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(Received for publication, October 2, 1995) From the
CD36 is an integral membrane glycoprotein expressed by several
cell types, including endothelial cells of the microvasculature,
erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36
is expressed during the late stages of differentiation in the bone
marrow, in circulating monocytes, and in some tissue resident
macrophages, and it is thought to mediate the phagocytosis of apoptotic
cells and the endocytic uptake of modified lipoproteins. Here we
analyze the synthesis, processing, and intracellular transport of CD36
in U937 and THP-1, two human cell lines representing different stages
of monocytic maturation. In both cell lines, phorbol 12-myristate
13-acetate induces the expression of CD36. A 74-kDa intracellular
precursor is first synthesized that has the hallmarks of a resident
protein of the endoplasmic reticulum. The precursor protein is later
processed into a mature form of 90-105 kDa which is transported
to the cell surface. The kinetics of processing differ significantly in
U937 and THP-1. These differences are specific for the CD36, as two
unrelated proteins (CD11b and CD45R) are processed and transported to
the surface at similar rates in the two cell lines. A 33-kDa
endoglycosidase H-sensitive glycoprotein specifically associates with
the 74-kDa precursor. Coprecipitation of gp33 correlates with slow
processing of CD36 precursor, suggesting that gp33 may play a role in
regulating the intracellular transport of CD36, during monocyte
maturation.
CD36 is a membrane glycoprotein expressed by erythrocyte
precursors, mature monocytes, platelets, endothelial cells of the
microvasculature, and mammary epithelial cells(1) . The CD36
cDNA predicts a polypeptide of 53-kDa with 10 potential N-linked glycosylation sites(2) . Depending on the
cell type, CD36 displays different molecular masses (78, 88, or 94 KDa)
corresponding to different glycoforms(1, 3) . Several
functions, all depending on the molecule being expressed at the cell
surface, have been ascribed to CD36. In platelets, the molecule acts as
a receptor for the extracellular matrix proteins collagen (4) and thrombospondin 1(5, 6) . CD36 also
mediates adherence of erythrocytes infected with Plasmodium
falciparum to capillary endothelial cells, a phenomenon that
contributes to the morbidity and mortality of malaria in
humans(7) . Recently, CD36 has been reported to be involved in
the phagocytosis of neutrophils and T lymphocytes undergoing apoptosis
by macrophages(8, 9, 10) , as a receptor for
oxidized low density lipoprotein on
macrophages(11, 12) , and for fatty acid binding and
transport in foam cells(13) . In many cell types including
platelets, endothelial cells, and monocytes the role of CD36 as a cell
surface receptor has been extended to that of a signal transduction
molecule(14, 15, 16, 17) . An
association with protein tyrosine kinases of the src gene
family has been described in platelets and endothelial cells
CD36(18, 19) . The regulation of CD36 expression
during monocyte differentiation in terms of gene activation,
post-transcriptional and post-translational modifications and
intracellular transport is still poorly understood. In this study, we
have exploited U937 and THP-1 cells, two human lines that immortalize
different steps of monocytic maturation, to investigate the synthesis,
processing and intracellular transport of CD36 molecule. We find that
in both cell lines the expression of the CD36 antigen is induced by
phorbol esters (PMA) (
The monoclonal antibodies (mAbs)
used in this study were NL07(3) , OKM5 (Ortho Diagnostics,
Milan, Italy), Mo91(20) , all specific for CD36; the L31
anti-HLA class I(21) ; the OKMI anti-CD11b (Ortho Diagnostics),
and the GAP 8.3 anti-CD45RA, B(22) . The IgG fraction was
prepared from 1207 rabbit anti-CD36 antiserum (20) or from
rabbit anti-mouse Ig µ-chain (23) by affinity
chromatography on protein A-Sepharose. HRP-conjugated rabbit anti-mouse
Ig and swine anti-rabbit Ig HRP were from Dako (Glostrup, Denmark).
Controls were class-matched irrelevant monoclonal antibodies or a
rabbit preimmune serum.
The molecular mass standards
included lysozyme (14 kDa), trypsin inhibitor (21 kDa), carbonic
anhydrase (30 kDa), ovalbumin (46 kDa), BSA (69 kDa), phosphorylase b (97 kDa),
After washing in 0.1% Nonidet
P-40 TBS, the immunocomplexes were eluted by treatment with SDS-PAGE
sample buffer for 5 min at 100 °C and resolved by SDS-PAGE. Gels
were either blotted to nitrocellulose or fixed and processed with
Amplify (Amersham Corp., Milan, Italy) before exposure to Kodak X-Omat
AR films or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Autoradiograms were analyzed by a computing densitometer (Molecular
Dynamics).
Figure 1:
Flow cytometry analysis of the surface
expression of CD36 on resting and differentiated THP-1 and U937 cell
lines. Indirect immunofluorescence tests were performed on untreated
cells or cells treated with PMA for 48 h using the NL07 anti-CD36 mAb.
The detection antibody was goat anti-mouse Ig-fluorescein
isothiocyanate. Shadowed peak indicates control mouse Ig as
fluorescence background. The ordinate shows cell numbers, and
the abscissa shows fluorescence
intensity.
Figure 2:
Kinetics of CD36 expression in the
PMA-differentiated THP-1 and U937 cell lines. Western blot analysis was
performed using Mo91 anti-CD36 mAb and L31 anti-HLA class I mAb on
whole extracts from U937 (a) or THP-1 (b) cells
treated with 40 nM of PMA for times indicated in the figures
and resolved on 7.5% SDS-PAGE. The arrowheads indicate the
74-kDa protein, the brackets indicate the broad band ranging
between 90 and 105 kDa. Molecular mass markers are as indicated under
``Materials and Methods.''
Noteworthy, PMA does not induce significant changes in the HLA class
I molecule steady state levels (Fig. 2, bottom panels).
Hence, induction of surface proteins does not appear to be a general
phenomenon generated by PMA. The kinetics of appearance of p74 suggests
that it might represent a precursor of CD36 whose synthesis is
activated by PMA. Immunoprecipitation and pulse-chase experiments were
thus designed to verify this possibility.
Figure 3:
Analysis of CD36 processing in U937 and
THP-1 cells by pulse-chase experiments. Cells treated for 12 h with PMA
were endogenously labeled with
[
Figure 4:
Western blot analysis of
immunoprecipitated CD36 from THP-1 differentiated cells.
Immunoprecipitation was performed with 1207 anti-CD36 rabbit serum from
extracts of the THP-1 cell line after 48 h of PMA differentiation or
from THP-1 cells surface-labeled with succinimidobiotin undergoing the
same treatment as described under ``Materials and Methods.''
Immunocomplexes were resolved on 7.5% SDS-PAGE and transferred to
nitrocellulose membrane (lanes 1207). Control
immunoprecipitations (lanes Ctrl) were performed with
nonimmune rabbit serum. Western blots performed with anti-CD36 Mo91 mAb
on immunocomplexes from unlabeled cells and on differentiated THP-1
whole extract fractionated on the same gel (lane Total Lys)
were developed with rabbit anti-mouse Ig HRP (RaMIg-HRP).
Immunocomplexes from cell surface-biotinylated proteins were detected
on a nitrocellulose membrane by reaction with avidin-HRP. The arrowhead indicates the 74-kDa protein, the bracket indicates the broad band ranging between 90 and 105 kDa. Molecular
mass markers are as indicated under ``Materials and
Methods.''
When intact THP-1 cells are labeled with
biotin prior to cell lysis, only the gp90-105 band is decorated
by avidin (see the right lane), suggesting that p74 is not
expressed on the cell surface. The faint band with an apparent
molecular mass of 74 kDa detected by avidin in the
immunoprecipitate(1207) might be due to the biotin labeling of the
intracellular precursor from a few dead cells permeabilized during
incubation. A weak band of 130-kDa molecular mass is present in the
immunoprecipitated material(1207). This band, that was never detected
in the total lysates, is likely to originate from artifactual
cross-linking by succinimidobiotin. If p74 were a precursor of
mature CD36, treatment with N-glycosidase F, an enzyme that
cleaves all types of N-linked glycosidic residues, should
yield a single band of about 57 kDa(28) . As shown in Fig. 5(lanes 1 and 2) this is indeed the case.
In PMA-differentiated U937 cells, both the gp90-105-kDa protein
band and the one of 74 kDa yield a unique protein band of 57 kDa when N-linked sugars are removed. Treatment with endoglycosidase H
allows one to distinguish high mannose (sensitive) from complex sugars.
As evident from lanes 3 and 4, gp74 is sensitive to
this glycosidase, while gp90-105 is largely resistant. The
partial digestion of gp90-105 is probably due to incomplete
processing of some of the CD36 glycans.
Figure 5:
N-Glycosidase F and
endoglycosidase H treatments. Cell extracts prepared from 48-h
PMA-differentiated U937 were treated (+) with N-glycosidase F or with endoglycosidase H or incubated without
enzyme(-) as described under ``Materials and Methods.''
Proteins were resolved on 7.5% SDS-PAGE and transferred to a
nitrocellulose membrane. Western blot analysis was performed using Mo91
anti-CD36 mAb. Upper arrowhead indicates the 74-kDa protein,
the bracket indicates the broad band ranging between 90 and
105 kDa, and the lower arrowhead indicates the 57-kDa protein.
Molecular mass markers are as indicated under ``Materials and
Methods.''
The PMA-induced accumulation
of gp74 depends on de novo mRNA and protein synthesis.
Consistent with gp74 being a precursor of CD36, both actinomycin D (a
mRNA synthesis inhibitor) and cycloheximide (an inhibitor of protein
synthesis) prevent the accumulation of the gp74 induced by PMA
treatment (not shown).
Nonreducing gels (not
shown) reveal that the p33 molecule is not disulfide-linked to CD36
precursor, while like gp74, p33 is endo-H-sensitive and is digested
into a 22-24-kDa molecule (Fig. 6).
Figure 6:
Endoglycosidase treatment of the 33-kDa
co-immunoprecipitated protein. 1207 anti-CD36 rabbit serum
immunoprecipitated samples from endogenously labeled U937 cells after
PMA treatment were incubated with (+) or without(-) 10
milliunits of endoglycosidase H for 12-18 h at 37 °C, in
appropriate digestion buffer; see ``Material and Methods.''
Treated samples were run on 5-15% gradient SDS-PAGE in reducing
conditions, and dried gels were analyzed on a PhosphorImager (Molecular
Dynamics). Black arrows indicate undigested gp74 and gp33, and white arrows indicate shifted proteins after endoglycosidase
treatment. Molecular mass markers are as indicated under
``Materials and Methods.''
The first possibility is excluded by
adsorption experiments. Adsorption of the 1207 anti-CD36 rabbit serum
with human platelets, which express abundant mature CD36 but no
gp74
The second hypothesis was excluded by two lines of
evidence. First, neither polyclonal rabbit serum anti-CD36 nor the Mo91
mAb detected gp33 in Western blots of whole lysates or on anti-CD36
immunoprecipitates (not shown). Second, different peptides are
generated by trypsin digestion from gel-purified gp74 and gp33. While
the gp74 yields four peptides of 27, 21, 18, and 14 kDa, respectively,
three peptides of 30, 25, and 23 kDa are generated by limited
proteolysis of gp33 (Fig. 7). Hence, these results suggest that
gp33 is a protein that associates with the 74-kDa CD36 precursor.
Figure 7:
Peptide mapping by limited proteolysis and
SDS-PAGE analysis of gp74 and gp33. Immunocomplexes obtained from
endogenously labeled U937 cells after 12 h of PMA treatment were
separated by 10% SDS-PAGE. The gp74 and gp33 protein bands (arrowheads) were eluted from the gel, acetone-precipitated,
and treated with 100 µg/ml trypsin protease as indicated under
``Materials and Methods.'' They were then loaded onto a 12%
acrylamide gel. Dried gels were exposed to a PhosphorImager or film.
Molecular mass markers (MW) are as indicated under
``Materials and Methods.''
In the present study, the U937 promonocytic and the THP-1
monocytic cell lines have been exploited to analyze the synthesis,
processing, and surface expression of CD36 during monocyte/macrophage
differentiation. Some of the events that characterize this
developmental program, including changes in the growth rate, adherence,
and expression of surface markers can be mimicked in these cell lines
by stimulation with PMA(26, 27) . Compared with blood
monocytes, in vitro differentiated macrophages and alveolar
macrophages showed a down-regulation of CD36 surface expression (29) that is consistent with the different degree of
differentiation and CD36 expression observed in the the two cell lines
used in the study. The most intriguing finding that emerges from our
experiments is that the intracellular processing of CD36 is faster in
U937 than in THP-1. Crucial to this conclusion is the identification of
different molecular glycoforms of CD36 that represent discrete stages
in the biogenesis of this membrane protein. That the 74-kDa protein is
a precursor of the mature CD36 (gp90-105) is demonstrated by the
following lines of evidence: (i) antibodies raised against the mature
CD36 recognize the gp74 both in Western blot and immunoprecipitation
assays, (ii) treatment with N-glycosidase-F reduces both the
mature CD36 and the 74-kDa form to a single protein band of 57 kDa, and
(iii) pulse-chase experiments clearly define a precursor product
relationship between gp74 and gp90-105. As in other
glycoproteins, these size differences are likely to reflect different
intracellular locations. The selective accessibility to biotin
indicates that only the mature CD36 (gp90-105) is expressed on
the plasma membrane. On the other hand, the sensitivity of gp74 to the
endoglycosidase H, an enzyme that cleaves the high mannose glycans
characteristic of protein that did not reach the medial Golgi apparatus (30) , suggests that the low molecular weight precursor is
localized in an early compartment of the secretory pathway. Hence, the
steady state ratio between the two bands can be taken as an indication
of the efficiency of intracellular processing and transport. With this
in mind, it appears that the transport of CD36 is slower in THP-1 than
in U937 at all time points after PMA admininstration and that the rate
of synthesis of the precursor has little influence on the subsequent
processing events. As reported by others (31) we confirmed that
PMA induces an increase in the rate of transcription and accumulation
of specific transcripts, ( An intracellular localization of CD36
has already been reported in platelets, where some mature CD36 is
stored within the Although the
rate of bulk flow membrane traffic can be regulated(33) ,
faster transport per se is not sufficient to explain the more
efficient processing of CD36 in U937. Other membrane molecules, either
PMA-inducible (CD11b) or constitutively expressed (CD45R), are indeed
processed and transported intracellularly at similar rates in U937 and
THP-1. It is well established that individual proteins are secreted
at different rates by the same cell(34) . An important limiting
step of intracellular transport occurs at the endoplasmic
reticulum-Golgi apparatus boundary and generally reflects the rates at
which the folding of the cargo protein takes place. Also integral
membrane proteins are subject to the same quality control events that
restrain transport to structurally mature molecules, providing an
additional step for regulating gene expression during development.
Perhaps the best examples of such posttranslational regulatory
mechanisms come from the stage-specific expression of antigen receptors
on T and B lymphocytes (see (35) and (36) for
reviews). As for these multimeric proteins, subunit assembly is
essential for negotiating transport to the plasma membrane; the absence
of a single subunit is generally sufficient to cause retention and
degradation of the other components (37) . However, there are
also cases in which the assembly of existing subunits is dynamically
regulated. For instance, for unknown reasons B lymphocytes are unable
to polymerize and secrete IgM(23) . Similarly, immature
thymocytes synthesize but fail to assemble T cell antigen receptor
An appealing
hypothesis is that the observed association with gp33 may regulate the
transport and processing of the CD36 precursor, determining its
intracellular retention or the expression on cell surface. Like the
CD36 precursor to which it is noncovalently bound, gp33, or at least
the fraction that associates with gp74, also is retained in a pre-Golgi
compartment, as demonstrated by sensitivity to endoglycosidase H. The
association between gp33 and gp74 inversely correlates with processing
of the latter. Hence, only in THP-1, where processing of CD36 is slow,
does gp33 coprecipitate at extended chase times. Lastly, in U937
dissociation of gp33 correlates with the acquisition of endo-H
resistance. The disappearance of radioactive gp33 upon chase indicates
that it is mostly newly synthesized gp33 that associates with the CD36
precursors. These results suggest that gp33 interact only once with the
CD36 precursor. This behavior differs from other known chaperone
molecules, such as BiP, that have been shown to associate sequentially
with more than one newly synthesized proteins (40) . It is of
note that a molecule that displays similar biochemical properties to
gp33 is coprecipitated from COS cells transfected with CD36. A CD36 deficiency has been described
as the absence of surface expression of CD36
molecule(41, 42) . Recently, it has been shown that
the substitution of proline-90 to serine directly leads to CD36
deficiency, impairing the maturation of the CD36 precursor and
addressing its intracellular degradation(43) . It will be of
interest to investigate whether gp33 associates with these mutants. In conclusion, it appears that, during their differentiation,
monocytic cell lines exploit many levels, including alterations in
intracellular transport, to regulate the expression of CD36. It remains
to be seen whether the regulatory mechanisms demonstrated here for
cultured neoplastic cells are exploited, as postulated by
others(44) , also in the normal myelomonocytic maturating
pathways.
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1770-1775
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)treatment, but with different
kinetics. Our results show that cells of the myelomonocytic lineage
have the capability of selectively modulating the rate of intracellular
transport of CD36, possibly by the transient association of the CD36
precursor with a glycoprotein of 33 kDa.
Reagents and Antibodies
PMA, agarose anti-mouse
Ig, bicinchoninic acid solution, actinomycin D, Staphylococcus
aureus V8 proteases, and cycloheximide were from Sigma; N-glycosidase F and endoglycosidase H from Boehringer
Mannheim; protein A-Sepharose CL-4B from Pharmacia Biotech Inc.; ECL
Western blotting detection reagent from Amersham Corp. Acetyl
avidin-biotinylated horseradish peroxidase complex and
-caproylamido-biotin-N-hydroxyl-succinimide ester were
from Bio-Division (Milan, Italy).
Cell Lines
The promonocytic line U937, the
monocytic line THP-1, the myelomonocytic line HL60, and the leukemic T
cell line Jurkat were maintained in culture as described
previously(3) . Cells were plated at 1 
10
/ml in Petri dishes and treated with PMA (40 nM)
for different times. In selected experiments, after 1 h of incubation
in the presence of PMA, actinomycin D (5 µg/ml) or cycloheximide
(50 µM) was added to cell cultures for different times
before cell lysis.Immunofluorescence Staining of Cells
If cells
adhered, as after PMA treatment, they were detached by scraping.
Aggregates were disrupted by vigorous pipetteting. Cells were washed
twice with phosphate-buffered saline, resuspended at 1 10 cells, and incubated for 30 min at 4 °C with the appropriate
dilutions of purified mAbs or hybridoma cell culture supernatants.
Bound antibody was revealed by F(ab`)
goat anti-mouse Ig
labeled with fluorescein isothiocyanate (Technogenetics, Milan, Italy).
The cells were then analyzed on a FACScan cytofluorograph (Becton
Dickinson, San Jose, CA).SDS-PAGE and Western Blot
3 10
cells were washed twice with tris-buffered saline (TBS) (10 mM Tris-HCl, pH 7.4, 150 mM NaCl) resuspended at
10/ml, and lysed with Nonidet P-40 (final concentration 1%)
for 30 min at 0 °C in the presence of protease inhibitors. After
centrifugation for 15 min at 12,000 g, aliquots of the
lysates were resolved by polyacrylamide gel electrophoresis in the
presence of SDS (SDS-PAGE) as described previously (24) on
vertical slab gels of 7.5, 10, or 12% acrylamide. In each experiment
samples were normalized so as to contain the same amount of total
protein determined by bicinchoninic acid assay (Sigma). Proteins were
electron-transferred (2 h at 60 V) to nitrocellulose sheets. The latter
were preincubated 1 h at 20 °C with 5% bovine serum albumin in TBS
and incubated with purified mAbs specific for CD36 (Mo91, final
dilution 1:400) or HLA class I (L31, final dilution, 1:1000). After 1
h, the blots were washed with 0.1% Tween 20 in TBS, incubated with HRP
rabbit anti-mouse Ig (1:1000) for 1 h, and processed for ECL according
to the supplier's instructions.
-galactosidase (116 kDa), and myosin (200
kDa), and were revealed by red Ponceau staining.Cell Surface Protein Labeling with
Succinimidobiotin
About 5 10 cells were
washed twice in labeling buffer (150 mM NaCl, 100 mM
Hepes, pH 8.0) and resuspended in 0.5 ml of the same buffer.
Succinimidobiotin was added at the final concentration of 0.25
mM, and cells were incubated at 20 °C for 30 min in the
presence of 0.002% sodium azide. The cells were then washed twice with
3% fetal calf serum in phosphate-buffered saline and lysed as described
previously.Endogenous Labeling
For pulse-chase experiments,
cells were preincubated for 15 min in methionine/cysteine-free medium
supplemented with 2% dialyzed fetal calf serum and 1 mM
glutamine and labeled at 2 10
/ml for 25 min with
500 µCi/ml TranS-label (ICN Radiochemicals, Milan,
Italy), specific activity >800 Ci/mmol. Pulse-labeled cells were
then washed and incubated in regular culture medium for various time
periods before lysis and immunoprecipitations.
Immunoprecipitation
After a preclearing step (2 h
at 4 °C) with normal rabbit serum adsorbed to protein A-Sepharose,
cell lysates were immunoprecipitated with protein A-Sepharose beads
coated with the relevant antibodies (1 h at 4 °C). In selected
experiments before protein A-Sepharose bead coating, the 1207 anti-CD36
rabbit serum and the anti-Ig µ-chain rabbit serum were adsorbed on
intact human platelets (obtained from the Blood Bank of the San
Raffaele Hospital, Milan, Italy) pretreated with preimmune rabbit serum
in order to block the Ig Fc receptors.Deglycosylation of Glycoproteins
About 20 µg
of Nonidet P-40-soluble total cellular proteins were boiled 2 min in
the presence of 0.2% SDS, resuspended in 100 µl of digestion buffer
as specified by the supplier, incubated with or without 1 unit of of N-glycosidase F or 10 milliunits of endoglycosidase H for
12-18 h at 37 °C, and resolved by SDS-PAGE. In some
experiments endoglycosidase treatments were directly performed on
Sepharose-bound immunocomplexes.Peptide Mapping by Limited Proteolysis
The basic
method of Cleveland et al.(25) was used with minor
modifications. Immunocomplexes were resolved by SDS-PAGE containing 10%
acrylamide. The protein bands of interest were eluted from the gel in
PBS containing 0.1% SDS. Proteins were precipitated twice in acetone (24) and resuspended in digestion buffer (0.125 M
Tris-HCl, pH 6.8, 0.1% SDS, 10% glycerol). Samples were then incubated
for 30 min at 37 °C in the presence of 100 µg/ml trypsin
(Fluka, Milan, Italy). The reaction was stopped by heating at 100
°C for 4 min after v/v dilution with reducing SDS-PAGE sample
buffer. SDS and 2-mercaptoethanol were added to a final concentration
of 2 and 10%, respectively. Samples were loaded on 12% acrylamide
SDS-PAGE. After electrophoresis gels were treated as previously
described and exposed to films.
CD36 Surface Expression Is Induced by PMA
Treatment
As determined by flow cytometry, THP-1 in culture have
undetectable levels of CD36 on the cell surface and are induced by PMA
to express low levels of CD36 (Fig. 1). In contrast, U937 cells
constitutively express low levels of CD36, which increase significantly
after 48 h of PMA treatment (Fig. 1). Treatment induces
morphological changes in both cell lines that resemble the progression
in differentiation steps of monocytic cells into macrophages,
consisting mainly in adhesion to plastic, a change in morphology, and
cell aggregation that are less pronounced in U937 than in THP-1, in
that the U937 cells are more immature than are THP-1 (data not
shown)(26, 27) . In both cell lines, these
morphological changes are accompanied by a decrease in the rate of cell
division(26, 27) .
Identification of a 74-kDa Precursor of CD36
The
synthesis of CD36 in response to PMA was followed by Western blot
analyses with the Mo91 mAb, which recognizes denatured
CD36(20) . A faint band of approximately 94 kDa is detected in
resting U937 (Fig. 2a, lane 0). This band,
that likely corresponds to the mature CD36 molecules detected by flow
cytometry on the cell surface, is absent in THP-1 cells (Fig. 2b, lane 0). The intensity of the 94-kDa
band increases with time as U937 cells are cultured with PMA, becoming
a broad band of 90-105 kDa (Fig. 2a). Moreover, a
new molecular species of 74 kDa appears 6 h after PMA addition and
steadily decreases thereafter (arrowhead). Similar changes in
the CD36 steady state levels are detected in THP-1, but are somewhat
delayed in time (Fig. 2b). The p74 band peaks at
12-24 h and is still easily detectable after 72 h of culture with
PMA. The gp90-105 region of the blot, that appears as a
continuous smear in U937, is constituted by a series of distinct bands
in THP-1, likely representing different glycoforms of CD36. A weak band
of 72 kDa is observed in some experiments in unstimulated THP-1 cells,
but not in U937 (Fig. 2b). The identity of this band,
which disappears a few hours after PMA stimulation and is not evident
in pulse-chase assays (see below), was not investigated further.
p74 Is the CD36 Precursor and Is Differentially Processed
in the Two Cell Lines
Pulse-chase experiments were performed
with THP-1 and U937 stimulated for 12 h with PMA (Fig. 3). In
both cell lines, p74 is the most abundant form detected after a 25-min
pulse (lanes 1 and 6). In U937, p74 rapidly converts
into the endo-H-resistant forms of 90-105 kDa. After chasing 14
h, virtually all p74 has been converted in the mature form,
demonstrating a clear precursor-product relationship. Also THP-1 cells
synthesize p74 (lane 6); however, in these cells the
conversion into gp90-105, the form that can be found on the cell
surface, is much slower than in U937. Densitometric tracings of the
autoradiograms reveal that >75% of CD36 has been processed in U937
after 2 h, while only 35% in THP-1. These results correlate well the
fact that, as drawn by immunofluorescence and Western blots ( Fig. 1and Fig. 2), there is very little CD36 on the
surface of THP-1 after 12 h of PMA stimulation. Moreover, in THP-1
neither p74 nor mature CD36 are present after a 14-h chase (lane
10), suggesting that they undergo intracellular degradation.
Hence, the low expression of CD36 on the surface of THP-1 largely
depends on posttranslational events. In all likelihood, the absence of
the 90-105-kDa form reflects a slow transport through the Golgi
apparatus. The differences could be due to a generalized slower rate of
transport in THP-1, or rather a specific feature of CD36. To
discriminate between these two possibilities we analyzed the maturation
of two other proteins, the constitutively expressed CD45R and the
PMA-inducible molecule (CD11b). The kinetics of processing and
dimerization of CD11b antigen is similar in THP-1 and U937 cells. A
mobility shift of the 
-chain is evident in both cell lines between
1 and 2 h of chase (Fig. 3b, lanes 3, 4, 8, and 9). Moreover,
co-immunoprecipitation of radioactive -chain of the heterodimer
occurs after the overnight chase in both cell lines (Fig. 3b, lanes 5 and 10), also
indicating that the kinetics of the molecular assembly of the dimer in
the two cell lines is identical. Taking into account that U937
expresses more isoforms of CD45R molecule than THP-1 (22) , it
also appears that the processing of the constitutive CD45RA and B
molecules is similar in the two cell lines (Fig. 3c).
In both cell lines almost 50% of the CD45R precursor is processed after
1 h of chase (Fig. 3c, lanes 3 and 8).
S]methionine/cysteine, then washed and
incubated in regular culture medium for various chase-time periods
before lysis and sequential immunoprecipitations with 1207 anti-CD36
rabbit serum, OKM1 mAb anti-CD11b, and GAP 8.3 mAb anti-CD45RA and B;
see ``Materials and Methods.'' The arrow indicates
the co-immunoprecipitated 33-kDa protein, the bracket indicates the mature CD36 protein, and the arrowhead indicates the gp74. Molecular mass markers are as indicated under
``Materials and Methods.''
p74 Is Antigenically Related to gp90-105 and Is Not
Expressed on the Plasma Membrane
Consistent with the results of Fig. 2b, similar amounts of p74 and gp90-105 are
detected by Mo91 in the lysates of THP-1 cells that were treated with
PMA for 48 h (Fig. 4, Total Lys). A rabbit antiserum
raised against CD36 molecules (Fig. 4, 1207), but not
preimmune serum (Fig. 4, Ctrl), is able to precipitate
both p74 and gp90-105, confirming that the two molecules are
antigenically related.
A 33-kDa Endo-H-sensitive Glycoprotein Is Associated with
the gp74 Protein Precursor
As evident from Fig. 3a, a protein with a molecular mass of 33 kDa
(p33) is coprecipitated by anti-CD36 in both U937 and THP-1 (lanes
1 and 6). The presence of this protein seems to correlate
with the inability of the 74-kDa precursor of being processed. While
the 33-kDa protein is no longer immunoprecipitable after 30 min of
chase in U937 cells (Fig. 3a, lanes 2 and 3) it is still detectable after 2 h of chase in THP-1 cells (Fig. 3a, lane 9).
gp33 Is Not a Degradation Product of gp74
The
co-immunoprecipitation of the gp33 might be explained by three
different hypotheses: (i) the rabbit serum anti-CD36 used for the
immunoprecipitation experiments contained antibodies against the gp33,
(ii) gp33 is a degradation product of gp74, and (iii) gp33 is a protein
that associates with gp74.
gp33 complexes, ()results not only in a
significant reduction of gp74 present in the immunoprecipitates, but
also in a similar reduction of gp33 (Table 1). As a control,
adsorption on platelets did not affect the efficiency of a rabbit serum
anti-IgM to immunoprecipitate radiolabeled mouse Ig µ-chain (Table 1).
)but does not alter the
posttranslational events that modulate the expression of CD36 on the
cell membrane in the two cell lines. At least two features seem to
retard the transport of CD36 to the surface of THP-1. First, transport
of the protein to the Golgi apparatus is severely impaired in these
cells, as indicated by the abundance of the endo-H-sensitive gp74 and
its slow conversion into the more mature forms. Second, as evident from
the pulse-chase experiments (Fig. 3) and confirmed by detergent
insolubility analyses (not shown), gp90-105 is degraded at a
faster rate in THP-1 cells.-granules and can be rapidly transported to the
plasma membrane upon activation(32) . In uninduced monocytic
cell lines, however, the intracellular pool of CD36 is small (Fig. 2). Moreover, the PMA-induced expression of CD36 is
dependent on RNA and protein synthesis, suggesting that the molecule is
neither recycled from intracytoplasmic stores, as observed for
platelets, nor does it represent a degradation product.- and -chains, degrading them in the endoplasmic
reticulum(38) . If multimeric proteins hence can be regulated
at the level of assembly, only the rate of folding would modulate the
transport of monomeric molecules. As most of the chaperones identified
so far that catalyze the folding of nascent proteins are ubiquitous,
abundant proteins, it is not easy to envisage a model that explains the
different rate of transport of CD36. The latter is thought to be a
monomeric receptor (see (1) ). However, in platelets the mature
CD36 transiently associates with proteins involved in transducing
signals (18) , while in endothelial cells, it is present as a
detergent-insoluble complex(39) . These interactions may be
responsible for the detergent ``insolubility'' of
gp90-105 observed in U937 and THP-1, but it is less likely that
they mediate the different processing of gp74. A conservation among species and cell types would suggest a
general role for this protein.
)))
The authors thank Dr. P. Dellabona for helpful
comments and suggestions, and Dr. G. A. Jamieson for critically reading
the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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