(Received for publication, June 10, 1994; and in revised form, November 9, 1994)
From the
Two multifunctional receptors, low density lipoprotein
receptor-related protein (LRP) and gp330, have been implicated in the
cellular uptake and degradation of a wide spectrum of functionally
diverse ligands including plasma lipoproteins, proteases, and
proteinase-inhibitor complexes. The two receptors show distinct
tissue-specific expression patterns, suggesting different physiological
functions. We have examined the cellular degradation of two serine
proteinase inhibitor (serpin)-protease complexes,
-antitrypsin-neutrophil elastase (
AT
NEL) and
-antichymotrypsin-cathepsin G
(
ACT
CathG) by normal murine fibroblasts (MEF)
expressing LRP, and by a mutant fibroblast cell line (PEA13) which is
genetically deficient for LRP.
AT
NEL complexes
bound to LRP on ligand blots and were degraded efficiently by the MEF
cells, but not by PEA13 cells. Degradation of the complexes was also
significantly reduced by antibodies directed against LRP, further
suggesting that fibroblasts require LRP for the cellular uptake and
degradation of
AT
NEL complexes. In contrast to
AT
NEL, MEF cells did not degrade
ACT
CathG complexes. However, these complexes
were rapidly degraded by the rat embryonal carcinoma cell line L2p58
which abundantly expresses gp330, raising the possibility that the
ACT
CathG complex might be recognized by gp330.
Both complexes were efficiently metabolized by the hepatoma cell line
HepG2, presumably involving the serpin-enzyme complex receptor. The
differential recognition of serpin-protease complexes by fibroblasts
and hepatoma cells, however, indicates that LRP, gp330, and the
serpin-enzyme complex receptor are distinct proteins.
The low density lipoprotein receptor-related protein (LRP) ()(1) and the large glycoprotein gp330 (2) are complex, multifunctional endocytic receptors which
mediate the cellular uptake of a heterogeneous spectrum of ligands,
including apoE-rich remnant lipoproteins(3, 4) ,
lipoprotein lipase (5) , proteinase- or methylamine-activated
-macroglobulin
(
M
)(6, 7) , plasminogen
activator-inhibitor complexes(4, 8, 9) , the
active protease tissue-type plasminogen activator(10) ,
pro-urokinase(11) , and exotoxin A from Pseudomonas
aeruginosa (PEA)(12) . Plasminogen activator inhibitor-1
which apparently binds to an independent site on LRP (13) belongs to the serine proteinase inhibitor (serpin)
superfamily. Recently, a role for LRP in the removal of tissue factor
pathway inhibitor has also been reported, thus further underscoring the
role of this receptor in the regulation of extracellular protease
activity(14) .
The spectrum of ligands that bind to LRP and
gp330 is very similar(4) , although binding of
M
and PEA to gp330 has so far not been
reported. LRP and gp330 both belong to the LDL receptor gene family
which also includes a recently described very low density lipoprotein
receptor(15) , and the LDL receptor itself(16) . The
activity of LRP and gp330 has been proposed to be modulated in vivo by a receptor-associated protein (RAP) of approximately 39 kDa
which blocks the binding of all known ligands to either receptor (4, 8, 9, 10, 11, 12, 17, 18, 19) .
The distinct tissue-specific expression patterns of the two receptors suggest different physiological functions. Whereas gp330 is highly expressed in the glomerular and proximal tubular epithelia of the kidney (20) and in the type II pneumocytes of the lung(21) , LRP is predominantly expressed in the hepatocytes, the neurons of the brain, fibroblasts, and macrophages(22, 23) . Targeted disruption of the LRP gene in mice prevents development of homozygous defective embryos beyond day 13.5 post coitum(24, 25) . Complete loss of all functions associated with LRP is obviously incompatible with survival beyond the embryonic stage, but loss or impairment of individual functions of LRP concerning a specific ligand might cause more limited damage in organs or tissues most dependent on this specific function. Identification of differential and/or tissue-specific recognition of a ligand by the two receptors LRP and gp330 might thus allow conclusions relating to their specific functions in vivo.
The high expression of gp330 in type II
pneumocytes (21) suggests a specific role for this receptor in
the lung and especially in the alveolar space. In contrast, in the lung
LRP is preferentially expressed in the alveolar
macrophages(23) . Because LRP and gp330 are expressed by
defined cell populations within the lung, we have examined their
possible role in the removal of complexes of the two closely related
serpins -antitrypsin (
AT) and
-antichymotrypsin (
ACT), both of
which are expressed by lung alveolar macrophages and detectable in the
bronchoalveolar lavage fluid in variable
concentrations(26, 27) .
AT and
ACT are 42% homologous to each other on the amino acid
level(28, 29) , and both are encoded by genes which
lie in close proximity to each other on chromosome
14q32.1(30) .
AT is known to protect lung
structures against proteolytic damage by neutrophil elastase (NEL),
which is specifically complexed and inactivated by
AT(31) . Severe genetic
AT
deficiency is associated with a 20-30-fold increased risk for the
development of chronic obstructive pulmonary disease, due to
insufficient inhibition of elastolytic
activity(32, 33) . Recently, defective mutants of
ACT have also been described in patients with chronic
obstructive pulmonary disease(34, 35) . Both
inhibitors,
AT and
ACT, are
apparently taken up and degraded together with the complexed proteases
via a distinct SEC receptor which has been described in hepatoma cells
and monocytes(36, 37) .
In the present study we
have investigated possible roles of LRP and gp330 for the binding and
cellular uptake of AT
NEL and
ACT
CathG complexes by exploring the recognition
of these ligands by cell types expressing gp330, LRP, or neither
receptor, as well as by HepG2 cells which contain the SEC receptor.
Wild type (MEF) and LRP-deficient (PEA13) murine embryonic
fibroblasts were isolated as described earlier(38) . The L2p58
rat yolk sac carcinoma cell line was kindly provided by Dr. Marilyn
Farquhar, UCSD, La Jolla. Human -macroglobulin
activated with methylamine (
M
) was a gift
from Dudley Strickland (American Red Cross, Rockville, MD). Recombinant
glutathione S-transferase or a fusion protein of glutathione S-transferase and rat RAP were produced in DH5
bacteria
as described(17) . Human NEL, human leukocyte cathepsin G
(CathG), human
AT, and human
ACT were
purchased from Sigma. Human plasma LDL was isolated and iodinated as
described (39) . All other
I-labeled ligands were
radiolabeled by the IODO-GEN method(40) .
Complexes of I-labeled NEL and CathG with
AT or
ACT, respectively, were formed in vitro immediately following the radioiodination of the
proteases as described under ``Materials and Methods'' and
analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 1). In
both cases the majority of the
I-labeled proteases (lanes 1 and 3) was converted to an SDS-stable
complex of higher molecular weight (lanes 2 and 4) as
is typical for complexes of serpins with their target proteases.
Preparations of rat liver (L) or rat kidney (K) membrane proteins were
separated by electrophoresis on nonreducing 3-10%
SDS-polyacrylamide gradient gels, transferred to nitrocellulose
filters, and incubated with the labeled complexes or with
I-labeled GST-RAP fusion protein (Fig. 2A,
lanes 1 and 2) to demonstrate the presence of LRP and
gp330 in the respective membrane preparations.
AT
I-NEL complex bound only to LRP (lane 3) but not to gp330 (lane 4) on ligand blots.
ACT
I-CathG complexes did not bind
to either protein on ligand blots (not shown). The in vitro binding properties of the
AT
I-NEL complex to LRP in
partially purified liver membrane fractions were similar to those of
other previously characterized LRP ligands (Fig. 2B),
inasmuch as binding was dependent upon the presence of Ca
and could be abolished by coincubation with EDTA (lane
2) or GST-RAP fusion protein (lane 4).
Figure 1:
Protease-inhibitor complexes. 5 ng of I-elastase (
I-NEL, lane 1), 20 ng
of
I-NEL complexed with
-antitrypsin
(
AT
I-NEL, lane 2), 5 ng
of
I-cathepsin G (
I-CathG, lane
3), and 5 ng of
I-cathepsin G complexed with
-antichymotrypsin
(
ACT
I-CathG, lane 4) were
subjected to 4-15% SDS-gel electrophoresis, after which the
proteins were transferred to nitrocellulose and exposed to DuPont
Cronex film for 16 h (lane 2) or 5 days (lanes 1, 3, and 4) at -70 °C. The position of migration of
the proteases, protease-inhibitor complexes, and molecular weight
marker proteins (rainbow marker, Amersham) is
indicated.
Figure 2:
Binding of
-antitrypsin-
I-elastase to rat liver
membranes. 700 µg of partially purified membrane proteins from rat
liver and rat kidney were subjected to preparative nonreducing
3-10% SDS-gel electrophoresis and transferred to nitrocellulose
membranes. The position of migration of LRP and gp330 (arrow),
myosin (200 kDa), and phosphorylase b (97.4 kDa) in the gel is
indicated. A, replicate nitrocellulose strips (approximately
50 µg of protein/strip) of liver (L) and kidney (K) membrane preparations were subjected to ligand blot
analysis as described under ``Materials and Methods'' using 1
10
cpm/ml of
I-labeled GST-RAP (lanes 1 and 2) or 1
10
cpm/ml
AT
I-NEL complex (lanes 3 and 4). Blots were exposed to DuPont Cronex film for 24 h
at -70 °C. B, replicate nitrocellulose strips
(approximately 50 µg of protein/strip) of liver membrane proteins
were preincubated at 22 °C for 30 min with either blotting buffer (lane 1) or blotting buffer containing 20 mM EDTA (lane 2), 30 µg/ml GST (lane 3), or 30 µg/ml
GST-RAP (lane 4). Each strip was then incubated for 1 h with
AT
I-NEL complex (1
10
cpm/ml), after which the strips were exposed to DuPont
Cronex film for 24 h at -70 °C.
Cellular
degradation experiments performed in cultured fibroblasts (Fig. 3) that either express LRP abundantly (MEF, open
circles) or are genetically deficient for this receptor (PEA13, closed circles) confirmed the results obtained from the ligand
blot experiments. ACT
I-CathG
complexes that did not bind to LRP on ligand blots were not degraded by
either cell line (Fig. 3A). In contrast,
AT
I-NEL complexes which bound
specifically to immobilized LRP were efficiently degraded by the LRP
expressing MEF cells, but not by the PEA13 cells lacking LRP (Fig. 3B). As shown previously, PEA13 cells completely
lack the ability to endocytose the LRP ligand
-macroglobulin (Fig. 3C) due to
absence of the receptor(38) , while they take up and degrade
LDL normally (Fig. 3D). LDL does not bind to LRP, but
to the related LDL receptor. Primary LRP-deficient fibroblasts isolated
from 10.5 day mouse embryos homozygous for the LRP gene defect did also
not degrade
AT
I-NEL complexes,
indicating that the inability of PEA13 cells to degrade this ligand
resulted from the LRP gene defect and not from an unrelated clonal
aberration (data not shown).
Figure 3:
Degradation of multiple ligands by wild
type and LRP-deficient murine embryonic fibroblasts. Replicate
monolayers of wild type (MEF) or LRP-deficient (PEA13) murine embryonic fibroblasts received 1 ml of DMEM
(without glutamine) containing 0.2% (w/v) bovine serum albumin and 500
ng/ml ACT
I-CathG complex (Panel A), 500 ng/ml
AT
I-NEL complex (Panel
B), 5 µg/ml
I-
M
(Panel C), or 5 µg/ml
I-LDL (Panel
D). After incubation at 37 °C for the indicated time, the
amount of radiolabeled degradation products secreted into the medium
was determined. Each value represents the mean of duplicate
incubations.
In contrast to the in vitro binding of AT
I-NEL complexes
to LRP which was blocked by coincubation with GST-RAP fusion protein,
degradation of
AT
I-NEL by MEF
cells (Fig. 4, lane 5) was reproducibly unaffected by
the presence of GST-RAP in the culture medium (lane 6), while
the degradation of
I-
M
(lane 1) was completely blocked by GST-RAP (lane
2). Rabbit polyclonal antibodies directed against LRP (lane
3), but not preimmune IgG (lane 4), also substantially
reduced the cellular degradation of
I-
M
as well as of
AT
I-NEL (lane 8), further
indicating that LRP mediates the degradation of both these ligands.
Degradation of
AT
I-NEL (lane
7) and
I-
M
(not shown)
was largely inhibited by chloroquine, indicating that lysosomal
processes are involved.
Figure 4:
Inhibition of ligand degradation by
anti-LRP IgG, GST-RAP, or chloroquine. Replicate monolayers of wild
type murine embryonic fibroblasts received 1 ml of DMEM (without
glutamine) containing 0.2% (w/v) bovine serum albumin and either 5
µg/ml I-
M
(lanes
1-4) or 500 ng/ml
AT
I-NEL complex (lanes
5-9). In addition, the medium contained either 30 µg/ml
GST (lanes 1 and 5), 30 µg/ml GST-RAP (lanes
2 and 6), 200 µg/ml anti-LRP IgG (lanes 3 and 8), 200 µg/ml preimmune IgG (lanes 4 and 9), or 100 µM chloroquine (lane 7).
After 24 h incubation at 37 °C, the amount of radiolabeled
degradation products secreted into the medium was determined. Each
value represents the mean of duplicate incubations of multiple
experiments. 100% values represent degradation rates in the absence of
added reagents and range from 350 to 460 ng/mg of protein/h for
M
and from 7 to 14.6 ng/mg protein/h for
AT
NEL complex.
We next explored the ability of four
different cell lines, MEF, PEA13, HepG2, and the rat yolk sac carcinoma
cell line L2p58, to degrade
ACT
I-CathG complexes. HepG2 cells
have been reported to express a not yet completely characterized SEC
receptor (36, 37) and L2p58 cells have been found to
express gp330 abundantly(42) . As shown in Fig. 5A,
ACT
I-CathG complexes were not
degraded by either MEF or PEA13 cells, however, they were efficiently
metabolized by HepG2 cells and L2p58 cells. In contrast,
I-
M
was degraded much more
efficiently by MEF and by L2p58 than by HepG2 cells, indicating the
presence of SEC receptors in HepG2 and L2p58 cells which are unrelated
to LRP (Fig. 5B).
Figure 5:
Degradation of
ACT
I-CathG complex and
I-
M* by various cell lines. Replicate
monolayers of HepG2 cells, L2p58 cells, and wild type (MEF) and
LRP-deficient (PEA13) murine embryonic fibroblasts received 1 ml of
DMEM (without glutamine) containing 0.2% (w/v) bovine serum albumin and
either 500 ng/ml
ACT
I-CathG
complexes (Panel A) or 5 µg/ml
I-
M
(Panel B).
After incubation at 37 °C for the indicated time, the amount of
radiolabeled degradation products secreted into the medium was
determined. Each value represents the mean of duplicate
incubations.
Similar to the degradation of
AT
I-NEL complexes by murine
fibroblasts (Fig. 4), GST-RAP also had no effect on the
degradation of
ACT
I-CathG
complexes by L2p58 cells (Fig. 6, lane 5), while it
effectively blocked
I-
M
degradation (lane 2). As in MEF cells, degradation of
these ligands by L2p58 cells was chloroquine sensitive (lanes 3 and 6).
Figure 6:
Inhibition of ligand degradation in L2p58
cells by GST-RAP and chloroquine. Replicate monolayers of L2p58 cells
received 1 ml of DMEM (without glutamine) containing 0.2% (w/v) bovine
serum albumin and either 5 µg/ml I-
M
(1500 cpm/ng, lanes
1-3) or 500 ng/ml
ACT
I-CathG complex (7000 cpm/ng, lanes 4-6). In addition, the medium contained 30
µg/ml GST (lanes 1 and 4), 30 µg/ml GST-RAP (lanes 2 and 5), or 100 µM chloroquine (lanes 3 and 6). After 9 h incubation at 37 °C,
the amount of radiolabeled degradation products secreted into the
medium was determined. Each value represents the mean of duplicate
incubations. 100% values represent degradation rates in the absence of
added reagents and are 20 ng/mg of protein/h for
M
and 8 ng/mg of protein/h for
ACT
CathG complex.
L2p58 cells had been reported to abundantly express gp330. In an attempt to correlate the expression levels of LRP and gp330 in MEF cells and in L2p58 cells, respectively, we performed a semiquantitative Western blot analysis (Fig. 7). Crude membrane proteins from MEF (lanes 1 and 3) or L2p58 cells (lanes 2 and 4) were separated by SDS-gel electrophoresis and transferred to nitrocellulose membranes. LRP (lanes 1 and 2) and gp330 (lanes 3 and 4) were then detected by incubation of the filters with specific polyclonal antibodies. While MEF cells express LRP abundantly (lane 1) L2p58 cells express only low amounts of this protein (lane 2). In contrast, murine fibroblasts are completely devoid of gp330 (lane 3), whereas L2p58 cells contain this protein (lane 4).
Figure 7: Western blot analysis of L2p58 and MEF cells. Partially purified membrane proteins (40 µg in lanes 1 and 2, 20 µg in lanes 3 and 4) from MEF cells (M, lanes 1 and 3) and L2p58 cells (L, lanes 2 and 4) were subjected to nonreducing 4-15% SDS-gel electrophoresis and transferred to nitrocellulose membranes. The proteins were detected using 5 µg/ml anti-LRP IgG (lanes 1 and 2) or 5 µg/ml anti-gp330 IgG (lanes 3 and 4). Binding of the IgG was detected using the ECL system. The position of migration of LRP and gp330 (arrow), myosin (200 kDa), and phosphorylase b (97.4 kDa) in the gel is indicated.
We have studied the role of LRP in the cellular uptake and
degradation of two homologous serpins, AT and
ACT, complexed to their respective target proteases,
NEL and CathG. We find that
AT
NEL, but not
ACT
CathG complexes, bind to LRP on ligand blots.
Likewise,
AT
I-NEL, but not
ACT
I-CathG complexes, are
efficiently internalized and degraded by fibroblasts expressing LRP.
This degradation is reduced by polyclonal antibodies directed against
LRP. Furthermore, murine embryonic fibroblasts genetically deficient
for LRP did not degrade
AT
NEL complexes
indicating that this ligand is indeed endocytosed and degraded by
normal murine fibroblasts in an LRP-dependent manner. HepG2 cells, in
contrast, which have previously been reported to express a SEC receptor
readily metabolized
ACT
CathG (Fig. 5) and
AT
NEL complexes (not shown) in agreement with
earlier publications(36, 37, 43) . Both
complexes were also taken up and degraded by L2p58 cells ( Fig. 5and data not shown) which besides LRP also express gp330 (Fig. 7). In contrast to other ligands recognized by LRP or
gp330, degradation of neither
AT
NEL nor of
ACT
CathG complexes was affected by
GST-RAP(4, 17) . Surprisingly, however, binding of
AT
NEL complexes to LRP on ligand blots was
effectively blocked by RAP. Although the reasons for this difference
are not entirely clear, it is possible that the
AT
NEL binding site of LRP immobilized on
nitrocellulose filters may not be in its native conformation making it
more sensitive to competition by RAP.
Our data support the existence
of a SEC receptor on HepG2 cells which is unrelated to LRP, because
HepG2 cells, but not fibroblasts, efficiently degrade
ACT
CathG complexes. Although both cell types
express LRP, as shown by their ability to degrade
I-
M
, fibroblasts do so more
effectively. This is a further indication for a physical distinction
between the SEC receptor and LRP.
An involvement of gp330 in the
metabolism of ACT
CathG complexes is possible,
but not proven at the present time. Clearly, the SEC receptor in
liver-derived cells is different from gp330 which is not expressed in
the liver(4) . However, it is possible that the uptake and
degradation of
ACT
CathG complexes by L2p58 cells
could involve gp330.
AT and
ACT both
belong to the serpin class of protease inhibitors which includes about
20 different proteins including plasminogen activator inhibitor-1. The
latter binds to both LRP and gp330 (4) and LRP can also bind
AT
NEL complexes (this study). It is tempting to
speculate that, for as yet unknown reasons, LRP and gp330 display a
selective, but partially overlapping, ligand binding spectrum where
some serpins might bind to one, but not to the other or to either
receptor.
There is growing evidence that proteinase-antiproteinase
complexes directly or indirectly initiate cellular signal transduction
events. For example, AT-proteinase complexes mediate
an increase of
AT gene expression in human monocytes
and macrophages (44) and are also a strong chemoattractant for
neutrophils (45) . Similarly,
ACT
CathG
complexes stimulate the synthesis of acute phase proteins via
interleukin-6(46) . Furthermore, receptor binding-competent
M
has been shown to elevate intracellular
second messengers in murine peritoneal macrophages (47) and
M
has been reported to substantially
increase the transforming growth factor-
1 mediated proliferative
response in smooth muscle cells(48) . There is no evidence that
these signal transduction events are directly mediated by LRP, although
such a possibility has been discussed for the SEC receptor. However,
internalization and lysosomal degradation of these complexes by LRP
and/or by another endocytic receptor (e.g. possibly gp330)
could be an important regulatory mechanism which turns off the signal
evoked by the binding of these complexes to another unrelated signaling
receptor on the cell surface.
In summary, our data provide evidence
for: 1) a role of LRP in the cellular metabolism of
AT
NEL complexes, and 2) for differences in the
mechanisms by which
AT
NEL and
ACT
CathG complexes are taken up and degraded by
cells.
AT
NEL, but not
ACT
CathG complexes are bound and internalized by
LRP which therefore appears to be distinct from the SEC receptor
expressed on HepG2 cells. The availability of genetically defined cell
lines which express or are devoid of LRP protein will continue to be
valuable for future studies aiming at the analysis of receptor-ligand
interactions of the two structurally and functionally closely related
multifunctional receptors LRP and gp330.