/without synthetic LH/CG R
peptides and then subjected to the stage 1 incubation (40 min at
30 °C) under conditions which do promote (plus hCG) or do not
promote (minus hCG) desensitization, followed by a 5-min AC assay.
Agonist-dependent hyperdesensitization of LH/CG
R-stimulated AC activity was readily demonstrated in membranes
incubated with hCG, GTP, and 8% ethanol during the stage 1 desensitization incubation (Fig.
2A, compare hatched
with solid bars). Preincubation of follicular membranes with
a synthetic peptide corresponding to the LH/CG R 3i loop resulted in a
concentration-dependent rise in hCG-stimulated AC activity
when hCG was present in stage 1 of the desensitization reaction (Fig.
2A, compare hatched bars) and a corresponding
reduction in the percentage of desensitization of hCG-stimulated AC
activity (Fig. 2B). The ED50 for LH/CG R 3i
peptide to increase hCG-stimulated AC activity was 10 µM.
Concentrations of LH/CG R 3i greater than 20 µM were less
effective and, at 114 µM, no longer reversed
desensitization (Fig. 2C). In contrast to its effect on
desensitization of agonist-dependent AC activity, when the
desensitization (stage 1) incubation was omitted, the LH/CG R 3i
peptide over the same concentration range did not affect basal or
hCG-stimulated AC activities and only minimally (~10%) reduced
forskolin-stimulated AC activities (Fig. 2D). Like the entire 3i loop of the LH/CG R, a peptide (3iTM6) corresponding to the
C-terminal 12 residues of 3i through the N-terminal 8 residues of
transmembrane domain (TM) 6 also reversed desensitization by preventing
the hCG-dependent fall in hCG-stimulated AC activity (Fig.
2E). The concentration dependence for LH/CG R 3iTM6 was very
similar to that of LH/CG R 3i. The maximum effect of LH/CG R 3iTM6 was
seen at 20 µM, and 3iTM6 peptide was ineffective at 114 µM (not shown). LH/CG R 3iTM6 did not significantly
(p > 0.05) affect basal or hCG-stimulated AC
activities in a 5-min AC assay (Fig. 2F).
-arrestin tightly bound to porcine ovarian follicular membranes
mediates agonist-dependent desensitization of LH/CG
R-stimulated adenylyl cyclase (AC) activity (Mukherjee, S., Palczewski,
K., Gurevich, V. V., Benovic, J. L., Banga, J. P., and
Hunzicker-Dunn, M. (1999) Proc. Natl. Acad. Sci. U. S. A.
96, 493-498). We now show that addition of a synthetic peptide
corresponding to the entire third intracellular loop (3i) of the LH/CG
R completely and specifically reverses desensitization of AC activity,
with an ED50 of 10 µM but does not modulate
basal, hCG-stimulated, or forskolin-stimulated AC activities.
-Arrestin binds selectively to the 3i peptide coupled to activated
Sepharose. Desensitization of LH/CG R-stimulated AC activity is rescued
when the 3i peptide is preincubated with exogenous
-arrestin. These
results show that endogenous
-arrestin participates in cell-free
desensitization of agonist-dependent LH/CG R-stimulated AC
activity in follicular membranes by interacting directly with the 3i
loop of the receptor, thereby preventing Gs activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (29) that prevents G protein activation. A hyperdesensitized
state characterized by ~80% desensitization can be achieved by
preincubating follicular membranes in the presence of 8% ethanol (31).
This hyperdesensitized state of the LH/CG R retains dependence on GTP and agonist for development as well as its ability to be reversed by
GDP
S, and like LH/CG R desensitization in the absence of ethanol is
unaffected by addition of exogenous G
or by sequestration of
endogenous G
(32, 33).2
-arrestin (34). Disruption of
-arrestin binding to the LH/CG R with monoclonal arrestin antibodies
that recognize common epitopes on all arrestins specifically blocks
agonist-dependent LH/CG R desensitization, and this effect
is reversed by a synthetic peptide corresponding to the
antibody-binding site on
-arrestin (34). Moreover, addition of
purified recombinant
-arrestin to follicular membranes mimics desensitization by reducing agonist-dependent AC activity
to nearly basal levels, with an ED50 value of 0.1 nM (34). The sites on the ligand-activated, phosphorylated
GPCRs with which arrestins interact have been investigated using
purified light-activated phosphorylated rhodopsin and synthetic
peptides to cytoplasmic domains of rhodopsin (35). Results showed that
arrestin bound with highest affinity to the third intracellular (3i)
loop as well as to portions of the second intracellular (2i) loop and the C-terminal tail of rhodopsin. Consistent with this result, arrestins were shown to bind with high affinity to recombinant 3i
subdomains of the m2- and m3-muscarinic and
2A/D-adrenergic receptors fused to glutathione
S-transferase (36). Based on our evidence that endogenous
-arrestin participates in
desensitization of the LH/CG R, in this report we sought to determine
whether the putative 3i loop of the LH/CG R could compete with LH/CG R for endogenous
-arrestin and reverse agonist-dependent
LH/CG R desensitization.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin was expressed and purified (38).
-Arrestin peptide (VFEDFARQRLKG) and indicated LH/CG R peptides (see
Fig. 1) were synthesized and purified to 95% purity by high pressure liquid chromatography by the Protein Chemistry Core Facility at Baylor
College of Medicine (Houston, TX), dissolved in water, and neutralized.
CNBr-activated Sepharose 4B was purchased from Amersham Pharmacia
Biotech; remaining sources are as described previously (34).
70 °C. Protein concentrations were
determined (39) using BSA as standard. For the two-stage
desensitization reaction, reagents (in 20 µl) for stage 1 desensitization reaction (BSA or hCG at 10 µg/ml in 8% ethanol, 25 mM 1, 3 bis-[tris(hydroxymethyl)-methylamino]propane, pH
7.2, 10 µM GTP, 0.4 mM EDTA, 1 mM
EGTA, 0.2 mg/ml creatine phosphokinase, 20 mM
phosphocreatine, 5 mM MgCl2, 1 mM
ATP (or if indicated AMP-PNP), and 1 mM
[3H]cAMP (~20,000 cpm)) were added to follicular
membranes (~30 µg in 20 µl, preincubated with water or indicated
synthetic peptide), and incubation was for 40 min at 30 °C. An assay
for AC activity (stage 2) was immediately performed at 30 °C for 5 min with addition of a 10-µl volume containing 100 µM
GTP, [
-32P]ATP (~5 µCi, 100-200 cpm/pmol), and 10 µg/ml BSA or hCG. When only the AC assay was conducted (and stage 1 was omitted), complete reaction mix was added to membranes (in a
30-µl volume, such that final incubation volume was 50 µl).
Reaction was stopped (23), and [32P]cAMP was purified and
quantified (24, 40). Final concentrations of reagents in a 50-µl
reaction volume are indicated throughout.
-Arrestin to LH/CG R Peptides--
50 mg of
CNBr-activated Sepharose 4B was swollen in 1 mM HCl,
coupled overnight (4 °C) to 0.1 mg/ml indicated synthetic LH/CG peptide in 0.1 M NaHC03 (pH 8.3) containing 0.5 M NaCl, then washed to remove unbound peptide, incubated
2 h in 0.1 M Tris-HCl (pH 8.0), washed with 0.1 M acetate buffer (pH 4.0) containing 0.5 M
NaCl, washed with 0.1 M Tris-HCl (pH 8.0) containing 0.5 M NaCl, equilibrated in 50 mM Tris-HCl (pH 7.4)
containing 100 mM NaCl (buffer A), and incubated with 0.2 ml of 0.2 mg/ml recombinant
-arrestin 1 h at room temperature.
Sepharose was then washed with buffer A; proteins were eluted with 0.25 ml of 3× SDS sample buffer, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to Hybond for Western blot analysis
using arrestin antibody (Transduction Laboratory) (34).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
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Fig. 1.
Locations in the porcine LH/CG R of regions
corresponding to peptides synthesized and amino acid sequence of each
peptide. Peptides corresponding to indicated regions of the
porcine LH/CG R (2i, 3i, 3iTM 6, N-terminal 13 amino acids of 4i,
N1-20, and scrambled (scb) 3i) were synthesized and
purified as described under "Experimental Procedures."
View larger version (41K):
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Fig. 2.
Effect of synthetic peptides corresponding to
indicated regions of the porcine LH/CG R on
agonist-dependent desensitization of LH/CG R-stimulated AC
activity in porcine ovarian follicular membranes. A,
preincubation of membranes with a synthetic peptide corresponding to the 3i loop of the LH/CG R resulted in a
concentration-dependent rise in hCG-stimulated AC activity
when membranes were incubated in stage 1 of the desensitization
incubation with hCG. Membranes were preincubated in the presence of
synthetic peptide or water, as indicated, at 4 °C for 30 min.
Following preincubation, the two-stage desensitization incubation was
conducted, consisting of a 40-min stage 1 reaction under conditions
that promote development of desensitization of hCG-stimulated AC
activity (+ hCG) or do not promote desensitization (+ BSA) followed by
a 5-min AC assay (± hCG; stage 2) as described under "Experimental
Procedures". The presence of BSA in stages 1 and 2 measured basal AC
activity; BSA in stage 1 and hCG in stage 2 measured full
hCG-stimulated AC activity; hCG in stages 1 and 2 measured hCG-induced
desensitization of AC activity. The percentage of reduction of full
hCG-stimulated AC activity above basal AC activity, expressed as the
percentage of desensitization, was used as a measure of the extent of
LH/CG R desensitization. Peptide concentrations refer to final
concentrations in 50-µl reaction volume. Results are the means ± S.E. of quadruplicate determinations from a single experiment and
are representative of one to four separate experiments. Equivalent
results were obtained when desensitization reaction contained 1 mM AMP-PNP instead of ATP. B, composite effect
of indicated concentrations of 3i LH/CG R peptide on the percentage of
desensitization of hCG-stimulated AC activity. Results are the
means ± S.E. of four (water, 15 µM peptide) or two
(6.5 µM peptide) separate experiments and include the
experiment in which AMP-PNP was substituted for ATP. *, differences
between the percentage of desensitization values with 3i peptide are
significantly different from water control (p < 0.05).
C, concentration-dependent effect of synthetic
peptides corresponding to LH/CG R loops 3i and 2i on the AC activity of
membranes preincubated with the indicated concentrations of peptides
then subjected to a 40-min desensitization incubation and a 5-min AC
assay both in the presence of hCG (i.e. hCG/hCG in stage
1/stage 2). Results are the means ± S.E. of quadruplicate
determinations and are representative of two experiments. D,
effect of synthetic peptides corresponding to 3i loop of the LH/CG R on
basal, hCG-stimulated, and forskolin-stimulated AC activities in
follicular membranes. Membranes were preincubated as described in
A in the presence of indicated concentrations (in final
assay reaction volume) of synthetic peptide corresponding to LH/CG R
loop 3i and then subjected to a 5-min AC assay (omitting the 40-min
desensitization reaction) in the presence of 10 µg/ml BSA or hCG or
10 µM forskolin. Results are the means ± S.E. of
quadruplicate determinations and are representative of two separate
experiments. E, effect of synthetic peptides corresponding
to 4i and 3iTM6 loops of the LH/CG R on agonist-dependent
desensitization of LH/CG R-stimulated AC activity in follicular
membranes. Membranes were incubated as in A. Results are the
means ± S.E. of quadruplicate determinations and are
representative of three separate experiments. F, effect of
synthetic peptides corresponding to 3iTM6 loop of the LH/CG R on basal,
hCG-stimulated, and forskolin-stimulated AC activities in follicular
membranes. For details see D. Results are the means ± S.E. of quadruplicate determinations and are representative of two
separate experiments. Forskolin-stimulated AC activities in the
presence of 0.1 and 114 µM 3iTM6 are significantly
different (p < 0.05). G, effect of
synthetic peptides corresponding to the 2i loop of the LH/CG R on
agonist-dependent desensitization of LH/CG R-stimulated AC
activity in follicular membranes. For details see A. Results
are the means ± S.E. of quadruplicate determinations and are
representative of two separate experiments. H, effect of
synthetic peptides corresponding to the N1-20 of the LH/CG R on
agonist-dependent desensitization of LH/CG R-stimulated AC
activity in follicular membranes. For details see A. Results
are the means ± S.E. of quadruplicate determinations.
I, effect of a synthetic peptide containing amino acids of
the 3i loop of the LH/CG R in a scrambled order on
agonist-dependent desensitization of LH/CG R-stimulated AC
activity in follicular membranes. For details see A. Results
are the means ± S.E. of quadruplicate determinations and are
representative of two separate experiments.
To determine whether the effect of the LH/CG R 3i peptide on
agonist-dependent LH/CG R desensitization was specific, the
effects of LH/CG R peptides corresponding to the entire 2i loop or the N terminus of 4i were tested. A synthetic peptide containing the 20 amino acids of 3i randomly scrambled was also tested. Neither LH/CG R
loops 2i (Fig. 2G) nor the N terminus of 4i (Fig.
2E) nor follicle-stimulating hormone receptor 3i loop (not
shown) at 15 µM affected agonist-dependent
LH/CG R desensitization. Higher concentrations of LH/CG R 2i also did
not modulate hCG-desensitized AC activity (Fig. 2C). A
peptide corresponding to N-terminal 20 amino acids of the LH/CG R
(located in the extracellular domain) also did not modulate
agonist-dependent LH/CG R desensitization (Fig.
2H). Scrambled 3i peptide did not modulate
agonist-dependent LH/CG R desensitization (Fig.
2I) and did not affect basal or hCG-stimulated AC activities
in a 5-min AC assay (not shown). Based on our previous evidence that
agonist-dependent desensitization of the LH/CG R is
mediated, at least in part, by the binding of endogenous -arrestin
to the LH/CG R (34), these results are consistent with the hypothesis
that the endogenous
-arrestin is binding primarily to the 3i loop of
the LH/CG R.
To determine whether -arrestin could bind directly to the 3i loop of
the LH/CG R, synthetic peptides corresponding to the LH/CG R 3i or 2i
loops or a peptide containing amino acids of the 3i loop randomly
scrambled were coupled to CNBr-activated Sepharose. The ability of
recombinant
-arrestin to bind to Sepharose-coupled peptides was then
evaluated.
-Arrestin bound only to authentic LH/CG R 3i synthetic
peptide and not to LH/CG R 2i or a 3i scrambled peptide (Fig.
3).
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If the synthetic peptide corresponding to the 3i loop of the LH/CG R is
blocking agonist-dependent LH/CG R desensitization by
binding to the endogenous membrane-associated -arrestin, then saturation of the LH/CG R 3i synthetic peptide with an exogenous arrestin should free endogenous
-arrestin and thereby revive agonist-dependent LH/CG R desensitization. Results in Fig.
4A show that when the 3i LH/CG
R peptide (at 7.5 µM) was incubated with 0.2 µM visual arrestin (4C, 30 min) and then membranes were added and preincubation was continued for 30 min at 4 °C, followed by the two-stage desensitization reaction,
agonist-dependent desensitization of LH/CG R was rescued to
levels seen in the water control (Fig. 4A, compare
hatched bars). Equivalent results were obtained when 3i
LH/CG R peptide was incubated with 40 nM
-arrestin (Fig.
4B). In agreement with previous results (34), addition of
unopposed
-arrestin reduced full hCG-stimulated AC activity (Fig.
4B, solid bars) to levels seen with hCG in stage
1 (Fig. 4B, hatched bars).
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DISCUSSION |
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These results comprise the first report, to our knowledge, of the
ability of a synthetic peptide corresponding to the 3i loop of a GPCR
to reverse completely agonist-dependent desensitization of
effector AC activity. This result was anticipated, based on evidence
that arrestin binds primarily to the 3i loop of phosphorylated, light-activated rhodopsin (35) to quench receptor signaling (42); that
ligand-activated, phosphorylated 2-adrenergic receptor requires
binding of an arrestin to uncouple the phosphorylated receptor from
Gs (43-45), resulting in receptor-dependent
effector desensitization; and that apparent
arrestin-dependent internalization of the muscarinic m2
receptor is lost with deletion of the 3i loop (46), which includes the
G protein receptor kinase 2 phosphorylation sites (47). However, in
contrast to these receptors,
-arrestin binding to the porcine
follicular membrane LH/CG R and consequent desensitization of
agonist-dependent LH/CG R-stimulated AC activity appear to
be independent of LH/CG R phosphorylation, based on the occurrence of
desensitization and ethanol-induced hyperdesensitization in the
presence of the ATP phosphorylation antagonist AMP-PNP (23, 27, 30,
34), the absence of detectable phosphate incorporation into
immunoprecipitated LH/CG R under experimental conditions that support
desensitization (30) and
hyperdesensitization,2 the
ability of the poorly hydrolyzable GDP analog GDP
S to completely reverse desensitization (29), and the ability to promote or abolish
agonist-dependent desensitization of the LH/CG R by
exogenous
-arrestin or by
-arrestin antibody, respectively, in
the presence of AMP-PNP (34).
Recombinant -arrestin binds directly to a synthetic peptide
corresponding to the 3i loop of the LH/CG R and not to peptides corresponding to the 2i loop or scrambled 3i. Moreover, reversal of
agonist-dependent LH/CG R desensitization is specific for
the 3i loop of the LH/CG R. These results suggest that membrane-bound follicular
-arrestin binds predominately to the 3i loop of the LH/CG
R to functionally uncouple the activated LH/CG R from Gs, perhaps by displacing Gs. Although it is well established
for a number of GPCRs that the 3i loop comprises the primary binding site for Gs (48-51), sites of Gs contact with
the active LH/CG R are less clear. Synthetic peptides corresponding to
the lower portion of TM6 and to C-terminally extended TM6 peptides
containing point mutations that promote constitutive LH/CG R activation
(5, 52) can partially activate Gs to stimulate AC activity
(53, 54), consistent with the possibility that critical
Gs-binding sites in this region of TM6 become exposed on
LH/CG R activation and are displaced by
-arrestin binding to the
juxtaposed 3i loop of the receptor.
In summary, our results show that endogenous -arrestin binding to
the C-terminal portion of the 3i loop of the LH/CG R promotes desensitization of LH/CG R-stimulated AC activity by blocking receptor
activation of Gs in a physiological membrane model. A synthetic peptide corresponding to the 3i loop of the LH/CG R prevented
development of desensitization of AC activity apparently by displacing
-arrestin from the receptor but did not uncouple receptor-stimulated
activation of Gs. These results suggest that a synthetic
peptide corresponding to the putative 3i loop of the LH/CG R may be
used to enhance signaling of LH/CG R by preventing desensitization,
whereas
-arrestin replacement has the potential to decrease
signaling of overactive LH/CG Rs. Synthetic peptides to other GPCRs
that interfere with arrestin binding are therefore predicted to block
desensitization of effector activities as well as other receptor
responses to arrestins, including receptor sequestration and possibly
down-regulation.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Marlene Hosey, Heidi Hamm, and Andrew Shenker of Northwestern University Medical School for critical discussions of this work.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants P01 HD 21921 (to M. H.-D.), R01 EY 09339 (to K. P.), and R01 EY 11500 (to V. V. G.).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.
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, Northwestern University Medical School, 303 East
Chicago Ave., Chicago, IL 60611. Tel.: 312-503-8940; Fax: 312-503-0566;
E-mail: mhd{at}nwu.edu.
2 M. Hunzicker-Dunn, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
LH/CG R, luteinizing
hormone/choriogonadotropin receptor;
GPCR, guanine nucleotide-binding
protein-coupled receptor;
AC, adenylyl cyclase;
AMP-PNP, adenylyl-imidodiphosphate;
BSA, bovine serum albumin;
TM, transmembrane
domain;
GDPS, guanosine 5'-O-(2-thiodiphosphate).
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