(Received for publication, April 28, 1995)
From the
Adenovirus vector-mediated transfer of the receptor-associated
protein (RAP) gene into low density lipoprotein (LDL)
receptor-deficient mice was shown to achieve plasma concentrations
ranging between 20 and 200 µg/ml and to result in the accumulation
of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and
Herz, J.(1994) Science 264, 1471-1474). Both this
finding and the observation that in addition to various other members
of the LDL receptor gene family, RAP binds to a yet unidentified
protein of apparent molecular mass of 105 kDa prompted us to examine
the effect of high concentrations of RAP on the lipolysis-stimulated
receptor (LSR). LSR is a receptor distinct from the LDL receptor and
the LDL receptor-related protein and is capable of binding apoB and
apoE when activated by free fatty acids. Data reported here show that
in fibroblasts isolated from a subject homozygous for familial
hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding
of
Dietary lipids, hydrolyzed and absorbed by the intestine, are
repackaged and secreted as chylomicrons. Upon release into the plasma,
these particles undergo lipolysis by lipases located on the surface of
the endothelium. This process generates chylomicron remnants
(CMR)
Significant progress
in the understanding of CMR removal stemmed from the study by Willnow et al.(12) using adenovirus vector-mediated gene
transfer into mice of a 39-kDa receptor-associated protein (RAP). This
protein, which copurifies with the 515-kDa subunit of
LRP(5, 13, 14) , binds to LRP in the presence
of Ca
Experiments were initially undertaken to determine the effect
of RAP on the LSR expressed in human FH fibroblasts. FH fibroblasts
incubated in the presence of 75 µg/ml RAP fusion protein displayed
a consistent reduction of
Figure 1:
Effect of RAP fusion protein on the
kinetics of
Figure 2:
Effect of increasing concentrations of RAP
fusion protein on LSR activity in rat liver membranes. Rat liver
membranes were incubated at 37 °C for 30 min with or without 800
µM oleate and then washed 3 times in buffer A. Following
this, the membranes were incubated at 4 °C for 30 min with the
indicated concentrations of RAP fusion protein, followed by incubation
at the same temperature for 1 h with 10 µg/ml
Curve-fitting analysis of the data in Fig. 2yielded an estimate of 20 µg/ml RAP needed to achieve
50% inhibition. To characterize the molecular mechanism responsible for
RAP's inhibitory effect on LSR activity, the binding of
increasing
Figure 3:
Effect of RAP fusion protein on the
binding of
Figure 4:
Binding of
RAP interaction with LSR protein was confirmed using four
different experimental approaches. First, RAP was found to partially
block the binding of
On the basis of the data currently available, it
appears that LSR represents yet another receptor inhibited by RAP.
Indeed, LSR apparent molecular mass clearly distinguishes it from the
LRP (600 kDa) or the gp330 (600 kDa) but not from the LDL-receptor (120
kDa) or the VLDL receptor (130 kDa). LSR is, however, genetically
distinct from the LDL-receptor; LSR is expressed in homozygous FH
fibroblasts with null alleles, and its activity is
Ca
Thus, our
observations add one more receptor to the series inhibited by RAP, i.e. the LRP(15, 16, 17) , LDL
receptor(18, 19) , VLDL receptor(20) , and
gp330(21) . Currently, information regarding the LSR protein
primary sequence is not available. Thus, it is yet to be determined
whether or not it bears sufficient homology to be considered as a
member of the LDL receptor gene family.
Half-maximum LSR inhibition
was achieved with RAP fusion protein concentrations of 20 µg/ml
(350 nM). Therefore, RAP affinity for LSR is much lower than
that for LRP; half-maximum inhibition of
Analysis of the mechanism of
the inhibition of LSR activity by RAP suggested that RAP does not
directly compete with
Previous studies have established
that CMR clearance was delayed in both normal and LDL-receptor
deficient mice overexpressing RAP(12) . Because gp330 and the
VLDL receptor are not expressed to a large extent in the
liver(22, 23, 24) , it was therefore
concluded that the LRP was responsible for most of CMR removal. The
finding of the RAP inhibitory effect on LSR, however, offers an
alternative interpretation to Willnow's data. Indeed, RAP
inhibition of the LSR could at least partially account for the
hyperlipidemic effect of RAP transfection in both LDL receptor knockout
and wild type mice. Additional information is needed to ascertain the
relative contribution of the LRP and/or the LSR to CMR removal.
We thank Valérie Bordeau, Flora Coulon, and
Evan Behre for their excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-LDL and the subsequent internalization and
degradation of the particles. Studies on the interaction of RAP with
LSR in isolated rat liver membranes revealed that at concentrations
10 µg/ml, RAP inhibited in a dose-dependent manner the binding
of LDL to LSR; half-maximum inhibition was obtained with 20 µg/ml
RAP. Ligand blotting studies revealed that RAP bound directly to two
rat liver membrane proteins of apparent molecular masses identical to
those that bind
I-LDL after preincubation with oleate.
However, unlike LDL, binding of
I-RAP to LSR did not
require preincubation with oleate. Preincubation of nitrocellulose
membranes with an excess of unlabeled RAP fusion protein decreased
oleate-induced binding of
I-LDL to LSR candidate
proteins, whereas preincubation with excess unlabeled LDL was unable to
prevent the subsequent binding of
I-RAP to the LSR
proteins. Both the latter data and analysis of the mechanism of
inhibition were consistent with the RAP inhibitory effect on LSR being
achieved by interference with a site distinct from the oleate-induced
LDL binding site. In conclusion, this study shows that at
concentrations reported to delay chylomicron remnant removal in LDL
receptor-deficient mice, RAP exerted a significant inhibitory effect on
LSR.
(
)that are removed from the circulation by
the hepatocyte through receptor-mediated endocytosis. It is generally
accepted that the low density lipoprotein (LDL) receptor accounts for
part of this process and works in synergy with a second genetically
distinct receptor(1, 2) . The molecular nature of this
second lipoprotein receptor remains disputed(3) . Indeed, two
proteins, the LDL receptor-related protein (LRP) and the
lipolysis-stimulated receptor (LSR), have been proposed as candidates
for this function. The LRP was initially identified by homologous
cloning as a member of the LDL receptor gene family (4) and
subsequently found to be identical to the
-macroglobulin receptor(5) . The LRP is a
600-kDa, Ca
-dependent protein that binds
-very
low density lipoprotein (VLDL) enriched with apoprotein (apo)
E(6) , lipoprotein lipase(7) , as well as a series of
ligands apparently not related to the lipoprotein system, e.g. activated
-macroglobulin (for review, see (8) and (9) ). The biochemical characterization of LSR
is far less advanced(10) . Its activity is attributed in human
cells to two membrane proteins of apparent molecular masses of 115 and
85 kDa(11) . In rat hepatocytes, the apparent molecular masses
of these two proteins are 115 and 90 kDa.
(
)LSR
activation by free fatty acids (FFA) induces a conformational shift
that unmasks a Ca
-independent apoB and apoE binding
site that displays the highest affinity for triglyceride-rich
lipoproteins(10, 11) .
That LSR might
significantly contribute to the removal of CMR is supported by the
observation of a strong inverse correlation between the level of
nonfasting plasma triglycerides and the apparent number of LSR
receptors expressed in rat liver.
and inhibits the binding of all LRP ligands
thus far identified (15, 16, 17) . RAP
overexpression in both wild type and LDL receptor-negative mice
increased plasma cholesterol and triglyceride concentrations, primarily
through an accumulation of apoB-48 and apoE-containing
particles(12) . These data were consistent with RAP's
hyperlipidemic effect resulting from the inhibition of the clearance of
CMR and pointed toward LRP as being responsible for the removal of
these particles. This interpretation assumes that the RAP inhibitory
effect is LRP-specific. However, it has been shown that at the
concentrations achieved in animals overexpressing RAP, this protein
inhibits various other membrane receptors: the LDL
receptor(18, 19) , VLDL receptor(20) , and the
gp330(21) . The possibility that the RAP hyperlipidemic effect
is due to interaction with these receptors was ruled out by the
observation of its effect in transgenic homozygous LDL receptor
knockout mice and by the fact that neither the gp330 nor the VLDL
receptor is expressed in the
liver(22, 23, 24) . However, the issue of the
specificity of the RAP inhibitory effect is further complicated by the
finding that it binds to a yet unidentified 105-kDa
protein(20) . This prompted us to reexamine the possibility
that RAP interacts with LSR. Previous experiments showed that RAP at
concentrations of up to 5 µg/ml had no significant effect on LSR
activity, while fully inhibiting the LRP-mediated uptake and
degradation of
-macroglobulin-methylamine(11) . However, RAP
concentrations achieved in the plasma of mice through transfection
exceeded this value and ranged between 20 and 200
µg/ml(12) . Here we report that, while ineffective at low
concentrations, at the concentrations that in vivo delay
remnant clearance, RAP bound LSR candidate proteins and inhibited LSR
activity in both intact cells and isolated liver membranes.
Materials
NaI was purchased from Amersham (Les Ulis,
France). Oleic acid, bovine serum albumin (A2153) (BSA), CHAPS, Triton
X-100, Hepes, leupeptin, benzamidine, and bacitracin were obtained from
Sigma (St. Quentin, Fallavier, France). Sodium suramin and
glutathione-Sepharose were obtained from FBA Pharmaceutical (West
Haven, CT) and Pharmacia (Orsay, France), respectively. Human plasma
thrombin was purchased from Calbiochem (Meudon, France), and
Dulbecco's modified Eagle's medium (DMEM), trypsin,
penicillin-streptomycin, glutamine, and fetal bovine serum were
purchased from Life Technologies, Inc. (Eragny, France).
Methods
Preparation and Radiolabeling of LDL
Human LDL
(1.025 < density < 1.055 g/ml) was isolated by sequential
ultracentrifugation of fresh plasma obtained from the local blood
bank(10, 25) . All preparations were used within 2
weeks of their isolation. LDL was radioiodinated as described
previously using Bilheimer's modified McFarlane's procedure (26) and used no more than 1 week after radiolabeling. I-LDL was filtered (0.22-µm filter, Gelman, Ann
Arbor, MI) immediately prior to use.
Preparation and Radiolabeling of RAP Fusion Protein and
RAP
Human RAP was expressed in bacteria as a fusion protein
associated with glutathione S-transferase (GST), as described
previously(16, 17) . RAP fusion protein was cleaved
from its GST moiety by thrombin treatment(17) . For ligand
blotting studies, RAP fusion protein was iodinated using IODO-BEADS
(Pierce, Asnieres, France), following the manufacturer's
instructions.
Cells
Human fibroblasts from a French-Canadian
patient homozygous for familial hypercholesterolemia (FH) were kindly
provided by Dr. J. Davignon (Montreal). These cells were plated in
36-mm dishes at a density of 1.2 10
cells/dish and
grown to confluence (4-5 days) in DMEM containing 20% (v/v) fetal
bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, and 2
mM glutamine. LSR-mediated cell surface binding, uptake, and
degradation of lipoproteins were measured as described
previously(10) .
Rat Liver Membrane Preparation
Membranes from rat
liver were isolated according to the procedure of Belcher et al.(27) . Membrane preparations were stored under N in the dark at 4 °C and used within 1 week of their
preparation.
Measurement of LSR Activity in Isolated Rat Liver
Membranes
LSR activity was measured in rat liver membranes using
a modification of the previously reported procedure(11) . Briefly, membranes were diluted to 1 mg of protein/ml in buffer A
(0.1 M phosphate buffer containing 350 mM NaCl and 2
mM EDTA, pH 8) and sonicated (Bioblock Scientific Vibracell,
30 s, 25% pulse, setting 2.5). Aliquots of the membranes (100 µg of
protein/tube) were incubated at 37 °C for 30 min in the absence or
presence of 800 µM oleate in a total volume of 250
µl/tube adjusted with buffer A. The membranes were then washed by
three series of centrifugation (35,000
g, 15 min, 4
°C) and resuspension in buffer A by sonication (5 s, 90% pulse,
setting 1). After this, the membranes were incubated at 4 °C for 30
min in the absence or presence of RAP fusion protein or RAP and then
incubated at the same temperature for 1 h with
I-LDL. To
remove unbound ligand, 200 µl of the incubation mixtures were
layered over 600 µl of 5% (w/v) BSA in buffer A. The samples were
then centrifuged (35,000
g, 25 min, 4 °C), the
supernatants were gently aspirated using Pasteur pipettes, and the
bottoms of the tubes containing the membrane pellets were cut and
counted in a
counter (Pharmacia, 1470 Wizard).
Ligand-blotting Studies
Protein was solubilized
from rat liver membranes and partially purified LSR fractions were
prepared by anion exchange chromatography essentially as described
elsewhere. Fractions enriched in LSR activity were
separated by SDS-PAGE (4-12% gradient) under non-reducing
conditions, transferred to nitrocellulose (0.45 µm, Schleicher and
Schuell), and tested for their ability to bind
I-LDL or
I-RAP fusion protein after incubation with or without
oleate.
Protein Determinations
Protein concentrations were
determined using Markwell's modified Lowry procedure (28) and BSA as standard.
I-LDL binding, uptake, and
degradation (Fig. 1,
) induced by 500 µM oleate when compared with the values measured in dishes incubated
with the same concentration of oleate but in the absence of RAP
(
). In contrast, RAP had no detectable effect on the low values
of
I-LDL binding, uptake, and degradation measured in FH
cells incubated in the absence of oleate (data not shown). Since in the
presence of RAP, all three parameters were reduced to a similar degree,
we hypothesized that its major effect was to inhibit the initial event, i.e. the binding of the lipoprotein particle to the LSR.
I-LDL binding, uptake, and degradation by LSR
in FH fibroblasts. Confluent FH fibroblasts were incubated at 37 °C
for 60 min in the absence (
) or presence (
) of 75
µg/ml RAP fusion protein, followed by incubation at 37 °C for
90 min in the absence or presence of 500 µM oleate and
increasing concentrations of
I-LDL (specific activity,
182 cpm/ng) in DMEM containing 0.2% BSA, 2 mM CaCl
, and 5 mM Hepes, pH 7.5. After this,
cells were washed 3 times in phosphate-buffered saline (PBS) containing
0.2% BSA, pH 7.4, followed by two washes in PBS alone. Cells were then
incubated at 4 °C for 1 h with 10 mM suramin in PBS (1
ml/dish). The media were removed and counted for radioactivity; this
represented the amount of
I-LDL bound (A). Cells
were recovered in 0.1 N NaOH containing 0.24 mM EDTA
and counted; this represented the amount of
I-LDL
internalized (B). Degradation products were measured as
trichloroacetic acid-soluble products after chloroform extraction (C). Results represent the difference between dishes incubated
with and without oleate; each point is the mean of duplicate
determinations.
To
further explore the mechanism of RAP inhibition of LDL binding to LSR,
we used a recently developed rat liver membrane binding assay. With this model, RAP was also found to inhibit LSR activity in a
dose-dependent manner (Fig. 2). However, in keeping with our
previous observations (11) , low RAP concentrations (
5
µg/ml) did not reproducibly nor significantly decrease
I-LDL binding to LSR ( Fig. 2and data not shown).
The inhibitory effect of RAP fusion protein was due to its RAP rather
than GST moiety. Indeed, 81% inhibition was observed in experiments
measuring LSR activity in rat liver membranes incubated in the presence
of 50 µg/ml RAP cleaved by thrombin treatment from the GST fragment
(data not shown). This degree of inhibition was similar to that of 79%
achieved with an equivalent concentration of RAP fusion protein (Fig. 2). Thus, there was no added benefit in systematically
obtaining cleaved RAP fusion protein.
I-LDL
(specific activity, 140 cpm/ng). Unbound
I-LDL was
removed by layering 200 µl of the incubation mixture onto a
600-µl 5% (w/v) BSA cushion and centrifugation. The supernatants
were removed by aspiration, and the tube bottoms containing the
membrane pellets were cut and counted for radioactivity. Results
represent the difference between membranes incubated with and without
oleate; each point is the mean of duplicate
determinations.
In these assays, the
incubation medium contained no Ca and was
supplemented with 2 mM EDTA. These conditions allow the
characterization of LSR activity independently of those of the LDL
receptor and LRP that are strictly
Ca
-dependent(4, 29) . Thus, both LSR
activity and RAP inhibitory effect on LSR did not require the presence
of divalent cations.
I-LDL concentrations to LSR was measured in
the absence or presence of 20 µg/ml RAP fusion protein. Both direct
examination (Fig. 3, panelA) and
Lineweaver-Burk transformation of the data (Fig. 3, panelB) showed that the RAP inhibitory effect resulted from a
change in maximal binding capacity rather than a change in affinity.
This is consistent with RAP not exerting its inhibitory effect through
a direct competition with
I-LDL for the putative LSR
lipoprotein binding domain.
I-LDL to LSR in rat liver membranes. Rat liver
membranes were incubated at 37 °C for 30 min with or without 800
µM oleate and then washed 3 times in buffer A. Rat liver
membranes were then incubated without (
) or with (
) 20
µg/ml RAP fusion protein and increasing concentrations of
I-LDL (specific activity, 140 cpm/ng) exactly as
described in Fig. 2. Results represent the difference between
membranes incubated with and without oleate; each point is the
mean of duplicate determinations (A). PanelB represents the Lineweaver-Burk transformation of data in panelA.
To test this, solubilized rat liver
membrane proteins were separated by anion exchange chromatography;
fractions exhibiting LSR activity were pooled, separated on 4-12%
gradient SDS-polyacrylamide gels, and transferred to nitrocellulose.
The proteins immobilized on the strips were then incubated in the
presence or absence of oleate and tested for their ability to bind
either I-LDL or
I-RAP fusion protein.
Preincubation of nitrocellulose strips with oleate induced
I-LDL binding to the two LSR candidate membrane proteins
of apparent molecular masses 115 and 90 kDa (Fig. 4, lane2).
I-RAP fusion protein also bound to two
major bands of similar apparent molecular mass (lanes4 and 5). The binding of RAP fusion protein to LSR
candidate proteins appeared not to require preincubation with oleate (lane4). In strips incubated with oleate, the
pattern of bands revealed by either
I-LDL or
I-RAP fusion protein was virtually superimposable (lanes2 and 5, respectively). Preincubation
of nitrocellulose strips with excess unlabeled RAP prior to incubation
with oleate and
I-LDL significantly decreased the binding
of
I-LDL to LSR (lane3). However, the
reverse experiment showed that preincubation with unlabeled LDL and 800
µM oleate had little to no inhibitory effect on the
subsequent binding of
I-RAP (lane6).
This latter observation was consistent with the notion that the RAP LSR
binding site is distinct from the oleate-induced LDL binding site.
I-LDL (A) or
I-RAP (B) fusion protein in
absence or presence of oleate to LSR-enriched solubilized protein
fraction separated on 4-12% SDS-PAGE and transferred to
nitrocellulose. The LSR-enriched fraction of solubilized liver membrane
protein was separated under non-reducing conditions on a 4-12%
gradient SDS-PAGE gel, and the separated proteins were transferred to
nitrocellulose. After this, the strips were incubated for 30 min in PBS
containing 3% (w/v) BSA and washed with PBS. PanelA,
the nitrocellulose strips were incubated at 37 °C for 1 h in the
absence (lanes1 and 2) or presence (lane3) of 1 mg/ml RAP fusion protein in buffer A.
After this, the membranes were washed twice in buffer A and the strips
were incubated at 37 °C for 15 min without (lane1) or with (lanes2 and 3) 800
µM oleate and then at 37 °C for 1 h with 20 µg/ml
I-LDL (specific activity, 398 cpm/ng). PanelB, the strips were incubated at 37 °C for 15 min
without (lane4) or with (lanes5 and 6) 800 µM oleate in buffer A. The strips
were then incubated at 37 °C for 1 h in the absence (lanes4 and 5) or presence (lane6)
of 1 mg/ml unlabeled LDL, followed by incubation at the same
temperature for 1 h with 20 µg/ml
I-RAP fusion
protein (specific activity, 2696 cpm/ng). All membranes were then
washed in PBS containing 0.5% Triton X-100, dried, and exposed for 1 h
to a phosphor screen.
I-LDL to LSR in human FH
fibroblasts, which led to a parallel reduction in their subsequent
uptake and proteolytic degradation. Second, RAP inhibited, in a
dose-dependent manner, the binding of LDL to LSR in isolated rat liver
membranes. Third, RAP was shown by ligand blotting to bind to two main
proteins with apparent molecular masses identical to those that are
considered as responsible for LSR activity. Fourth, an excess of RAP
significantly reduced the binding of
I-LDL to partially
purified LSR proteins separated by SDS-PAGE and transferred to
nitrocellulose.
-independent. Circumstantial evidence also suggests
that LSR is distinct from the VLDL receptor. In living animals, LSR is
expressed to a large extent in the liver, while both in rabbits and
mice, VLDL receptor mRNA is at the limit of detectability in this
tissue(24, 30) . In addition, retinoic acid appears to
increase the expression of the VLDL receptor(30) , while
decreasing LSR activity(31) . Also, unlike LSR, the VLDL
receptor is inhibited by very low RAP concentrations (half-maximum
inhibition at approximately 4 µg/ml; (20) ).
-macroglobulin binding to LRP was obtained with RAP
concentrations of 0.4 µg/ml (10 nM)(17) . It is
not surprising then that at low RAP concentrations, LRP activity in
cultured cells is fully inhibited while LSR activity remains
unchanged(11) . On the basis of the affinity of RAP for its
various currently identified target receptors, two types of
interactions can be evidenced. The first is the high affinity group
with K
ranging between 0.7 and 20
nM; this group includes the LRP, the VLDL receptor, and gp330.
The second group, which includes the LDL receptor and LSR, is inhibited
only at much higher RAP concentrations.
I-LDL for binding to LSR. This
notion is further strengthened by the observation that unlike LDL, RAP
binding to LSR does not require the FFA-induced conformational shift of
the receptor. Furthermore, the observation that binding of LDL to LSR
did not prevent subsequent binding of RAP to the receptor is consistent
with RAP and LDL interacting at different sites on the LSR proteins.
The RAP inhibitory effect might therefore result from an interference
with the FFA-induced shift in LSR conformation or from another yet
unidentified regulatory mechanism.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.