(Received for publication, May 19, 1995)
From the
We previously showed that two subpopulations of
asialoglycoprotein receptors (ASGP-Rs), designated State 1 and State 2
ASGP-Rs, are present in intact cells and that State 2 ASGP-Rs can be
inactivated in permeable rat hepatocytes in a temperature- and
ATP-dependent manner. These inactivated ASGP-Rs can be quantitatively
reactivated by the addition of palmitoyl-CoA (Weigel, P. H., and Oka,
J. A.(1993) J. Biol. Chem. 268, 27186-27190). Here we
show that 50% of purified rat ASGP-Rs are inactivated by treatment
with hydroxylamine under mild conditions. The activity of
affinitypurified ASGP-Rs was assessed by measuring the specific binding
of
I-asialo-orosomucoid (ASOR) in a dot-blot assay after
immobilization onto nitrocellulose. Treatment of ASGP-Rs in solution
with 0.0125-1.0 M NH
OH, pH 7.4, at 4 °C
for 4 h resulted in a progressive loss of ASOR binding activity. ASGP-R
inactivation with NH
OH occurred more readily at basic pH or
at room temperature. Similar treatment with Tris had no effect on
ASGP-R activity. The kinetics of ASGP-R activity loss and the
dose-response for this inactivation were both biphasic, indicating the
presence of two equal populations of ASGP-Rs with different
sensitivities to NH
OH. The more sensitive population of
ASGP-Rs (
50%) was inactivated by treatment with 0.2 M
NH
OH (4 °C, 4 h) or with 1.0 M
NH
OH (4 °C, 1 h) without detectable peptide cleavage as
assessed by SDS-polyacrylamide gel electrophoresis. State 1 ASGP-Rs,
purified from chloroquine- or monensin-treated hepatocytes, showed
significantly less sensitivity to NH
OH treatment (both in
kinetics and dose dependence). Furthermore, under mild conditions
NH
OH caused dissociation and inactivation of
50% of
the total ASGP-Rs (State 1 and State 2) that were prebound to
ASOR-Sepharose, whereas the same treatment caused dissociation of only
<20% of State 1 ASGP-Rs from such preformed complexes. As shown in
the accompanying paper (Zeng, F. Y., Kaphalia, B. S., Ansari, G. A. S.,
and Weigel, P. H.(1995) J. Biol. Chem. 270, 21382-21387)
all three RHL subunits of active ASGP-Rs, in fact, contain covalently
attached palmitate and stearate. In cultured cells,
[
H]palmitic acid is metabolically incorporated
into all three subunits. These radiolabeled fatty acids are completely
released from purified ASGP-Rs by mild NH
OH treatment. We
conclude that the NH
OH-sensitive subpopulation of ASGP-Rs
corresponds to the previously described State 2 ASGP-Rs and that these
receptors require fatty acylation for their ligand binding activity.
The hepatic ASGP-R ()mediates the endocytosis of
desialylated glycoproteins containing terminal galactose or N-acetylgalactosamine(1, 2) . In isolated rat
hepatocytes, ASGP-Rs endocytose ligand by two functionally different
receptor populations; ligand is then intracellularly processed via two
distinct pathways(2, 3) . We have designated these two
receptor populations as the State 1 and State 2 ASGP-Rs. Previous
studies from our laboratory and others have shown that treatment of
hepatocytes with functionally diverse agents such as metabolic energy
poisons, microtubule depolymerizing drugs, lysosomatropic amines,
vanadate, or proton ionophores causes an inactivation and/or
redistribution of cell surface State 2 ASGP-Rs without affecting the
State 1 ASGP-Rs(4, 5, 6, 7) .
Studies with intact cells indicate that the State 2 ASGP-Rs are
inactivated and then reactivated during receptor recycling(4) .
In digitonin-permeabilized cells, the State 2 ASGP-Rs can be
inactivated in the absence of cytosol in a temperature- and
ATP-dependent manner(8) . Recent investigations demonstrated
that the ATP-inactivated receptor population, which corresponds to the
State 2 receptors, was rapidly and quantitatively reactivated in the
absence of cytosol when fatty acyl-CoAs were added (9) . These
studies demonstrated the occurrence of a novel ASGP-R
inactivation-reactivation cycle that could regulate receptor activity
during endocytosis and receptor recycling(10) . Although these
above results support the existence of two functionally different
populations of ASGP-Rs and the occurrence of an
inactivation-reactivation cycle for the State 2 ASGP-R population, the
structural basis for the two receptor states and the molecular
mechanism(s) responsible for this cycle are unknown.
The molecular
mass of the rat ASGP-R is approximately 264 kDa in nonionic detergent
with a ligand-binding domain of 105-148
kDa(11, 12) . Based on SDS-PAGE(13) , the rat
ASGP-R contains three subunits (RHL1, 2, and 3) with molecular masses
of 42, 49, and 54 kDa, respectively. These three subunits are the
products of two different genes. RHL2 and RHL3 have the same core
protein, but differ in the type and extent of post-translational
carbohydrate modification(14) . The stoichiometry and subunit
composition of native rat ASGP-Rs are still unknown, although we have
suggested that ASGP-Rs are hetero-hexamers composed of four RHL1
subunits and two subunits of either RHL2 and/or RHL3(15) . That
previous studies also demonstrated that the surface and internal
ASGP-Rs of the two functionally distinct State 1 and State 2 receptor
populations have the same hetero-oliogometric subunit
composition(16) , indicates the two populations of ASGP-Rs may
not be attributed to different subunit compositions.
The finding
that fatty acyl-CoAs such as palmitoyl-CoA can regulate the activity of
one receptor population (State 2 ASGP-Rs) led us to suggest that this
receptor population may be fatty acylated in vivo(9, 10) . A palmitoylation/depalmitoylation cycle
might regulate the ligand binding activity of State 2 ASGP-Rs. A
growing number of membrane proteins have been found to be modified by
fatty acids. Fatty acids are often covalently attached to proteins
through an amide bond to an N-terminal glycine residue or through an
ester or thioester bond to internal serine, threonine, or
cysteine(17, 18) . The thioester linkages of fatty
acids such as palmitic acid, are very labile to mild alkaline or
NHOH treatment. NH
OH, a chemical usually used
to release thioester-linked fatty acids from acylated
proteins(17, 18) , has been used to study the role of
palmitate in the function of rhodopsin and the influenza virus spike
glycoprotein(19, 20) . Here we report that
50% of
the activity of affinity-purified ASGP-Rs is lost by treatment with
NH
OH under mild conditions and this
NH
OH-sensitive receptor population corresponds to the State
2 ASGP-Rs. A preliminary report of these results has been
presented(21) .
To exclude the
possible effects of other factors such as pH changes and temperature on
ASGP-R activity, the treatment of ASGP-Rs with NHOH or Tris
was carried out on ice in BIC20 containing 0.025% Triton X-100 at pH
7.4. In initial studies, we found that purified total ASGP-Rs
progressively lost up to
50% activity after storage in BIC10
containing 0.05% Triton X-100 at 4 °C for more than 1 week.
Furthermore, the rate of ASGP-R activity loss rapidly increased when
the temperature was
20 °C. Freezing and thawing also caused
loss of activity. We suspect that the State 2 ASGP-R activity is being
lost under these conditions. For this reason, we used only freshly
purified ASGP-Rs in all the experiments reported here (<12 h old) to
minimize any initial loss of ASGP-R activity.
Treatment of ASGP-Rs
with 1 M NHOH caused ASGP-R inactivation in a
time-dependent manner, whereas similar treatment with milder
nucleophiles such as 1 M Tris or methylamine (not shown) had
no significant effect on ASGP-R activity (Fig. 1A). The
kinetics of ASGP-R inactivation in the presence of 1 M NH
OH were quite biphasic. The rate of inactivation
during the initial 60 min was greater than that during incubation after
60 min. About 50% of the ASGP-Rs were inactivated within 1 h; extended
incubation times of up to 18 h resulted in a progressive inactivation
of the remaining
50% of ASGP-Rs. A possible reason for ASGP-R
inactivation might be that under these conditions NH
OH
could cause cleavage of ASGP-R subunits that results in the loss of
activity. To assess this possibility, the NH
OH-treated
ASGP-Rs were analyzed by SDS-PAGE under reducing conditions (Fig. 1B). Within the first 2 h (during which time
50% ASGP-R inactivation occurs), NH
OH treatment did
not degrade any of the three ASGP-R subunits (Fig. 1B).
Prolonged incubation times after 2 h indeed caused a progressive
increase of ASGP-R degradation: prominent fragments of 40 and 24 kDa
were observed. This result indicates that inactivation of the first
50% of ASGP-Rs was not due to peptide cleavage, whereas
inactivation of the second
50% ASGP-Rs could be explained by the
observed peptide degradation.
Figure 1:
Kinetics
of ASGP-R inactivation by NHOH. Freshly purified, total
active ASGP-Rs were incubated in BIC20 containing 0.025% Triton X-100,
and either 1 M NH
OH (
) or 1 M Tris
(
) on ice for 0-18 h. A, at the indicated times,
samples were diluted with 40 volumes of BIC20 and loaded onto
nitrocellulose (0.5 µg of ASGP-R/spot) using a dot-blot manifold.
I-ASOR binding activity for each sample was determined as
described under ``Experimental Procedures.'' B, at
the indicated times, samples were immediately mixed with an equal
volume of 2
Laemmli sample buffer (30) with 5%
-mecaptoethanol, boiled for 1 min, and then analyzed by SDS-PAGE
using a 3.9% stacking gel and a 12.5% resolving gel. The protein bands
were visualized by silver staining.
In another experiment, purified total
ASGP-Rs were incubated with 1 M NHOH on ice for 1
h (this treatment caused an inactivation of
50% ASGP-Rs as shown
in Fig. 1A), and NH
OH was then removed by
repeated dilution of the mixture and subsequent concentration using a
Centricon-10 device. Active ASGP-Rs were then separated from inactive
ASGP-Rs by purification on ASOR-Sepharose. About 50% of the total
ASGP-Rs could still bind to ASOR, the other
50% had lost their
ligand binding activity without detectable peptide cleavage (data not
shown). This result supports the conclusion that under mild conditions,
NH
OH inactivates one population of receptors without
affecting the other population. Hydroxylamine sensitivity of ASGP-Rs
was identical whether a solid-phase or solution-based assay was used to
assess ligand binding activity.
Another possibility is that the
observed ASGP-R inactivation by NHOH is not really due to
reactivity of NH
OH, but rather to other minor components
present in commercial NH
OH preparations, such as heavy
metal ions that could activate minor contaminating proteases that
copurify with ASGP-Rs. To exclude this and several alternative
possibilities, other controls were performed. Incubation of purified
ASGP-Rs with divalent metal ions including Mg
,
Zn
, Ni
, Ca
, and
Mn
did not change their ligand binding activity.
Furthermore, NH
OH from various sources (Sigma, Aldrich,
Fluka, and MC/B) all showed very similar kinetics of ASGP-R
inactivation (not shown). The addition of the divalent metal ion
chelater EGTA did not affect the ability of NH
OH (1 M, 20 h) to inactivate up to
50% of total ASGP-Rs, but
did prevent further inactivation and degradation of the remaining 50%
of ASGP-R subunits (Fig. 2, A and B). This
observation suggests that the activity loss of the less sensitive
ASGP-R population is the consequence of subunit cleavage by
NH
OH. Addition of protease inhibitors such as PMSF,
pepstatin, and leupeptin did not reduce the effectiveness of
NH
OH to inactivate these ASGP-Rs. Furthermore, after
incubation of ASGP-Rs with Tris in the presence of Ca
for more than 24 h, no detectable protein degradation could be
observed (not shown).
Figure 2:
Effect of EGTA on inactivation and
cleavage of ASGP-R by NHOH. A, freshly purified
active ASGP-Rs were incubated with 1 M NH
OH in
buffer 1 containing different concentrations of EGTA or CaCl
on ice for 2 h (
) or 20 h (
). ASOR binding activity
was then measured using a dot-blot assay as described under
``Experimental Procedures.'' ASGP-R activity is expressed as
a percentage of control samples incubated with 20 mM CaCl
and without NH
OH. B, one
portion of treated ASGP-R samples (20 h) was subjected to SDS-PAGE
analysis as described in Fig. 1B. Lanes: 1, 20
mM CaCl
; 2, 1 mM
CaCl
; 3, 2.5 mM EGTA; 4, 10
mM EGTA. Note that degraded receptor bands at 40 kDa (open
arrow) and 24 kDa (solid arrow) decrease as the EGTA
concentration increases.
The inactivation of ASGP-Rs by
NHOH treatment (4 °C for 4 h) is also dependent on
NH
OH concentration and this dose-response is biphasic (Fig. 3). The extent of ASGP-R inactivation was proportional to
NH
OH concentration in the range of 0-0.1 M,
corresponding to 0 to
40% inactivation. ASGP-R inactivation was
50% with 0.2 M NH
OH. Increasing
NH
OH concentration in the range of 0.2-1.0 M only slightly increased ASGP-R inactivation. SDS-PAGE analysis
showed no degradation products or decrease in size of RHL1, RHL2, and
RHL3 after treatment with 0 to 0.2 M NH
OH at 4
°C for 4 h (not shown).
Figure 3:
Effect of NHOH concentration
on the inactivation of ASGP-Rs. Freshly purified, active total (State 1
plus State 2) ASGP-Rs were incubated in BIC20 containing 0.025% Triton
X-100, and either 0.0125-1.0 M NH
OH (
)
or 0.0125-1.0 M Tris (
) on ice for 4 h. The
I-ASOR binding activity of samples was then assessed as
described in the legend to Fig. 1.
Biphasic kinetic and dose-responses were
also observed for ASGP-R inactivation by NHOH using
receptor immobilized on nitrocellulose, but inactivation occurred more
slowly than in solution (not shown). The inactivation of ASGP-Rs was
also pH- and temperature-dependent (Fig. 4). At pH 6.0 and 4
°C, no significant inactivation was observed with 0.5 M NH
OH for 1.5 h. A sharp increase in ASGP-R
inactivation was seen between pH 6 and 7 and the extent of inactivation
remained constant from pH 7 to 11. In comparison with treatment at 4
°C, ASGP-R inactivation occurred more readily at 25 °C,
especially at basic pH (Fig. 4).
Figure 4:
Effect of pH and temperature on ASGP-R
inactivation by NHOH. Freshly purified, total active
ASGP-Rs were incubated on ice or at 25 °C for 90 min with 0.5 M NH
OH (
,
) or 0.5 M Tris (
)
in 10 mM Hepes at the indicated pH containing 142 mM NaCl, 6.7 mM KCl, 20 mM CaCl
, and
0.025% Triton X-100. The treated samples were diluted with 100 volumes
of BIC20, loaded onto nitrocellulose, and
I-ASOR binding
activity was assessed as described in the legend to Fig. 1.
To confirm that the NHOH-sensitive ASGP-R population
corresponds to State 2 ASGP-Rs, we purified State 1 ASGP-Rs, after
first inactivating State 2 ASGP-Rs in intact cells with either
chloroquine or monensin as described under ``Experimental
Procedures.'' Treatment of isolated rat hepatocytes with
chloroquine (1 mM, 60 min, at 37 °C) or monensin (50
µM, 60 min, at 37 °C) resulted in 40-60%
inactivation of total ASGP-Rs as assessed by
I-ASOR
binding with permeable cells. In comparison with untreated cells, about
40-50% of active ASGP-Rs could be purified from treated cells,
verifying that about half of the ASGP-Rs were indeed inactivated. The
purified State 1 ASGP-Rs showed very similar subunit patterns to that
of total ASGP-Rs by SDS-PAGE under reducing conditions (not shown). We
then compared the sensitivity of State 1 ASGP-Rs and total (State 1
plus State 2) ASGP-Rs purified in parallel, to NH
OH
treatment. Kinetically, State 1 ASGP-Rs showed significantly less
sensitivity to NH
OH inactivation than the total ASGP-R pool (Fig. 5). During the first 30 min, 30-35% of the total
ASGP-Rs were inactivated, whereas State 1 ASGP-R activity remained
essentially unchanged. After 30 min incubation, a slow progressive loss
of State 1 ASGP-R activity occurred that reached up to
40%
inactivation at 180 min. The difference in the rate of activity loss
between State 1 versus State 1 plus State 2 ASGP-Rs was
significant within 1 h (p < 0.05). However, the difference
became smaller after 3 h. This latter observation is in agreement with
the finding noted above, that extended incubation causes protein
degradation.
Figure 5:
Kinetic difference between State 1 and
total (State 1 plus State 2) ASGP-R inactivation by NHOH
treatment. Active State 1 ASGP-Rs (
,
), freshly purified
either from chloroquine-treated (
), or monensin-treated
hepatocytes (
), and active total ASGP-Rs (
), freshly
purified from nontreated cells, were incubated in BIC20 containing
0.025% Triton X-100 and 0.5 M NH
OH on ice for
0-3 h. At the indicated times, the
I-ASOR binding
activity of samples was assessed. Each point is the mean ± S.E
of four to six independent experiments. Values significantly different
from the control (total ASGP-Rs) are indicated: *, p <
0.001; +, p < 0.005; #, p <
0.05.
The dose dependence for ASGP-R inactivation by
NHOH also showed a marked difference between the State 1 versus State 1 plus State 2 ASGP-R populations (Fig. 6). At
50 mM NH
OH, no significant
loss of State 1 ASGP-R activity was observed after incubation at 4
°C for 4 h, whereas
25% of the activity of the total receptors
was lost. The difference in the ability of NH
OH to cause
inactivation was more marked at a lower concentration range than at
higher concentration (
400 mM). The purified State 1
ASGP-Rs prepared from either chloroquine- or monensin-treated
hepatocytes, showed essentially identical sensitivity to
NH
OH (both in kinetics and dose dependence; Fig. 5and 6), indicating that treatment of isolated cells with
chloroquine or monensin inactivated the same ASGP-R population (State
2), as reported previously(26) . These results strongly support
the conclusion that the NH
OH-sensitive population is the
State 2 receptor population.
Figure 6:
State
1 and total (State 1 plus State 2) ASGP-Rs show different
dose-responses to NHOH. Active State 1 ASGP-Rs (
,
), freshly purified either from chloroquine-treated (
)
or monensin-treated hepatocytes (
) and total ASGP-Rs (
)
were incubated with 0-400 mM NH
OH in BIC20
containing 0.025% Triton X-100 on ice for 4 h. The
I-ASOR
binding activity of individual samples was assessed as described under
``Experimental Procedures.'' Each point is the mean ±
S.E. of four to six experiments. Values significantly different from
the control (total ASGP-Rs) are indicated: *, p < 0.001;
+, p < 0.005; #, p <
0.05.
Figure 7:
Effect
of NHOH on preformed ASGP-R
ASOR complexes. Preformed
ASGP-R
ASOR-Sepharose complexes, prepared as described under
``Experimental Procedures,'' were incubated with BIC20
containing 0.05% Triton X-100, and either 0.1 M Tris or 0.1 M NH
OH at 4 °C for 18 h. After centrifugation
the supernatant (s) and pellet (s), were washed once
with BIC20-0.05% Triton X-100, and then analyzed by SDS-PAGE and
silver staining as described in the legend to Fig. 1.
In contrast
to total purified ASGP-Rs, only 14-19% of the State 1 ASGP-Rs
were dissociated and inactivated by treatment with NHOH
under the same conditions (Table 1). The difference in
inactivation between total versus State 1 ASGP-Rs was about
25% (p < 0.005) for both treatment conditions tested (0.1 M NH
OH, 18 h; 1 M NH
OH, 1 h).
That only 14-19% of receptors were still released from State 1
ASGP-R
ASOR complexes by NH
OH under conditions that
removed almost 50% of total ASGP-Rs, is in agreement with the results
shown in Fig. 5and Fig. 6, and could be due to the
presence of a small amount of State 2 ASGP-Rs in our State 1 ASGP-R
preparations. We find that the extent of ASGP-R inactivation in
isolated rat hepatocytes treated with chloroquine or monensin varies
(40-60%) from cell preparation to preparation, even using the
same conditions(26) . At present, we cannot determine the
percentage of State 2 ASGP-Rs that copurifies with State 1 ASGP-Rs by
this method. Nonetheless, our results demonstrate the existence of two
receptor populations in purified ASGP-Rs.
One potential disadvantage
of using the dot-blot assay to quantitate changes in ASGP-R activity
after treatment with NHOH is that only about 10% of
immobilized ASGP-Rs are capable of binding ASOR (assuming that one
ASGP-R binds one ASOR).
The majority (
90%) of
immobilized receptors are unable to bind ASOR, perhaps due to
conformational inflexibility of immobilized molecules or
inaccessibility to ligand. Differential adsorption or preferential
conformational changes of active or inactivated ASGP-Rs during
immobilization onto nitrocellulose could complicate interpretation of
the results. Several controls were performed to address this
possibility. Using specific antireceptor antibody to quantitate
nontreated or NH
OH-treated ASGP-Rs after immobilization, we
found that NH
OH treatment did not change the adsorption of
receptor. Most importantly, the finding that
50% of ligand-bound
ASGP-Rs are more readily dissociated and inactivated by
NH
OH (Table 1) corroborates the results obtained from
the dot-blot assay. These results also argue against a difference in
adsorption ability or in conformational changes of immobilized active versus inactivated ASGP-Rs.
Figure 8:
Mild treatment with NHOH
releases all of the metabolically incorporated
[
H]palmitate from ASGP-Rs. Cultured hepatocytes
(2
10
cells/dish) were labeled with 400 µCi/ml
[
H]palmitate for 4 h and active ASGP-Rs were
purified as described under ``Experimental Procedures.'' The
purified ASGP-Rs were incubated with BIC20 alone (lane 1), or
containing 1 M Tris (lane 2), or 1 M NH
OH (lane 3) on ice for 2 h prior to
SDS-PAGE. The gel was subjected to fluorography (26) for 25
days.
Our previous studies have demonstrated that rat hepatocytes
contain two functionally different receptor populations: State 1
ASGP-Rs and State 2 ASGP-Rs(2) . Although present in
approximately equal numbers, the State 2 ASGP-Rs mediate the large
majority (80%) of ligand processing (i.e. endocytosis,
segregation, and degradation) in hepatocytes. Functionally therefore,
State 2 receptors are roughly about 4 times more active than State 1
receptors. In contrast to State 1 ASGP-Rs, the State 2 ASGP-Rs can be
modulated in their cellular distribution and/or ligand binding activity
by treating cells with low temperature or with metabolic energy
poisons, microtubule drugs, monensin, chloroquine, or
vanadate(4, 5, 25, 32, 33) .
Many of these agents cause selective inactivation of the State 2
ASGP-Rs, which led us to propose that this receptor population
undergoes an intracellular inactivation-reactivation cycle during
receptor-mediated endocytosis and receptor recycling(34) .
Since the inactivation of State 2 ASGP-Rs is transient and
reversible, we suggested that receptor inactivation could be the
mechanism by which cells achieve efficient ligand dissociation and
subsequent segregation of ligand from receptor(34) . During
receptor-mediated endocytosis, the concentration of ligand in endosomes
can be increased up to 10-fold over the extracellular
concentration(2) . Cell surface ASGP-Rs are also concentrated
about 50-fold in endosomal membranes. Under these conditions of such
high concentrations, dissociated ligand molecules would likely rebind
to active receptors even at the lower pH of early endosomes. Any ligand
rebound to receptor would not be shuttled to lysosomes for degradation,
but rather nonproductively recycled back to the cell surface. Such
``futile'' receptor cylces would, of course, be wasteful.
Most significantly, futile ligand recycling would increase as the
extracellular ligand concentration increased, and the endocytic
machinery would function less efficiently when the need for ligand
clearance was greatest. This situation, which could be physiologically
deleterious, is avoided by inactivating ASGP receptors so that ligand
rebinding does not occur.
The cumulative evidence indicates that the
ASGP-R is a hetero-oligomeric complex, composed of RHL1 and RHL2/3
subunits. (i) Both RHL1 and RHL2/3 gene products are required for
expression of a functional ASGP-R capable of binding ASOR in
transfected cells(35) . (ii) Subunit-specific antibodies
coimmunoprecipitate all three RHL subunits of the
ASGP-R(16, 36) . (iii) Studies with a chemical
affinity derivative of I-ASOR showed identical
cross-linking patterns for State 1 or State 2 ASGP-Rs(16) .
(iv) Affinity-purified ASGP-Rs from cells with active State 1 or State
1 plus State 2 ASGP-Rs show identical subunit patterns in SDS-PAGE. (
)These above results indicate that functional differences
between State 1 and State 2 ASGP-R are not due to different subunit
compositions.
This and the accompanying paper (28) establish
for the first time a structural difference between the two populations
of receptor and the possible role of this structural difference in the
functional differences between State 1 and State 2 ASGP-Rs. The
molecular basis for the inactivation-reactivation cycle of State 2
ASGP-Rs is related to fatty acylation. We previously showed that
palmitoyl-CoA rapidly and quantitatively reactivates inactivated State
2 ASGP-Rs, suggesting that either ASGP-R subunits or other unknown
regulatory proteins are palmitoylated and that a
palmitoylation-depalmitoylation cycle may regulate ASGP-R
activity(9) . These results prompted us to examine more
directly if ASGP-Rs are modified by fatty acylation. As detailed in the
accompanying paper(28) , all three RHL subunits contain
covalently attached palmitate and stearate. These fatty acids were
found using gas chromatography-mass spectroscopic analysis of purified
RHL subunits after SDS-PAGE of active ASGP-Rs. Our results in the
present study provide further evidence to support this conclusion;
metabolic labeling with [H]palmitate also
confirms that RHL1, RHL2, and RHL3 are fatty acylated in intact cells.
Surprisingly, only the State 2 ASGP-Rs, not State 1 ASGP-Rs, are palmitoylated in a metabolic labeling experiment. Essentially all of the palmitic acid metabolically incorporated into RHL1, RHL2, and RHL3 is released by mild hydroxylamine treatment that inactivates only the State 2 ASGP-Rs. We conclude that State 2 ASGP-Rs are clearly fatty acylated. However, further studies are needed to determine whether State 1 ASGP-Rs are fatty acylated; it is quite possible that they are not.
NHOH treatment decreases ASGP-R activity in a time-
and dose-dependent manner ( Fig. 1and Fig. 2).
Inactivation of ASGP-Rs is kinetically biphasic;
50% of ASGP-Rs
are very sensitive to NH
OH and the other 50% are much less
sensitive. During the loss of NH
OH-sensitive ASGP-R
activity, no protein degradation occurs, indicating that loss of
activity is not due to peptide cleavage. Although the percentage of
NH
OH-sensitive ASGP-Rs ranged from 30 to 60% with different
ASGP-R preparations, the phenomenon of biphasic ASGP-R inactivation by
NH
OH was always observed. This observation is in agreement
with our previous results in intact cells, that the percentage of State
2 ASGP-Rs that are sensitive to treatment with diverse agents varies
from 40 to 60% with different hepatocyte
preparations(32, 33) . The studies using purified
State 1 ASGP-Rs confirm that the NH
OH-sensitive population
corresponds to the State 2 ASGP-Rs. Furthermore, the purification
process may also cause inactivation of some State 2 ASGP-Rs, as we find
that a small percentage (5-10%) of ASGP-R activity is lost after
elution from ASOR-Sepharose. At present, we cannot quantitate the
amount of receptor that may be inactivated during purification, due to
the lack of specific antibodies that can differentiate between active
and inactivated ASGP-Rs.
Our results in this study suggest that
ASGP-Rs are acylated in vivo. Many membrane proteins and
receptors are palmitoylated, often at Cys residues near transmembrane
domains(37, 38, 39, 40, 41, 42) .
The subunits of the rat and human ASGP-Rs have a Cys-Ser sequence close
to the cytoplasm membrane junction(14, 43) . This same
sequence in the transferrin receptor (44) and the
HLA-D-associated invariant chain (45) is palmitoylated at Cys.
It is possible, therefore, that the same position in one or more of the
ASGP-R subunits is also palmitoylated. Inactivation of ASGP-R by
NHOH is likely due to the removal of covalently bound fatty
acids from one or more of the ASGP-R subunits. Since all three RHL
subunits contain covalently bound fatty acids (palmitic acid and
stearic acid) and ASGP-Rs are hetero-oligomeric, there are many
possible partially deacylated receptor species whose ligand binding
activity could be affected.
We also find that NHOH
treatment, but not Tris treatment, results in the formation of dimeric
RHL subunits (based on SDS-PAGE analysis under nonreducing conditions)
indicating that deacylation with NH
OH generates free thiol
groups that then form disulfide bonds in a time-dependent way. (
)This formation of new disulfide bonds upon
NH
OH treatment has been demonstrated in other palmitoylated
proteins such as vesicular stomatitis virus G glycoprotein (38) and human tissue factor(46) . NH
OH has
been widely used to remove thioester bond-linked palmitate from many
palmitoylated proteins, such as transferrin receptor(37) ,
rhodopsin (19) , and virus glycoproteins(38) . Under
mild conditions, depalmitoylation by NH
OH did not greatly
affect the conformational structure of rhodopsin as determined by
circular dichroism(39) . Although NH
OH has usually
been used as a specific chemical to release ester and thioester-linked
fatty acids from fatty acylated proteins(40, 41) , it
also has a number of other chemical effects on proteins.
NH
OH (usually
1 M, pH
9.0;
37 °C)
cleaves susceptible Asn-Gly bonds in many proteins(47) . 2 M NH
OH at pH 9 and at 45 °C has been used
specifically to cleave at the C-terminal side of succinimides in
proteins(48) . NH
OH is also known to remove O-acetyl groups from tyrosine(49) . Our results also
show that 1 M NH
OH cleaves all three RHL subunits
after extended incubation (>4 h). Loss of the ligand binding
activity of the less NH
OH-sensitive State 1 ASGP-R
population is most probably the consequence of this subunit cleavage.
The functional significance of palmitoylation is not known for the
majority of proteins containing this
modification(41, 42) . Since the turnover of fatty
acyl groups is generally much faster than that for the protein, the
potential exists to have a cyclic, or reversible, alteration of some
function of the protein tied to its fatty acylation.
Acylation/deacylation cycles may serve as a general mechanism for
controlling the subcellular distribution and/or function of many
proteins. For example, the activation-induced depalmitoylation results
in activationinduced translocation of the G-protein subunit into
cytoplasm(50) . Regulation of the palmitoylation state of
p21
, a key component of growth factor receptor
signaling pathways, affects its avidity for binding to membranes and
therefore regulates its ability to interact with other
proteins(51) . Palmitoylation of the neuronal growth cone
protein, GAP43, alters its ability to stimulate G
protein(52) . Palmitoylation of the transferrin receptor
may regulate the receptor-mediated endocytosis of diferric
transferrin(53) .
Based on the results here, in the accompanying paper(28) , and our previous results that palmitoyl-CoA reactivates inactive State 2 ASGP-Rs(9, 10) , we propose that a dynamic deacylation/acylation cycle in vivo is the molecular basis for the inactivation/reactivation cycle that State 2 ASGP-Rs undergo during receptor mediated endocytosis.