SolubleLDL-Rareformedbycellsurfacecleavagein response
to phorbol esters
Michael J. Begg
1,
*, Edward D. Sturrock
1
and Deneys R. van der Westhuyzen
2
1
Division of Medical Biochemistry, University of Cape Town, South Africa;
2
Department of Internal Medicine, University of Kentucky
Medical Center, and Department of Veteran Affairs Medical Center, Lexington, Kentucky, USA
A 140-kDa soluble form of the low density lipoprotein
(LDL) receptor has been isolated from the culture medium
of HepG2 cells and a number of other cell types. It is
produced from the 160-kDa mature LDL receptor by a
proteolytic cleavage, which is stimulated in the presence of
4b-phorbol 12-myristate 13-acetate (PMA), leading to the
release of a soluble fragment that constitutes the bulk of the
extracellular domain of the LDL receptor. By labeling
HepG2 cells with [
35
S]methionine and chasing in the pres-
ence of PMA, we demonstrated that up to 20% of LDL-
receptors were released into the medium in a 2-h period.
Simultaneously, the level of labeled cellular receptors was
reduced by 30% in those cells treated with PMA compared
to untreated cells, as was the total number of cell surface
LDL-receptors assayed by the binding of
125
I-labeled anti-
body to whole cells. To determine if endocytosis was
required for cleavage, internalization-defective LDL-recep-
tors were created by mutagenesis or deletion of the NPXY
internalization signal, transfected into Chinese hamster
ovary cells, and assayed for cleavagein the presence and
absence of PMA. Cleavage was significantly greater in the
case of the mutant receptors than for wild-type receptors,
both in the absence and presence of PMA. Similar results
were seen in human skin fibroblasts homozygous for each of
the internalization-defective LDL receptor phenotypes.
LDL receptor cleavage was inhibited by the hydoxamate-
based inhibitor TAPI, indicating the resemblance of the
LDL receptor cleavage mechanism to that of other surface
released membrane proteins.
Keywords: internalization signal; LDL-R; low density lipo-
protein; ectodomain shedding.
The low density lipoprotein (LDL) receptor (LDL-R) is a
cell surface protein that mediates the uptake and clearance
of the cholesterol-rich lipoprotein LDL from the plasma [1].
The LDL-R plays an important role in regulating plasma
LDL and cellular cholesterol levels and the activity of this
receptor has a direct bearing on plasma cholesterol levels.
The LDL receptor is regulated at the transcriptional level
in responseto intracellular sterol levels by means of sterol
sensitive transcription factors SREBP1 and SREBP2 [2,3],
however, it can also be upregulated by numerous cytokines
[tumor necrosis factor (TNF)-a, interleukin (IL)-1, trans-
forming growth factor (TGF)-b, oncostatin M, platelet-
derived growth factor and basic fibroblast growth factor
(bFGF) [4–8], hormones (insulin and estradiol) [9,10] and
second messenger systems [11,12]. Although much is known
about the transcriptional mechanisms that control LDL-R
expression, little is known about the mechanism(s) that
control the turnover and degradation of this important
protein. Earlier studies established that the LDL-R degra-
dation mechanism(s) is a nonlysosomal process that is
dependant on short lived mediator protein(s) and unaffected
by the presence of ligand and/or sterol [13,14]. Some
evidence suggests that degradation may also be able to
modulate LDL-R number. Ness et al.[15]showedthat
degradation of LDL-Rin hepatocytes is increased in the
presence of cholesterol synthesis inhibitors. Kraemer et al.
[16] reported that the half-life of LDL-Rin rat adipocytes is
decreased by 40% in the presence of insulin. The proteolytic
mechanism responsible for such regulated LDL-R degra-
dation is not known.
A regulatory mechanism common to many cell surface
proteins is a proteolytic cleavage of the membrane anchor
that releases a soluble form into the extracellular medium
[17]. Examples include TGF-a,TNF-a, TNF-receptor,
angiotensin converting enzyme (ACE), amyloid precursor
protein,
L
-selectin, IL-6 receptor and also members of the
LDL receptor gene family, LRP and VLDL-R [17–22]. In
addition, this process has been observed to be highly
regulated by second messenger systems [23]. A common
feature is the release of the extracellular domain via a single
proteolytic cleavage at a site just extracellular to the
membrane spanning domain. In some cases further proteo-
lytic processing of the extracellular domain occurs. The
function of these soluble forms varies. In the cases of ACE,
TGFa and other mitogens the soluble protein has the
Correspondence to E. D. Sturrock, Division of Medical Biochemistry,
University of Cape Town, Observatory 7925, South Africa.
Fax: + 27 21 406 6470, Tel.: + 27 21 406 6312,
E-mail: sturrock@curie.uct.ac.za
Abbreviations: LDL, low density lipoprotein; LDL-R, low density
lipoprotein receptor; sLDL-R, soluble low density lipoprotein
receptor;PMA,4b-phorbol 12-myristate 13-acetate; LPDS, lipopro-
tein deficient serum; CHO, Chinese hamster ovary; HSF, human skin
fibroblasts; TAPI, TNF-a protease inhibitor; TNF, tumor necrosis
factor; IL, interleukin; TGF, transforming growth factor; ACE,
angiotensin converting enzyme; PKC, protein kinase C.
*Present address: Ribotech Pty Ltd, Biopolymer Unit, 15 A Mail
Street, Western Province Park, Good wood 7460, South Africa.
(Received 3 September 2003, revised 27 November 2003,
accepted 2 December 2003)
Eur. J. Biochem. 271, 524–533 (2004) Ó FEBS 2004 doi:10.1046/j.1432-1033.2003.03953.x
potential of carrying out its function at sites remote from the
original cellsurface [19]. Another function may be the rapid
downregulation of the cell associated protein. In other cases
the released soluble protein has been associated with
pathological conditions such as Alzheimer’s disease where
aberrant cleavage of the amyloid precursor protein forms
the soluble b-amyloid peptide which deposits in plaques
resulting in neuronal degeneration. In almost all cases the
formation of these soluble proteins is greatly enhanced by
phorbol esters and inhibited by hydroxamate-based inhi-
bitors such as TAPI (TNF-a protease inhibitor) and
batimastat.
In this report, we have investigated the degradation of
LDL-R in HepG2 cells and show that LDL-Rs are degraded
in part by a proteolytic mechanism which cleaves the receptor
close to the transmembrane domain, resulting in the release
of the extracellular domain as a solubleLDL-R (sLDL-R).
Like the release of many other transmembrane proteins, the
cleavage of LDL-R is markedly activated byphorbol esters.
Investigations using mutant LDL-R and protease inhibitors
suggest that cleavage takes place at the cellsurface and that
the mechanism is closely related to that which generates
soluble derivatives of other transmembrane proteins.
Experimental procedures
Materials
All tissue culture media was from Highveld Biological,
Kelvin, South Africa. Fetal bovine serum was from delta
bioproducts, Kempton Park, South Africa. Human LDL
(density, 1.019–1.063 gÆmL
)1
) and lipoprotein deficient
serum (LPDS) (density > 1.25 gÆmL
)1
) were prepared
from whole, male blood, and iodinated by the iodine
monochloride method as described previously [24]. IgG-C7
was prepared as described [24] from hybridoma cells
obtained from the American Type Culture Collection
(CRL 1691). Goat anti-mouse (IgG subfraction) was
from Cappel Research Products (Durham, NC, USA.)
[
35
S]Methionine as Tran
35
Slabel
TM
, and methionine/cys-
teine free media were from ICN Radiochemicals (Irvine,
CA, USA). Protease inhibitors and 4b-phorbol 12-myristate
13-acetate (PMA) were from either Sigma Chemical Co. or
Boehringer Mannheim. TAPI was from Immunex. Mutant
fibroblasts GM2408 (HSF-JD) and FH683 (HSF-792stop)
and the monoclonal antibodies 4A4 (raised against LDL-R
cytoplasmic domain) and HL-1 (raised against LDL-R
ligand binding domain) were a kindly supplied by M. S.
Brown and J. L. Goldstein (Dallas, TX, USA).
Generation of the cDNA constructs
Construction and expression of LDL-R mutants. To
investigate whether endocytosis plays a role in the ecto-
domain shedding of the LDL receptor, three cytoplasmic
domain mutants were made (Fig. 1). The mutant 792-LDL-
R has a truncated cytoplasmic domain of only two amino
acids due to a single base substitution (TGG fi TGA),
which converts Trp792 to a stop codon. The mutant
receptor, JD-LDL-R (Tyr807Cys), has a single amino acid
substitution that disrupts the NPVY internalization signal.
To assess whether other signals in the LDL-R cytoplasmic
tail influence cleavage, we constructed a third mutant (812-
LDL-R), which contains a functional NPVY internalization
sequence but lacks the last 27 amino acids of the
cytoplasmic domain.
Mutagenesis of the LDL-R cytoplasmic tail was carried
out according to the method described [25]. Briefly, a 2.1-kb
EcoR1/Sma1 fragment was subcloned from the LDL-R
expression plasmid pLDL-R2 into the bacteriophage vector,
M13mp18. Mutagenic oligonucleotides and primer exten-
sion were used to generate the mutated double-stranded
vector according to the method of Kunkel et al.[26].After
sequencing, to confirm the mutations, a 1.1-kb BglII–SmaI
fragment containing the cytoplasmic, transmembrane and
O-linked sugar domains was subcloned into pLDL-R2.
Fig. 1. LDL-R constructs. LDL-R mutations JD-LDL-R, 812-LDL-R and 792-LDL-R are depicted in relation to the wild-type (WT) receptor.
Each of the mutants is shown as an expanded view of the juxtamembrane, transmembrane and cytoplasmic domains in which all these mutations
occur. Above the linear bar diagram of the WT-LDL-R is the amino acid sequence with a cysteine which has been substituted for Tyr807 in
JD-LDL-R indicated by an asterisk.
Ó FEBS 2004 Cellsurfacecleavage of LDL-R (Eur. J. Biochem. 271) 525
Transfection of the mutant plasmids into LDL-R-negative
CHO cells (CHO-A7) and selection of positive clones
were carried out according to the procedures described by
Davis et al.[27].
Metabolic labeling with [
35
S]methionine
Semi-confluent HepG2 cells were seeded into 35-mm dishes
at a split ratio of 1 : 4 and cultured at 37 °CinMEM
containing 10% (v/v) fetal bovine serum. After 36 h, LDL-
R activity was upregulated by replacing medium with
MEM containing LPDS (2.5 mgÆmL
)1
) and culturing for a
further 12 h at 37 °C [24]. Cells were then metabolically
labeled with [
35
S]methionine by incubating in methionine/
cysteine-free EMEM/LPDS for 30 min and then for
2 h in methionine/cysteine-free EMEM/LPDS containing
50 lCiÆmL
)1
[
35
S]methionine. The medium was changed
to complete MEM/LPDS containing 200 l
M
unlabeled
methionine and incubated at 37 °C for various chase times
in the presence or absence of PMA. After the indicated
chase periods, the medium was removed from the cells
and spun at 15 000 g for 10 min before adding one-tenth
volume of 100 m
M
Hepes pH 7.4, 500 m
M
NaCl, 20 m
M
MgCl
2
,0.5m
M
leupeptin, 10 m
M
phenylmethanesulfonyl
fluoride and 10% (v/v) Triton X-100. The cells were then
washed in buffer A (10 m
M
Hepes pH 7.4, 150 m
M
NaCl,
2m
M
CaCl
2
), and cell associated LDL-R solubilized in
buffer B (10 m
M
Hepes pH 7.4, 200 m
M
NaCl, 2 m
M
CaCl
2
,2.5m
M
MgCl
2
,1m
M
phenylmethylsulphonyl flou-
ride, 0.02 m
M
Leupeptin, 1% (v/v) Triton X-100). Both
medium and cells were immunoprecipitated using pre-
formed immune complexes of the monoclonal antibody
IgG-C7 as described [28]. The immunoprecipitates were
separated by SDS/PAGE, enhanced with salicylate and
visualized by fluorography, or direct detection on a Packard
Instant Imager 2024 (Packard Instrument Company).
Surface LDL-R binding of
125
I-labeled LDL or
125
I-labeled IgG-C7
Human LDL (density, 1.019–1.063 gÆmL
)1
)andthe
monoclonal antibody IgG-C7 were prepared and labeled
with
125
I as described [24]. HepG2 cells were seeded into
35-mm dishes at a split ratio of 1 : 4 and cultured in
MEM containing 10% (v/v) fetal bovine serum. After
36 h, LDL-R activity was upregulated by incubating cells
in MEM/LPDS (2.5 mgÆmL
)1
) for a further 12 h. Surface
LDL-R activity was measured by incubating cells with
1 mL ice cold MEM/LPDS buffered with 20 m
M
Hepes
pH 7.4, containing either
125
I-labeled LDL (10 lgÆmL
)1
)
or
125
I-labeled IgG-IgG-C7 (1 lgÆmL
)1
)for2hat4°Cas
described [24]. This medium was removed and cells were
washed four times with 2 mL NaCl/P
i
containing 0.2%
BSA, followed by three times with 2 mL NaCl/P
i
.The
specifically bound fraction of
125
I-labeled LDL that
remained cell associated after washing was removed by
incubating cells with 0.4% heparin for 1 h at 4 °Cand
counted. Bound
125
I-labeled IgGC7 was determined by
solubilizing cells in 1
M
NaOH and measuring the
associated counts. Nonspecific counts, determined by
adding excess unlabeled ligand, was subtracted from total
counts to give specific binding.
Results
Phorbol ester enhances the release of sLDL-R
The LDL receptor typically has a half-life of 10–12 h in
fibroblasts and Chinese hamster ovary (CHO) cells [13,27].
To determine if LDL-R is cleaved in a similar fashion to
certain other cellsurface proteins, HepG2 cells were pulse-
labeled with [
35
S]methionine and chased in the absence or
presence of PMA. The cells (C) and medium (M) were then
immunopreciptated with LDL-R-specific antibody IgGC7
and subjected to SDS/PAGE and autoradiography (Fig. 2).
Immunoprecipitates of labeled HepG2 cells with the IgG-
C7 antibody showed the characteristic 120-kDa precursor
LDL receptor present at the start of the chase period but not
at later time points when only the 160-kDa mature receptor
is seen. The addition of PMA to labeled cells resulted in a
marked increase in the levels of a 140-kDa immunopreci-
pitable protein in the extracellular medium. The presence of
this protein in the medium of untreated cells was hardly
noticable (Fig. 2). Recognition of this protein by immune-
complexes of the IgG-C7 antibody suggest that it is a
soluble form of LDL-R. Immunecomplexes of a second
monoclonal antibody HL-1 demonstrated equivalent results
to the IgG-C7 antibody (data not shown).
Human skin fibroblasts from a receptor negative familial
hypercholesterolemia (FH) patient (NS) were analyzed in
the same manner for LDL-Rcleavage (Fig. 3). The
140-kDa protein was absent from the culture medium of
PMA-treated FH fibroblasts but present in the medium
of HepG2 cells and normal fibroblasts. Downregulation
of LDL-Rin the normal cells by pretreatment with
25-hydroxycholesterol resulted in the absence of any
detectable 140-kDa soluble protein (Fig. 3). The normal
LDL-R expressed in transfected CHO cells (CHO-715)
displayed significant 140-kDa protein in the medium while
the parent LDL-R-negative cells (CHO-A7) showed no
immunoprecipitable protein in the chase medium. Taken
together, these data clearly suggest the identity of the
140-kDa protein as a soluble form of LDL-R.
Fig. 2. Characterization of the soluble form of LDL-R induced by
PMA. HepG2 cells were metabolically labeled for 2 h with
[
35
S]methionine (lane 1) followed by a 4-h chase period (lanes 2–4) in
the presence or absence of PMA (100 ngÆmL
)1
). Both cells (C) and
medium (M) were collected and immunoprecipitated with IgG-C7,
followed by SDS/PAGE and autoradiography.
526 M. J. Begg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Based on the mobility of the soluble protein on SDS/
PAGE (140 kDa for sLDL-R vs. 160 kDa for the mature
LDL-R), and the fact that a monoclonal antibody (4A4)
directed against the cytoplasmic domain of LDL-R does not
recognize the 140-kDa protein (data not shown), it is likely
that the sLDL-R consists of the bulk of the extracellular
domain of the LDL receptor. This protein could be
generated either by alternative splicing of LDL-R mRNA,
eliminating sequences responsible for anchorage in the
membrane, or by proteolytic cleavage of the transmembrane
LDL-R. mRNA splicing is less likely as sLDL-R was
detected only in the medium and not in the associated
cells either during or after the pulse. Furthermore, soluble
receptors were only readily detected once PMA was added
to the medium and this occurred even when labeling of
newly synthesized receptors had ceased, i.e., during the
chase period. Incorporation of [
35
S]methionine into newly
synthesized receptors ceased within 15 min of commencing
the chase as no more 120-kDa precursor LDL receptor
protein could be detected after this time period. Even after
a 4-h chase period the addition of PMA stimulated the
generation of labeled soluble receptors (data not shown).
This would not be the case if mRNA splicing was the source
of the truncated sLDL-R.
The rate at which sLDL-R was released from PMA-
treated and untreated HepG2 cells was assessed as shown in
Fig. 4. Cells treated with PMA (closed symbols) released
sLDL-R at a rate significantly faster than untreated cells
(open symbols). The accelerated release induced by PMA
lasted for about 2 h after which the rate of release tended to
slow down. The slowdown inLDL-Rcleavage was not due
to sLDL-R degradation in the medium as no significant
loss of sLDL-R was detected during a 20-h incubation of
sLDL-R-containing medium at 37 °C (data not shown). By
2 h, 18.7% (± 3.5; n ¼ 5) of the total labeled LDL-R was
detected as soluble receptor in PMA-treated cells, compared
to 4.8% (± 2.1; n ¼ 5) released from untreated cells in the
same time period.
The regulation of cleavage was further characterized by
determining the effect of PMA on sLDL-R release (Fig. 5).
No effect was detected at PMA concentrations
<1 ngÆmL
)1
and sLDL-R release was increased between
1ngÆmL
)1
and 10 ngÆmL
)1
PMA. No significant increase in
sLDL-R is seen > 10 ngÆmL
)1
, although in one experiment
maximum release was achieved only at 30 ngÆmL
)1
.The
response of cells to the different PMA concentrations varied
between experiments as indicated by the relatively large
error bars. This variation is thought to be systematic as all
values were either high or low depending on the experiment.
The requirement for an active protein kinase C (PKC) in the
PMA response was established by using the PKC inhibitor
staurosporine (10 l
M
) which almost completely abolished
the enhanced release at 100 ngÆmL
)1
PMA (closed dia-
mond, Fig. 5).
The number of surfaceLDL-R is affected by PMA
To assess whether PMA-stimulated release of sLDL-R
alters the number of LDL-Rs on the cell surface, HepG2
cells were incubated for various times in the presence of
PMA after which the number of cellsurface receptors was
assessed by binding of an anti-LDL-R monoclonal Ig (IgG-
C7)at4°C. A 1-h treatment of HepG2 cells with PMA
resulted in a 30% decrease in the number of cell surface
LDL-R as measured by labeled antibody (Fig. 6). Incuba-
ting the cells with PMA for longer periods at 37 °Cresulted
in a reversal of the decreased receptor number seen at 1 h,
such that by 4 h the number of cellsurface LDL-Rs had
doubled. This increase is in all likelihood due to PMA
stimulation of the PKC dependent, p42/44
MAPK
induction
of LDL-R transcription in HepG2 cells [29,30]. The initial
Fig. 4. Kinetics of sLDL-R release induced by PMA. HepG2 cells,
metabolically labeled for 2 h with [
35
S]methionine, were chased in
unlabeled MEM/LPDS for the indicated periods in the presence (d)or
absence (s)ofPMA(100ngÆmL
)1
). After the indicated chase periods,
the medium was removed, immunoprecipitated with IgG-C7, subjec-
ted to SDS/PAGE and followed by autoradiography and quantifica-
tion. These data represent the mean (± SEM) of duplicates from three
experiments.
Fig. 3. Production of sLDL-R by different cell types. HepG2 cells,
normal HSF and FH HSF, were either upregulated in DMEM/LPDS
(up) or downregulated in DMEM/fetal bovine serum + 1 lgÆmL
)1
25OH-cholesterol (down) for 24 h prior to metabolic labeling with
[
35
S] methionine. CHO cells transfected with human LDL-R (715) and
its LDL-R negative parent cell line (A7) were maintained in full
medium. All cells were pulse-labeled with [
35
S]methionine for 2 h and
chased in DMEM/LPDS in the presence of PMA (100 ngÆmL
)1
)for
4 h. The medium was removed from the dishes and subjected to
immunoprecipitation and autoradiography described.
Ó FEBS 2004 Cellsurfacecleavage of LDL-R (Eur. J. Biochem. 271) 527
decrease incellsurface receptor number following PMA
treatment was also seen in CHO cells transfected with
human LDL-R (CHO-715); however, in these cells the
number of surface receptors remained below the control
for an extended period and no reversal of this effect was
detected (data not shown). This is probably because the
transfected gene is not under the control of its native
promoter. The loss in LDL receptor surface binding was
supported by degradation studies, which demonstrate that
PMA enhances the loss of [
35
S]methionine-labeled receptors
significantly (Fig. 7), such that by 2 h, PMA-treated cells
have 30% less labeled receptors than untreated cells. Given
the lack of steady state conditions in this experiment, it was
not possible to determine accurately the half-life of LDL-R
following PMA treatment. As an estimate, the apparent
half-life of LDL-R was 2 h in PMA-treated cells
compared to 5–6 h in untreated cells. The discrepancy
between soluble receptor detected in the medium (18.7%) in
2 h vs. the increased loss of total receptor following PMA
treatment (40% less labeled receptors in treated cells vs.
untreated cells after 2 h) is an indication that other
proteolytic pathways are also stimulated by PMA. Other
degradative pathways may include the generation of the
125-kDa Band X as reported by Lehrman et al.[31].
Internalization deficient LDL-R undergo increased
cleavage
While sLDL-R of 140 kDa was readily detected in the
medium of PMA-treated cells, no protein of this size was
detected incell lysates, suggesting that receptor cleavage
takes place at or near the cell surface, possibly in the
endosomal compartment. In order to ascertain if endo-
cytosis plays a role in cleavage, two mutant LDL-Rs
(792-LDL-R and JD-LDL-R) were constructed which are
unable to undergo endocytosis via coated pits. The muta-
tions were confirmed to be internalization defective as
assayed by
125
I-labeled LDL uptake, with the rates of
internalization being 10% and 25% of normal for
792-LDL-R and JD-LDL-R, respectively. The transfected
CHO cell lines, CHO-792 and CHO-JD, were pulse-labeled
with [
35
S]methionine and chased in the presence or absence
of PMA for 2 h (Fig. 8A). Both 792-LDL-R and JD-LDL-R
were cleaved to a significantly greater extent than the wild-
type LDL-R. After the 2-h chase period in the presence of
PMA, 70% of the total population of labeled 792-LDL-R
was present as sLDL-R in the medium, compared to 42% of
JD-LDL-R and 20% of wild-type receptors (Fig. 8B). In
the absence of PMA, the degree of cleavage for 792-LDL-R,
JD-LDL-R and wild-type LDL-R was 40%, 11% and 6%,
respectively. These results indicated that endocytosis via
coated pits is not required for cleavage and in fact it may
inhibit cleavage of LDL-R. In addition it suggested that
cleavage takes place on the cell surface.
The LDL-R mutant 812-LDL-R that is truncated after
Thr811 (Fig. 1) was used to investigate whether other
Fig. 6. Effect of PMA on surfaceLDL-R number. HepG2 cells
upregulatedfor12hinMEM/LPDSwereincubatedat37°Cinthe
presence (d) or absence (s)ofPMA(100ngÆmL
)1
) for the indicated
time period. They were then cooled to 4 °C and incubated for 2 h in
the presence of
125
I-labeled monoclonal antibody IgGC7. After sub-
stantial washing the remaining cell associated label was determined
and normalized tocell protein in each dish. The graph represents the
level of IgG-C7 binding to the cellsurface as a percentage of the zero
hour value. Error bars represent the SEM of four values.
Fig. 5. Effect of PMA dose on sLDL-R production. HepG2 cells were
pulse-labeled with [
35
S]methionine and chased for 4 h in the presence
(d) or absence (s) of indicated doses of PMA, or in the presence of
100 ngÆmL
)1
PMA and 10 l
M
staurosporine (r). The medium was
immunoprecipitated and subjected to SDS/PAGE as described. The
dried gels were then exposed to electronic autoradiography and
quantitation. The error bars represent the range of four data points
from two experiments, with the symbol representing the mean.
528 M. J. Begg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
signals in the LDL-R cytoplasmic tail influence cleavage.
The last 27 amino acids of the cytoplasmic domain are
required for receptor dimerization [32] and also contain a
phosphorylation site at Ser833 [33]. These receptors were
shown to be internalization competent (data not shown).
Deletion of this domain had no significant effect on cleavage
(Fig. 8B) showing that neither the phosphorylation site,
nor receptor dimerization appear to play any role in the
formation of the 140-kDa sLDL-R.
Two of the three cytoplasmic-mutants, JD-LDL-R and
792-LDL-R occur in patients with FH. To confirm the
cleavage process observed in the transfected cells, assays
were carried out using human skin fibroblasts (HSF) from
patients homozygous for these mutations. Both HSF-JD
(GM2408) and HSF-792 (FH683) behaved in a very
similar fashion to their transfected CHO cell counterparts
when stimulated with PMA. The percentage of labeled
receptors released as sLDL-R in a 2-h chase period
following PMA treatment was 85.9% (± 6.0; n ¼ 4) for
HSF-792 compared to 70.1% (± 10.9; n ¼ 6) for CHO-
792. Likewise the percentage release of the JD mutant
receptors was 42.2% (± 1.2; n ¼ 4) in human skin
fibroblasts and 41.6% (± 8.9; n ¼ 8) in CHO cells. Wild-
type LDL receptor release was also very similar – 18.7%
for HSF and 20.7% for CHO cells. The most notable
difference between CHO cells and HSF cells was that the
unstimulated release was much lower in HSF cells than in
their CHO counterparts. For example, unstimulated wild-
type CHO cells released 6.6% of their LDL-Rin 2 h
compared to 0.9% for wild-type HSF cells. Similarly,
CHO-792 released 40.4% compared to 18.5% HSF. The
phenotype of the cytoplasmic domain mutations is thus a
direct result of the mutation itself and cannot be ascribed
as an artifact of transfection and receptor overexpres-
sion. Furthermore, the cellsurfacecleavage mechanism
appeared more sensitive to PMA regulation in HSF than
in CHO cells.
Enzymes responsible for ectodomain shedding of cell
surface proteins have remained largely unidentified except
for a few proteases such as ADAM 10 and ADAM 17
(TNF-a converting enzyme or TACE) [34]. From protease
inhibitor studies these proteinases fall into two main classes:
(a) elastase-like serine proteinases as for c-kit receptor
ligands KL-1 and KL-2 [23] and (b) metalloproteinases as
for ACE and TNF-a [35,36]. To characterize the protease
responsible for the generation of sLDL-R, HepG2 cells were
pulse-labeled with
35
S-methionine and chased in the pres-
ence of various protease inhibitors. Table 1 shows the
effects of multiple inhibitors on sLDL-R release. Only the
metalloproteinase inhibitors showed any significant inhibi-
tion of release. TAPI, a hydroxamate-based metallopro-
teinase inhibitor, shown to inhibit the cleavage of TNF-a,
IL-6R, ACE and others [37], inhibited sLDL-R production
by as much as 90% (Table 1). The other metalloproteinase
inhibitors, EDTA and EGTA inhibited the release by
50–70%.
Fig. 8. Effect of cytoplasmic mutations on LDL-R cleavage. LDL-R
negative CHO cells (CHO-A7) were transfected with pLDL-R2 con-
taining the requisite mutations, and stable clones were selected and
seeded into 35-mm dishes. After 24 h, cells were labeled with
[
35
S]methionine for 2 h and chased in unlabeled medium in the pres-
ence or absence of PMA for a further 2 h. Immunoprecipitates of cells
and medium were subject to SDS/PAGE and fluorography.
(A) Autoradiographs of respective mutants labeled as above; for each
cell type: (lane 1) cells after 2-h pulse; (lanes 2 and 3) medium after 2-h
pulse and 2-h chase in the absence (lane 2) or presence (lane 3) of PMA.
(B) The medium bands from B were quantified and expressed as a
percent of total receptor label at time zero. The data represents the
mean (± SEM) of duplicates from four experiments.
Fig. 7. Effect of PMA on cellular LDL-R turnover. HepG2 cells labeled
for 2 h with [
35
S]methionine, were first chased in unlabeled MEM/
LPDS for 1 h followed by a further chase in the presence (d)or
absence (s) of PMA (100 ngÆmL
)1
) for the indicated times, after which
the cells were solubilized, immunoprecipitated and subject to electronic
autoradiography and quantification. The points reflect the quantity of
labeled LDL-R remaining in the cell as a percentage of the 0 time point
at the start of the chase period. These data represent the mean
(± SEM) of duplicates from four experiments.
Ó FEBS 2004 Cellsurfacecleavage of LDL-R (Eur. J. Biochem. 271) 529
Discussion
In this study we have investigated LDL-R degradation in
HepG2 cells, and report that a 140-kDa sLDL-R is released
into the medium by a proteolytic mechanism sensitive to
phorbol-ester induction and inhibited by TAPI, a metallo-
protease inhibitor. Such solubilizing proteolysis occurs for
a number of transmembrane proteins, including TGF-a,
TNF-a,TNF-R,ACE,amyloidprecursorprotein,
L-selectin and IL-6 receptor [17–20]. The release of sLDL-
R into the medium after phorbol ester induction is
accompanied by a decrease in both surfaceLDL-R number
(Fig. 6) and total cellular LDL-R (Fig. 7). A similar loss of
LDL-R binding after PMA treatment has been reported in
U937 cells but the mechanism responsible for this is
unknown [38].
The formation of sLDL-R was found in various cell types
when stimulated with phorbolesters (Fig. 3). Soluble LDL-
Rs have previously been reported to be in the medium of
CHO cells that are defective in O-linked glycosylation [39],
and to be produced by cells inresponseto interferon [40]. In
the latter case, the 28-kDa soluble receptor, which consists
of the N terminus of the receptor, has marked antiviral
activity by interfering with vesicular stomatitis virus assem-
bly and budding. This 28-kDa N-terminal domain is
contained within the 140-kDa soluble receptor reported in
this study. Other members of the LDL receptor family, LRP
and VLDL-R also undergo surface proteolysis to generate
soluble ectodomains. In the case of VLDL-R, the soluble
fragment, as well as the corresponding region of the LDL-
R, binds minor group rhinoviruses and inhibits viral
infection in HeLa cells [21,22]. The important question is
whether the cleavage mechanism responsible for the
140 kDa sLDL-R is operative in vivo,andifso,whatis
the function of such soluble receptors? Apart from potential
antiviral activity, a possible function of these sLDL-R
wouldbetobindligand(LDL)andthusinterferewithits
uptake and clearance from the plasma bycell bound LDL-R.
This occurs in the case of soluble growth factor receptors
and soluble cytokine receptors, where the soluble receptors
have been shown to act as antagonists by binding to their
respective ligands and thereby reducing their effects [19].
Such soluble complexes of receptor and ligand are reported
to stabilize the cytokine or growth factor in the extracellular
fluid [41]. On the other hand, some cytokines and their
receptors, such as IL-6 and its receptor, can act as potent
agonists on cells [42]. Cleaved membrane proteins are also
involved in various diseases, such as Alzheimer disease and
Heymann nephritis [43]. In both of these the pathology is a
result of deposition of solublized membrane proteins in
plaques. It is tempting to speculate that sLDL-R could
become deposited in atherosclerotic plaques and act as a
trap for LDL at these sites. Further work is needed to
establish to what extent this pathway is operative in vivo
and what the potential in vivo activators of this pathway
might be.
Transferrin receptors and asialoglycoprotein receptors
also display a decrease incellsurface number inresponse to
PMA treatment of HepG2 cells [44]. In the case of the latter
two receptors, reduced binding is due to redistribution of
receptors to intracellular compartments [44]. This redistri-
bution scenario does not hold true for the LDL-R as the loss
of surface binding is equivalent to the loss of total receptors;
also, the mutant LDL-R that is unable to undergo
endocytosis and is thus restricted to the cellsurface is more
susceptible tosurfacecleavage than the wild-type LDL-R.
These mutant receptors also demonstrate an equivalent loss
of surface binding (data not shown), indicating that
redistribution from the cellsurface is not required for the
loss insurface receptor number. Furthermore, the kinetics
of LDL-R binding loss do not match the much faster
kinetics of transferrin receptor and asialoglycoprotein
receptor redistribution [44].
The cytoplasmic domain of LDL-R appears to contain
elements that are able to modulate cleavage. Receptors with
a deletion of the entire cytoplasmic domain (792-LDL-R) or
Table 1. The effect of protease inhibitors on the release of sLDL-R into the medium. HepG2 cells were labeled for 2 h with [
35
S]methionine and then
chased in DMEM/LPDS plus 100 ng mL
)1
PMA for a further 2 h in the presence of various protease inhibitors. The medium was immuno-
precipitated and subjected to SDS/PAGE and quantitative autoradiography as indicated in the methods section. The degree of release was
calculated as a percentage of the zero inhibitor control. NA, not applicable; n, Number of experiments performed; for each experiment duplicate
dishes were used for each inhibitor; SEM, Standard error of the mean.
Protease inhibitors
class Protease
Concentration
of inhibitor
sLDL-R release
(% of control) SEM n
None – – 100 0 9
ALLN Cysteine 260 l
M
116 22 4
Leupeptin cysteine/serine 200 l
M
128 27 6
E-64 Cysteine 280 l
M
91 9 3
Pepstatin Aspartic 1 l
M
96 7 3
PMSF Serine 1 m
M
79 7 4
Pefabloc Serine 1 m
M
92 NA 1
Aprotinin Serine 1.5 l
M
101 NA 2
3,4-dichloroisocoumarin Serine 100 l
M
128 NA 2
TLCK Serine 130 l
M
98 8 4
EDTA Metallo 10 m
M
33 18 3
EGTA Metallo 10 m
M
50 29 3
Phosphoramidon Metallo 500 l
M
95 NA 2
TAPI Metallo 10 l
M
10 NA 2
530 M. J. Begg et al. (Eur. J. Biochem. 271) Ó FEBS 2004
an amino acid subsitution in the NPVY internalization
signal (JD-LDL-R) become hypersensitive to solubilizing
cleavage (Fig. 8), presumably due to prolonged residence
times on the cellsurface and thus more frequent exposure to
a cellsurface cleaving protease. This hypothesis is supported
by an inverse correlation between the ability to internalize
via coated pits (internalization index) and the sensitivity of
LDL-R to cleavage. 792-LDL-R which has the lowest
internalization index (10% of normal) is cleaved at the
highest rate, whereas JD-LDL-R is internalized more
efficiently than 792-LDL-R (25% of normal) and is less
sensitive to ectodomain cleavage than 792-LDL-R, but still
cleaved to a greater extent than wild-type receptors. We
cannot, however, rule out that changes in the tertiary
structure of the cytoplasmic domain are responsible for the
enhanced release. In any case, the modulatory element(s)
must reside in the N-terminal 22 amino acids of the
cytoplasmic domain (Lys790–Glu812) containing the
NPVY internalization signal, as a deletion of the last 27
amino acids of the cytoplasmic tail (812-LDL-R) has no
effect on cleavage. The modulatory element may in fact
be the NPXY motif itself, as cytosolic adaptor proteins
containing phospho-tyrosine binding domains are known to
interact with the NPXY motif. ARH and Disabled 1 are
two cytosolic proteins that have been shown to bind to the
LDL-R cytoplasmic domain [45,46]. Recent studies have
demonstrated the interaction of numerous signal transduc-
tion proteins with the cytoplasmic domains of members of
the LDL-R gene family [47].
In contrast to the large release measured for 792-LDL-R
in both CHO and HSF cells, Lehrman et al.[31]didnot
detect any soluble receptor in the medium of HSF-792. This
may be due to the immunoprecipitation protocol used,
as we have found that immunoprecipitation with IgG-C7
and Protein A sepharose does not precipitate any product
from the extracellular medium whereas precipitation with
immunecomplexes of IgG-C7 and goat antimouse IgG, or
IgG-C7 linked to sepharose beads precipitates significant
amounts of sLDL-R.
Of the proteases responsible for the solubilizing of
surface proteins, the best characterized is TACE [48–50].
In the main, the solubilizing enzymes responsible for the
large array of ectodomain cleavages have not been
identified. Two potential classes of solubilizing proteases
appear to exist, a serine class with elastase-like sequence
specificity [23], and a metalloproteinase class, of which
TACE is an example. In our study, only metalloprotein-
ase inhibitors inhibited cleavage, with TAPI inhibiting
cleavage by up to 90%. TAPI and other closely related
hydroxamate-based inhibitors have proved useful in
inhibiting the cleavage of proteins such as TNF-a,
L
-selectin, p60 TNF receptor and IL-6 receptor as well
as a number of other cellsurface proteins [36,51–54]. The
specificity with which TAPI inhibits the cleavage of LDL-R
suggests a close relationship between the LDL-R cleaving
protease and the family of protease’s of which TACE is a
part. This family belongs to a subgroup of adamalysin-like
metalloproteinases known as ADAMs, which contain both
a disintegrin and a metalloproteinase domain [55,56].
Our data are supported by Guo et al.[57]whodemonstrate
the accumulation of amongst others, sLDL-R in the
medium of TACE–/– DRM cells transfected with TACE.
No sLDL-R is detected in the medium of the untransfected
TACE–/– cells [57].
Recent evidence also suggests that reactive oxygen
species, including nitric oxide (NO) are responsible for
mediating the PMA induced activation of enzymes such as
TACE, thus providing a potential natural mechanism for
inducing cleavage of LDL-Rin vivo [58,59].
Turnover of LDL-Rin HepG2 cells is rapid with a half-
life in the order of 4 h (Fig. 7) compared to turnover in
CHO cells and fibroblasts (t
½
, 10–12 h) [13]. This difference
may be significant in the overall regulation of cholesterol
homeostasis within the liver, as the faster the rate of
turnover of a protein, the more rapid is the response to
transcriptional downregulation. In contrast to our findings,
Tam et al. [60] measured the half-life of LDL-Rin HepG2
cells to be 9–10 h. We are unable to explain the differences
in half-life measured in the same cell type; however, evidence
from circadian rhythms in rat liver LDL-R expression
suggests a LDL-R half-life of 6 h in the liver [61]. The
enhanced LDL-R degradation measured in Fig. 7 in the
presence of phorbol ester, could not be completely accoun-
ted for by the increased release of receptors into the
medium, as the increase in [
35
S]methionine labeled sLDL-R
in the medium was approximately 2.5-fold less than the
decrease of [
35
S]methionine labeled cellular LDL-R over the
same time period. In addition there was no significant
degradation of sLDL-R in the medium. We therefore
conclude that PMA induces more than one degradation
mechanism for LDL-R, of which the formation of sLDL-R
contributes about 50%. Another degradation pathway may
involve the production of a 125 kDa LDL-R degradation
intermediate referred to a band X, reported by Lehrman
et al. [31], which is not recognized by the anti-
N-terminal Ig (IgG-C7) used in this study, and is presum-
ably due tocleavage of the N terminus from the remainder
of the receptor. This potential pathway requires further
investigation.
In conclusion, LDL receptor number, at least in HepG2
cells, can be regulated not only by transcriptional control
but also by enhanced turnover inresponseto unknown
stimuli that signal via PKC. This enhanced turnover
involves at least two proteolytic systems, one of which
involves cellsurfacecleavage and the release of soluble
receptors into the extracellular medium. Cellsurface clea-
vage of the LDL-R represents the first described mechanism
that can regulate LDL-R expression post-translationally.
Acknowledgements
We thank S. Jones and L. Gatterdam for excellent technical assistance.
This work was supported by American Heart Association grant
9750284 N (D. R. v.d.W.), and by the South African Medical Research
Council (E. D. S and D. R. v.d.W).
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. compared
to untreated cells, as was the total number of cell surface
LDL-receptors assayed by the binding of
125
I-labeled anti-
body to whole cells. To. adaptor proteins
containing phospho-tyrosine binding domains are known to
interact with the NPXY motif. ARH and Disabled 1 are
two cytosolic proteins that