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J. Biol. Chem., Vol. 278, Issue 36, 34219-34225, September 5, 2003
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¶
From the
Departments of Internal Medicine and
Molecular and Biomedical Pharmacology,
University of Kentucky, Lexington, Kentucky 40536-0298
Received for publication, April 3, 2003 , and in revised form, June 12, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1–55) greatly reduced receptor protein
abundance. Retaining the six amino acids proximal to the membrane
(SR-A1–49) restored receptor protein
abundance. Although SR-A1–49 localized to
the cell surface, cells expressing this receptor failed to internalize the
ligand acetylated low density lipoprotein. Replacing the cytoplasmic tail of
SR-A with that of the transferrin receptor (TfR/SR-A) resulted in retention of
the chimeric receptor in the endoplasmic reticulum suggesting a specific role
for the membrane-proximal amino acids in trafficking SR-A from the endoplasmic
reticulum to the Golgi. Like SR-A expressing cells, cells expressing
SR-A1–49 displayed increased spreading and
adhesion, demonstrating that the membrane-proximal amino acids were sufficient
for SR-A-mediated cell adhesion. Together, our results indicate a critical
role for the membrane-proximal amino acids in SR-A trafficking and demonstrate
that SR-A-mediated adhesion and internalization require distinct cytoplasmic
domains. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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SR-A was originally identified on macrophages as the receptor that binds and internalizes modified lipoproteins via clathrin-coated pit-mediated endocytosis (6, 7). Consequently, most studies on SR-A have focused on its ability to internalize ligand. However, SR-A also mediates cell adhesion (8). Several components of the extracellular matrix, including modified types of collagen and certain proteoglycans present at sites of inflammation, have been identified as adhesion substrates for SR-A (9–12). Therefore, SR-A-mediated adhesion might play an important role in macrophage retention, specifically at sites of tissue injury.
It has not been defined whether SR-A-mediated adhesion results from an attempt to internalize an immobilized ligand or involves functional processes that are distinct from those involved in receptor internalization. Previous studies have indicated that SR-A-mediated adhesion and internalization have similar extracellular requirements (8–11). Consequently, it has been postulated that these functions involve a common mechanism (13). However, our recent results showed that clathrin does not co-localize with SR-A in the filopodia-like projections associated with the enhanced adhesion of SR-A expressing cells and this suggests that SR-A-mediated adhesion is a process that is distinct from that of receptor endocytosis (14).
In general, receptor-mediated endocytosis requires specific internalization motifs located within the cytoplasmic tail. For example, the transferrin receptor (TfR), which like SR-A, is a type II transmembrane receptor, has a well defined cytoplasmic motif (YTRF) that is required for receptor internalization (15). Previous studies with SR-A have identified specific amino acids in the cytoplasmic tail that are involved in regulating receptor internalization and cell-surface expression (13, 16, 17). However, the importance of cytoplasmic domains in SR-A-mediated adhesion has not been addressed. To investigate the role of the cytoplasmic tail in SR-A-mediated cell adhesion and to determine whether adhesion and internalization require distinct cytosolic domains, various SR-A constructs were stably expressed in human embryonic kidney (HEK 293) cells under the control of a tetracycline-inducible promoter.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1–55),
and SR-A lacking the first 49 amino acids of the cytoplasmic tail
(SR-A1–49) were amplified by PCR from SR-A
type II murine cDNA (GenBankTM accession number L04275
[GenBank]
). Deleting the
cDNA encoding the amino-terminal amino acids eliminated the translation start
codon. Therefore, the 5' PCR primers used to amplify
SR-A1–55 and
SR-A1–49 were designed to insert a
methionine translation start codon and a spacer alanine codon. To replace the
cytoplasmic tail of SR-A with that of the transferrin receptor (TfR/SR-A), the
cDNA encoding the amino-terminal 57 amino acids of the murine transferrin
receptor (GenBankTM accession X57349
[GenBank]
) was amplified by RT-PCR (Access
RT-PCR System, Promega, Madison, WI) from RNA isolated from mouse liver and
inserted 5' to the cDNA encoding the SR-A transmembrane and
extracellular domains. These cDNAs inserts were cloned into the pcDNA5/FRT/TO
expression vector (Invitrogen, Carlsbad, CA). The sequence of the inserts was
confirmed at the Macromolecular Structure Analysis Facility at the University
of Kentucky. The amino acid sequences of the receptor constructs are shown in
Table I.
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Generation of HEK Cells Expressing SR-A Constructs and Cell Culture—To generate cells that express the different SR-A constructs, we used the Flp-InTM T-RExTM system (Invitrogen). This system allows tetracycline-inducible expression after integration of the cDNA into a specific genomic site. An important advantage of this system is that similar levels of expression can be obtained upon induction of cells transfected with the different SR-A constructs. Flp-InTM T-RExTM HEK 293 host cells were used, as HEK 293 cells do not endogenously express SR-A and have been used previously to study SR-A function (14). HEK 293 host cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing penicillin (10 units/ml), streptomycin (10 µg/ml), heat-inactivated fetal bovine serum (10% fetal bovine serum; Invitrogen) (DMEM/fetal bovine serum), and the selection antibiotics blasticidin (15 µg/ml) and zeocin (100 µg/ml). Cells were co-transfected with pcDNA5/FRT/TO containing the SR-A constructs and pOG44 encoding Flp recombinase, which mediates insertion of pcDNA5/FRT/TO into the genomic integration site, using the TransIT-293 transfection reagent according to the manufacturer's protocol (Mi-rus, Madison, WI). Integration of pcDNA5/FRT/TO eliminates zeocin resistance and confers hygromycin B resistance. Thus, transfected cells were selected for stable integration of the SR-A constructs with hygromycin B (200 µg/ml). Receptor expression was induced by adding tetracycline to the culture media for 16 h unless stated otherwise. Concentration response studies showed that a concentration of 0.5 µg/ml tetracycline induced maximal protein expression (data not shown).
RT-PCR—Total RNA was isolated using the SV total RNA isolation system (Promega, Madison, WI). The Access RT-PCR system (Promega) was used to assess mRNA expression of the SR-A constructs. The primers used (5'-cgcgggaagcttgcaatggctgccctcattgctctc-3' and 5'-gggctcgagttatactgatcttgatccgcc-3') amplify a 897-bp product corresponding to the SR-A transmembrane and extracellular domains (SR-A nucleotides 171–1067) common to all SR-A constructs. The parameters for the thermocycle reaction were: 48 °C for 45 min, 94 °C for 2 min, and 40 cycles of 94 °C for 30 s, 60 °C for 60 s, and 68 °C for 120 s. Reactions performed in the absence of RNA or of reverse transcriptase were used as negative controls. RT-PCR products were analyzed by agarose gel electrophoresis and visualized following ethidium bromide staining using a Kodak Image Station 440.
Preparation of Cell Lysates—Cell lysates were prepared by incubating cells in lysis buffer (25 mM MES, 150 mM NaCl, 60 mM octylgluco-pyranoside, 1% Triton X-100, pH 6.4) for 30 min on ice. Protein concentrations were determined using bovine serum albumin as standard (DC Protein Assay, Bio-Rad).
Western Blotting—Cell lysates were resolved by SDS-PAGE on a 7.5% gel under reducing or non-reducing conditions and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). In some experiments, SR-A receptors were immunoprecipitated from cell lysates or culture media using the rat SR-A monoclonal antibody, 2F8 (3 µg/ml; Serotec, Raleigh, NC) and anti-rat IgG-coated magnetic beads. Receptor proteins resolved under reducing conditions were detected using a guinea pig antiserum developed to a fusion protein of the extracellular SR-A domain as described previously (18). To detect oligomeric forms of the receptors, cell lysates were resolved under non-reducing conditions and receptor proteins were detected with 2F8 (3 µg/ml, Serotec), which also recognizes an extracellular epitope of SR-A (18). Primary antibody binding was detected with horseradish peroxidase-labeled anti-guinea pig (1:10,000 dilution; Jackson, West Grove, PA) or anti-rat (1:5,000 dilution; Santa Cruz) secondary antibodies. Horseradish peroxidase activity was visualized by chemiluminescence (Pierce) using a Kodak Image Station 440.
N-Glycanase (PNGase F) and Endoglycosidase H Digestion—To
cleave all N-linked oligosaccharides, cell lysate protein (10 µg)
was digested with N-glycanase (PNGase F; Sigma) in buffer (50 µl:
50 mM NaHPO4, 0.1% SDS, 0.05 M
-mercaptoethanol, pH 7.5) according to the manufacturer's protocol.
Briefly, proteins were denatured by heating to 100 °C for 5 min. After
cooling, Triton X-100 (0.75%, v/v) and N-glycanase were added and the
reaction was incubated for 3 h at 37 °C. To cleave N-linked
precursor oligosaccharides, cell lysate protein (10 µg) was digested with
endoglycosidase H (EndoH, Sigma) in buffer (50 µl: 50 mM
NaHPO4, 0.1% SDS, 0.05 M -mercaptoethanol, pH 5.5)
according to the manufacturer's protocol, which was identical to that used for
N-glycanase digestion with the exception of the addition of Triton
X-100.
Tunicamycin and Brefeldin A Incubation—To inhibit N-linked glycosylation in the endoplasmic reticulum (ER), cells were cultured in the presence of tunicamycin (5 µg/ml, Sigma). To inhibit maturation of N-linked oligosaccharides in the Golgi apparatus, cells were cultured in the presence of brefeldin A (5 µg/ml, Sigma). These inhibitors were added 60 min before inducing cells with tetracycline and were present throughout induction. Cell lysates were prepared 6 h after addition of tetracycline.
Biotinylation of Cell-surface Proteins—Cells were cultured and induced in 6-well plates (0.5 x 106 cells/well), or to study the effect of cell adhesion on cell-surface localization of receptor, in ultra-low attachment plates (Costar, Corning, NY). Induced cells were trypsinized, washed, and cell-surface proteins were biotinylated in suspension (1 mg/ml EZ-Link sulfo-NHS-LC-Biotin in PBS, Pierce) at 4 °C for 30 min according to the manufacturer's instructions. To remove non-bound biotin, cells were washed with ice-cold Tris-buffered saline (pH 7.4) followed by PBS. Subsequently, cell lysates were prepared and biotinylated surface proteins were precipitated from lysates using streptavidin-coated magnetic beads. Cell-surface proteins (bead pellet) and intracellular proteins (supernatant) of the precipitation were resolved under reducing conditions by SDS-PAGE and SR-A was detected by Western blotting.
AcLDL Internalization—Cells were cultured and induced in
6-well plates (0.5 x 106 cells/well), or to study the effect
of cell adhesion on receptor-mediated ligand uptake, cells were cultured in
ultra-low attachment plates (Costar). Following incubation of induced cells in
serum-free DMEM for 2 h, fluorescently labeled AcLDL (2.5 µg/ml, Alexa
Fluor 488-AcLDL, Molecular Probes) was added to the media and incubation was
continued for another 2 h. Adherent cells were trypsinized, washed, and
resuspended in PBS (4 °C). AcLDL internalization was assessed by
quantifying cell-associated fluorescence (Flow Cytometry Core Facility,
University of Kentucky). Fluorescence was gated for individual live cells and
the fluorescence associated with 
20,000 cells was determined for each
sample. To assess nonspecific AcLDL cell association, polyinosine (10
µg/ml) was added 5 min before addition of fluorescently labeled AcLDL.
Microscopy—Cells were plated on 2-chamber (5 x 104 cells/chamber) LAB-TEK glass slides (Nalge Nunc International, Naperville, IL) and receptor proteins were detected as previously described (14). Briefly, cells were induced where indicated, washed twice with phenol-red free DMEM (37 °C), fixed in paraformaldehyde (4% w/v), and permeabilized with Triton-X (0.1% in PBS). Nonspecific binding sites were blocked with bovine serum albumin (1% in PBS). To detect expressed receptors, cells were incubated with 2F8 (3 µg/ml, Serotec), followed by incubation with an Alexa Fluor 488-labeled goat anti-rat antibody (2 µg/ml; Molecular Probes). Cell nuclei were stained using 4,5-diamidino-2-phenylindole (Molecular Probes). Cells were mounted in Mowiol embedding medium containing n-propyl gallate (1%, w/v) and dried overnight at 4 °C. Images were captured digitally by fluorescence microscopy with exposure times kept constant.
Cell Adhesion Assay—Cells were plated in 96-well plates (2 x 104 cells/well) in DMEM/fetal bovine serum and induced where indicated. Adhered cells were incubated in EDTA solution (0.2 g/liter, 37 °C) for 10 min to eliminate divalent cation-dependent adhesion. After washing with PBS (37 °C), cells remaining adhered were quantified using the CyQUANT assay (Molecular Probes) according to the manufacturer's instructions. Cell adhesion is expressed as a percentage of total cells plated.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1–55) greatly diminished SR-A protein
abundance compared with that of SR-A full-length expressing cells
(Fig. 1A). SR-A
protein was not detectable in the culture media indicating that the decreased
cellular SR-A1–55 protein abundance was not
because of secretion into the media (data not shown). In contrast to complete
deletion of the cytoplasmic tail, retaining the membrane-proximal six amino
acids by deleting amino acids 1 to 49
(SR-A1–49) restored protein abundance of
SR-A (Fig. 1A). SR-A
mRNA abundance was similar for all SR-A constructs
(Fig. 1B). Thus, our
results demonstrate that the six membrane-proximal amino acids are required
for post-transcriptional processing of SR-A.
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The Membrane-proximal Amino Acids of the Cytoplasmic Tail of SR-A Are
Sufficient for Cell-surface Localization but Not for Receptor
Internalization—To study the role of the membrane-proximal amino
acids in regulating SR-A function, we analyzed cell-surface localization of
SR-A1-49. Cell-surface localization of
SR-A1–49 was greater than that of
full-length SR-A (Fig. 2).
Surface localization of SR-A1–49 was
similar in cells grown adhered to tissue culture plates or in suspension
demonstrating that the increased surface localization of
SR-A1–49 was independent of SR-A-mediated
adhesion. To address the role of the membrane-proximal amino acids for SR-A
internalization, we examined internalization of the SR-A ligand AcLDL by
quantifying cell-associated fluorescence after incubation with fluorescently
labeled AcLDL for 2 h (Fig. 3). As shown previously, HEK 293 cells expressing full-length SR-A cells
efficiently internalized AcLDL
(14). However, cell-associated
fluorescence detected in cells expressing
SR-A1–49 was 14% of that detected in cells
expressing full-length SR-A (Fig.
3B). The small amount of fluorescence associated with
SR-A1–49 expressing cells most likely
reflects AcLDL binding to cell-surface receptors. The increased
cell-associated fluorescence observed in SR-A and
SR-A1–49 expressing cells was completely
blocked by the SR-A antagonist polyinosine demonstrating specificity for SR-A.
As shown for cell-surface localization, AcLDL internalization by cells
expressing SR-A1–49 was similar for both
adherent and suspended cells (Fig.
3C), demonstrating that the failure of this receptor to
internalize ligand was not the result of increased receptor-mediated adhesion.
It is likely that the lack of SR-A1–49
internalization results in the increased cell-surface localization of
SR-A1–49
(Fig. 2).
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The Membrane-proximal Amino Acids of SR-A Are Required for Trafficking
from the Endoplasmic Reticulum to the Golgi Apparatus—The ability
of the membrane-proximal amino acids to restore protein expression and
cell-surface localization suggest an important role for this motif in SR-A
trafficking. Previous studies have associated SR-A cell-surface localization
and receptor internalization with a common cytoplasmic motif
(17). However, the finding
that SR-A1–49 localized to the cell
surface, but did not internalize ligand, suggests that SR-A trafficking to the
cell surface and internalization have distinct cytoplasmic requirements. To
address the specific role of the membrane-proximal amino acids in SR-A
trafficking and whether an internalization motif is sufficient to mediate
cell-surface localization, we generated a chimeric receptor in which the
entire cytoplasmic tail of SR-A was replaced by 57 amino acids of the TfR
cytoplasmic tail (TfR/SR-A; Table
I) omitting a similar membrane-proximal domain. Like SR-A, the TfR
is a type II transmembrane receptor that mediates internalization via
clathrin-coated pits. However, whereas the cytoplasmic tail of SR-A is still
poorly characterized, the cytoplasmic tail of TfR has been studied extensively
and contains a well characterized internalization motif (YTRF; Ref.
15).
To study the trafficking and intracellular processing of the chimeric TfR/SR-A, we assessed the ability of TfR/SR-A to form oligomers by resolving cell lysates from induced cells under non-reducing conditions. TfR/SR-A was expressed and assembled into oligomers similar to SR-A. Monomers of TfR/SR-A and SR-A were detected at a molecular weight higher than predicted from the amino acid sequences (about 38 kDa) suggesting that both receptors were post-translationally modified (Fig. 4A). However, the apparent molecular weight of TfR/SR-A was lower than SR-A, indicating that the post-translational processing was different.
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To examine the post-translational processing of the SR-A receptor
constructs, we digested cell lysates prepared from induced cells with
N-glycanase (Fig.
4B), which cleaves all N-linked
oligosaccharides. The deglycosylated monomeric proteins were detectable at 38
kDa for both SR-A and the chimeric TfR/SR-A indicating that the differences in
apparent molecular weight resulted from differences in N-linked
oligosaccharides. To test whether the N-linked oligosaccharides of
the receptors differ in Golgi processing, lysates prepared from induced cells
were digested with EndoH (Fig.
4C). EndoH cleaves the high-mannose precursor
N-linked oligosaccharides attached in the ER, but not the low-mannose
mature oligosaccharides that are formed after processing in the Golgi. In
contrast to SR-A (Fig.
4C) and SR-A1–49 (data
not shown), TfR/SR-A (Fig.
4C) was EndoH-sensitive indicating that TfR/SR-A was not
processed into the EndoH-resistant mature form by the Golgi apparatus.
The EndoH sensitivity of TfR/SR-A suggests that the oligosaccharides of TfR/SR-A are analogous to that of the EndoH-sensitive SR-A precursor. To confirm this, cells were treated before induction either with brefeldin A, which disrupts the Golgi apparatus thereby preventing conversion of the precursor into the mature form, or with tunicamycin, an inhibitor of N-linked glycosylation (Fig. 4D). We found that the apparent molecular weight of the SR-A precursor detected in brefeldin A-treated cells was the same as that of TfR/SR-A in untreated cells but was greater than that of the unglycosylated receptor in tunicamycin-treated cells. Together, the data demonstrate that TfR/SR-A is translated and subsequently glycosylated in the ER, but the glycosylated precursor is not further processed in the Golgi apparatus. Because TfR/SR-A assembles into trimers, it seems unlikely that TfR/SR-A is retained in the ER because of misfolding of the protein. As expected, TfR/SR-A was not detected on the cell surface using the biotinylation protocol described above (data not shown).
Overall, our results from experiments studying the trafficking and intracellular processing of the different SR-A constructs demonstrate that an internalization motif is not sufficient to mediate trafficking to cell surface. Furthermore, the membrane-proximal amino acids of SR-A are sufficient for cell-surface localization and are specifically required for trafficking of SR-A from the ER to the Golgi apparatus.
The Membrane-proximal Amino Acids of SR-A Are Sufficient to Mediate
Cell Spreading and Adhesion—SR-A is involved in both ligand
internalization and cell adhesion. As we described previously, SR-A-mediated
cell adhesion involves an increase attachment of cells and distinct changes in
cell morphology (14). To
determine whether SR-A1–49 mediates cell
adhesion, we investigated the morphology of cells expressing the SR-A
constructs (Fig. 5). Consistent
with our previous findings
(14), SR-A expressing HEK
cells exhibited a cell morphology characterized by filopodia-like projections
and increased cell spreading (Fig.
5, panels A and B). Consistent with the results
indicating a lack of surface expression, cells expressing TfR/SR-A (panels
C and D) exhibited an intracellular immunostaining pattern and
no increased cell spreading. Interestingly,
SR-A1–49 expressing cells (panel E
and F) exhibited filopodia-like projections and increased cell
spreading that was similar to that observed for cells expressing full-length
SR-A.
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As the presence of the filopodia-like projections and increased cell
spreading are associated with increased cell adhesion of SR-A expressing
cells, we analyzed cell adhesion of cells expressing the different SR-A
constructs. Consistent with increased spreading of SR-A and
SR-A1–49 expressing cells, both receptors
were able to increase cell adhesion to the same extent
(Fig. 6), demonstrating that
the membrane-proximal amino acids are sufficient for SR-A-mediated adhesion.
As expected from the lack of surface expression, neither TfR/SR-A
(Fig. 6) nor
SR-A1–55 (not shown) increased cell
adhesion.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The trafficking and post-translational processing of SR-A have been
described previously (19). An
initial step in the post-transcriptional processing of SR-A is insertion of
the nascent protein into the membrane of the ER. The process whereby
transmembrane proteins are inserted into the ER membrane and adopt a type I
(carboxyl-terminal cytoplasmic tail) or type II (amino-terminal cytoplasmic
tail) topology is not fully understood
(20). It is generally thought
that type I and II transmembrane proteins are targeted to the ER and then
inserted and anchored in the membrane via a process involving signal-anchor
sequences located in the hydrophobic amino acids of the transmembrane domains.
However, we found that the transmembrane domain of SR-A
(SR-A1–55) was not sufficient for the
post-transcriptional processing of SR-A. Retaining the membrane-proximal amino
acids of the cytoplasmic tail (SR-A1–49)
restored intracellular processing and trafficking to the cell surface. Three
of the six retained SR-A membrane-proximal amino acids (KLKSFK) are positively
charged. Charged membrane-proximal amino acids have been shown to affect the
topology of transmembrane proteins
(21). Thus, the
membrane-proximal charged residues might be critical during ER membrane
insertion of SR-A. However, replacing the cytoplasmic tail of SR-A with that
of the transferrin receptor (TfR/SR-A), omitting a similar charged
membrane-proximal domain, was sufficient for protein expression,
glycosylation, and oligomerization in the ER, but not for ER to Golgi
trafficking. Therefore, the membrane-proximal amino acids of SR-A are
specifically required for trafficking from ER to the Golgi.
After translocation of the carboxyl-terminal extracellular domains into the ER, the monomeric SR-A protein is N-linked glycosylated and the glycosylated monomers assemble into trimers. These "precursor" SR-A trimers are transported through the Golgi apparatus, where further processing of the N-linked oligosaccharides converts the EndoH-sensitive precursor into the EndoH-resistant mature form that is expressed on the cell surface. The mechanism by which proteins traffic from the ER to Golgi remains controversial (22). The bulk-flow model proposes that proteins are transported from the ER to the Golgi by default. In contrast, the selective export model proposes that proteins are selectively exported based on the presence of ER-export signals (23). Several cytosolic proteins are involved in trafficking of newly synthesized transmembrane proteins. Thus, the membrane-proximal amino acids might provide a critical recognition site for proteins involved in selective ER export.
Although SR-A1–49 was expressed
primarily on the cell surface, SR-A1–49
failed to internalize ligand. A well defined internalization motif is not
present in the cytoplasmic tail of SR-A. However, Morimoto et al.
(17) have suggested that a
single motif, VXFD, is required for both SR-A internalization and
cell-surface localization. In contrast, our results show that the
membrane-proximal amino acids are sufficient for cell-surface localization,
but that receptor internalization depends on a distinct motif, possibly the
VXFD motif.
It has been hypothesized that SR-A-mediated adhesion and internalization are regulated by the same mechanism and that SR-A-mediated adhesion results from the attempt to internalize an immobilized ligand (13). The inability to separate SR-A-mediated adhesion from ligand internalization using various approaches supports this hypothesis (8–11). For example, structure-function studies have shown that SR-A-mediated adhesion depends on the same region in the extracellular collagen-like domain that mediates binding of soluble ligands during internalization (11). In contrast to data supporting a common mechanism for adhesion and internalization, we recently showed that clathrin does not co-localize with SR-A in the filopodia-like projections associated with the enhanced adhesion of SR-A expressing cells (14). This observation provided indirect evidence that SR-A-mediated adhesion is a process that is distinct from clathrin-coated pit internalization. The results of the current study demonstrating that SR-A-mediated adhesion and internalization require distinct domains of the receptor provide direct evidence to support this hypothesis.
The mechanism by which SR-A mediates cell adhesion remains unclear. It has
been shown that SR-A-mediated adhesion involves changes in the actin
cytoskeleton and the formation of focal adhesions. The ability of
SRA1–49 to enhance cell adhesion suggests
that the membrane-proximal amino acids of SR-A are sufficient to mediate these
processes. Likewise, the cytoplasmic membrane-proximal amino acids of the
adhesion molecule L1 have been shown to be sufficient for association of this
receptor with the cytoskeleton
(24). Thus, the
membrane-proximal amino acids of SR-A might be a binding site for cytosolic
proteins involved in formation of focal adhesions.
SR-A-mediated adhesion may have important physiological roles, particularly
at sites of tissue inflammation. For example, we recently demonstrated a role
of SR-A-mediated adhesion in an in vivo model of inflammation using
SR-A overexpressing mice (25).
Macrophage-specific SR-A overexpression enhanced granuloma formation after
subcutaneous injection of carrageenan. Enhanced granuloma formation was
associated with an increase in macrophage numbers suggesting that SR-A
enhanced macrophage recruitment and/or retention. SR-A mediates adhesion
specifically to modified extracellular matrix proteins such as collagen types
I, III, and IV (9,
10). Such modifications of the
extracellular matrix may occur during certain pathophysiological conditions
such as hyperglycemia or inflammation. Recently, SR-A also has been shown to
mediate adhesion to proteoglycans of the extracellular matrix that are present
in atherosclerotic lesions
(12). Furthermore,
SR-A-mediated adhesion of microglial cells to -amyloid fibrils might
play a role in Alzheimer's disease
(26). Thus, SR-A-mediated
adhesion might be a mechanism of macrophage retention specifically at sites of
tissue injury associated with inflammation.
In summary, we have shown that the cytoplasmic amino acids proximal to the
membrane are required for SR-A posttranslational processing and play a
critical role in SR-A trafficking to the cell surface. Furthermore, these
amino acids were sufficient for SR-A-mediated adhesion, but not for ligand
internalization. These are the first data to demonstrate that SR-A-mediated
adhesion and internalization require distinct cytoplasmic domains and
therefore must be distinct cellular processes. The development of a SR-A
mutant such as SRA1–49 that specifically
mediates cell adhesion will make it possible to specifically study the role of
SR-A-mediated adhesion in various physiological and pathophysiological
processes.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dept. of Molecular and Biomedical Pharmacology, UK Medical Center-MS305, Lexington, KY 40536-0298. Tel.: 859-323-3996 (ext. 293); Fax: 859-257-9166; E-mail: spost{at}uky.edu.
1 The abbreviations used are: SR-A, Class A scavenger receptor; AcLDL,
acetylated low density lipoprotein; EndoH, endoglycosidase H; ER, endoplasmic
reticulum; HEK, human embryonic kidney; RT, reverse transcriptase; TfR,
transferrin receptor; DMEM, Dulbecco's modified Eagle's medium; PBS,
phosphate-buffered saline; MES, 4-morpho-lineethanesulfonic acid.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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 Y. Chen, X. Wang, J. Ben, S. Yue, H. Bai, X. Guan, X. Bai, L. Jiang, Y. Ji, L. Fan, et al. The Di-Leucine Motif Contributes to Class A Scavenger Receptor-Mediated Internalization of Acetylated Lipoproteins Arterioscler. Thromb. Vasc. Biol., June 1, 2006; 26(6): 1317 - 1322. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. B. Fulton, R. H. Reeves, M. Takeya, and A. De Maio A Quantitative Trait Loci Analysis to Map Genes Involved in Lipopolysaccharide-Induced Inflammatory Response: Identification of Macrophage Scavenger Receptor 1 as a Candidate Gene J. Immunol., March 15, 2006; 176(6): 3767 - 3773. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ricci, G. Sumara, I. Sumara, I. Rozenberg, M. Kurrer, A. Akhmedov, M. Hersberger, U. Eriksson, F. R. Eberli, B. Becher, et al. Requirement of JNK2 for Scavenger Receptor A-Mediated Foam Cell Formation in Atherogenesis Science, November 26, 2004; 306(5701): 1558 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jozefowski and L. Kobzik Scavenger receptor A mediates H2O2 production and suppression of IL-12 release in murine macrophages J. Leukoc. Biol., November 1, 2004; 76(5): 1066 - 1074. [Abstract] [Full Text] [PDF]
|
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