1 Departments of Biochemistry and 2 Medicine, Division of Gastroenterology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In polarized cells, the delivery of numerous membrane proteins from the trans-Golgi network to the basolateral surface depends on specific sequences located in their cytoplasmic domain. We have previously shown that the insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) exhibits a polarized cell surface distribution in the human colon adenocarcinoma (Caco-2) cell line in which there is a threefold enrichment on the basolateral surface. To investigate the role of residues in the cytoplasmic region of the receptor that facilitates its entry into the basolateral sorting pathway, we generated stably transfected Caco-2 cell lines expressing various mutant bovine IGF-II/MPRs. The steady-state surface distribution of mutant receptors was analyzed by subjecting filter-grown cell monolayers to incubation with iodinated IGF-II/MPR-specific antibody or to indirect immunofluorescence and visualization by confocal microscopy. Together, these results demonstrate that the sorting of the IGF-II/MPR to the basolateral cell surface depends on recognition of sequences located in its cytoplasmic region that are distinct from the Tyr-based internalization and dileucine-dependent endosomal trafficking motifs.
intracellular trafficking; insulin-like growth factor receptor; polarized cells
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
EPITHELIAL CELLS CARRY OUT a variety of vectorial transport and secretory processes that depend on the polarized distribution of proteins and lipids on their cell surface. The plasma membrane of these cells is divided into two morphologically, functionally, and biochemically distinct cell surface domains: 1) an apical domain that faces the exterior of the organism and 2) a basolateral domain that faces the internal environment. Epithelial cells are able to selectively direct newly synthesized membrane or secretory proteins to either of these domains, and polarity is maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways (48). Sorting signals that specify basolateral surface expression have been localized to the cytoplasmic domain of numerous membrane proteins (22, 29, 44). A number of these basolateral sorting signals are colinear with the signals for coated pit localization and belong to two subgroups: 1) an essential tyrosine residue in the context of NPXY or YXXØ (where Ø is a bulky hydrophobic residue) (28, 41, 42) and 2) a dileucine motif (13, 18, 41, 43). In addition to directing basolateral expression, signals from both classes have been shown, in some cases, to also function in endocytosis from the plasma membrane and in mediating lysosomal sorting from the trans-Golgi network (TGN) to endosomes and lysosomes (18, 29, 43).
The 300-kDa insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a multifunctional protein that delivers newly synthesized lysosomal enzymes to the lysosome and regulates the circulating levels of IGF-II by mediating its uptake and lysosomal degradation. In higher eukaryotic cells, newly synthesized soluble acid hydrolases acquire mannose 6-phosphate (Man-6-P) residues on their N-linked oligosaccharides. In the Golgi, phosphomannosyl residues serve as high-affinity ligands for binding to the IGF-II/MPR. The removal of acid hydrolases from the secretory pathway occurs when the receptor-lysosomal enzyme complex enters into clathrin-coated pits and vesicles for delivery from the TGN to an acidified late endosomal compartment. The acidic pH of this compartment induces the complex to dissociate. Released lysosomal enzymes are then delivered to lysosomes, whereas the receptors either return to the Golgi to repeat the process or move to the plasma membrane where the IGF-II/MPR functions to internalize extracellular ligands via a recapture pathway (7, 23, 33, 45). Numerous studies have been performed to identify those signals in the cytoplasmic region of the IGF-II/MPR that mediate its intracellular trafficking in nonpolarized cells. A conserved casein kinase II site followed by a dileucine motif (DDSDEDLL) at the COOH terminus of the IGF-II/MPR is important for sorting lysosomal enzymes to the lysosome (5, 6, 21, 27), whereas an aromatic-based sequence (YKYSKV) located 24-29 residues from the transmembrane region is essential for rapid internalization of the receptor (4, 20, 27).
Our recent studies have demonstrated that in the human intestinal epithelial cell line Caco-2, the steady-state polarized secretion of lysosomal enzymes into the apical medium is facilitated by the IGF-II/MPR selectively endocytosing lysosomal enzymes from the basolateral surface (47). We have previously demonstrated that the IGF-II/MPR is enriched threefold on the basolateral surface relative to its expression on the apical cell surface (9). This polarized surface expression of the IGF-II/MPR to the basolateral surface is not limited to Caco-2 cells, because studies by Prydz et al. (40) report that the IGF-II/MPR could be detected on the basolateral, but not the apical, surface of Madin-Darby canine kidney (MDCK) cells. However, nothing is known about the structural determinants of the receptor that mediate its polarized distribution in epithelial cells. To determine whether sequences in the cytoplasmic region of the IGF-II/MPR serve as a targeting signal to the basolateral cell surface, mutant forms of the bovine IGF-II/MPR containing truncations in its COOH-terminal cytoplasmic region were expressed in polarized human Caco-2 cells and the surface distribution of the bovine MPRs was determined. Our results demonstrate that residues essential for entry of the IGF-II/MPR into the basolateral sorting pathway are located within residues 36-50 of the cytoplasmic domain and are distinct from both the aromatic-based motif required for rapid internalization and from the dileucine motif required for endosomal sorting.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials. The following reagents were obtained commercially as indicated: EXPRE35S35S 35S-protein labeling mix (1,200 Ci/mmol, NEN Life Science); fetal bovine serum (FBS; HyClone Laboratories); DMEM and trypsin-EDTA (GIBCO-BRL Life Technologies); and protein A-Sepharose, glucose 6-phosphate (Glc-6-P), and Man-6-P (Sigma). Caco-2 cells were kindly provided by Dr. Ward Olsen of Veterans Administration Hospital (Madison, WI).
Cell culture. Caco-2 cells (passages 76-96) were grown in DMEM (25 mM glucose) supplemented with 20% heat-inactivated FBS, 4 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 5% CO2. To determine the cell surface distribution of the IGF-II/MPR, cells were grown as epithelial layers by high density seeding (3.4 × 105 cells/cm2) onto Transwell polycarbonate membrane filter inserts (Costar). The formation and integrity of monolayers were assessed by the development of a significant transepithelial electrical resistance of 250-300 ohms/cm2 over the resistance of the filter alone. Resistance readings were measured with a Millicell-ERS Voltohmeter (Millipore). All polarity studies were performed >10 days after plating.
Transfection of Caco-2 cells. Generation of the bovine wild-type and mutant (YAYA, Val8, Asn21, Leu75, and Arg124) IGF-II/MPR constructs was described previously (27). The construct encoding the Asp36 mutant IGF-II/MPR was described previously (4). A mutant IGF-II/MPR lacking the COOH-terminal 113 residues was generated by substituting Ala51 of the cytoplasmic region with a stop codon using a single-step polymerase chain reaction-based method [forward primer, 5'-CGTCCATCACGGGCTCCAGCA3'; reverse mutagenic primer, 5'-CGACGCGTTCATCACGGCGGCTGGATCTCCTCCATC3' (stop codon underlined)]. The stop codon at Ala51 was followed by a second stop codon and an Mlu I site. The resulting PCR product was digested with BsrG I and Mlu I and cloned into the corresponding sites of the IGF-II/MPR. The region of the construct synthesized by PCR was confirmed by DNA sequencing. These constructs were placed in the SFFV-neo vector (14) that uses the Friend spleen focus-forming virus 5'-long terminal repeat to promote transcription of the cDNA and also contains the neomycin resistance gene that confers resistance to the antibiotic G418. Caco-2 cells were seeded in 100-mm dishes and transfected 48 h after plating with 35 µg of Xba I-linearized plasmid DNA using a modification of the calcium phosphate method. Briefly, precipitates were formed by adding equal volumes of DNA in 250 mM CaCl2 and 2× HeBS buffer [1× HeBS consists of (in mM) 140 NaCl, 5 KCl, 0.75 Na2HPO4, 6 dextrose, and 25 HEPES, pH 7.05] and the mixture was incubated at room temperature for 30 min. The precipitated DNA was added to Caco-2 cells in DMEM, and cells were incubated for 6 h at 37°C. Cells were then treated with 15% DMSO in HeBS for 1 min, rinsed, and then placed in DMEM containing 20% FBS. After 72 h, cells were passaged and seeded to 100-mm dishes using serial dilutions. Selection was started 48 h later using 500 µg/ml of G418 sulfate (GIBCO-BRL Life Technologies) and continued for 10-14 days. Clones were isolated either by using cloning rings or by serial dilution in 96-well plates, and cells were grown in the continuous presence of 350 µg/ml G418. The expression level of the recombinant bovine IGF-II/MPR in each of the clones was determined by quantitative Western blot analysis (see below).
Metabolic labeling. Caco-2 cells were starved for 15 min in DMEM lacking methionine and cysteine (GIBCO-BRL Life Technologies) containing 10% heat-inactivated FBS (DMEM-FBS). Cells were then incubated in DMEM-FBS containing EXPRE35S35S 35S-protein labeling mix (50 µCi/ml) for 19 h. Cells were solubilized for 1 h on ice in buffer containing 0.1 M Tris, pH 8.0, 0.1 M NaCl, 10 mM EDTA, Triton X-100 (1% vol/vol), sodium deoxycholate (0.1% wt/vol), aprotinin (1% vol/vol), antipain (4 µg/ml), benzamidine (20 µg/ml), and 2 µg/ml each of leupeptin, chymostatin, and pepstatin. The IGF-II/MPR was purified from the resulting cell lysates by pentamannosyl phosphate-agarose affinity chromatography (8, 17). Protein-encoding domains 1-13 of the extracytoplasmic region of the bovine IGF-II/MPR was purified from transiently transfected COS-1 cells metabolically labeled with EXPRE35S35S 35S-protein labeling mix as described previously (10).
Immunoprecipitations. Purified IGF-II/MPRs were incubated at 4°C for 16-24 h with protein A-Sepharose plus anti-IGF-II/MPR polyclonal antibody (B2.5) or monoclonal antibodies (32g, 56f, 70h, and 86f7) plus rabbit anti-mouse IgG. IGF-II/MPRs were purified from [35S]methionine-labeled cells by pentamannosyl phosphate-agarose affinity chromatography (8, 17). After recovery by centrifugation, the protein A-Sepharose beads were washed four times with buffer containing 0.1 M Tris (pH 8.0), 0.1 M NaCl, 10 mM EDTA, and 1% Triton X-100 and washed once in buffer containing 20 mM Tris (pH 8.0) and 20 mM NaCl. Bound proteins were eluted by the addition of Laemmli sample buffer and analyzed on 7.5 or 9% SDS polyacrylamide gels under reducing conditions. The radiolabeled bands were analyzed using a PhosphorImager (Storm 860; Molecular Dynamics) with ImageQuant (version 4.1) software.
Western blot analysis. Caco-2 cells were solubilized for 1 h on ice in buffer containing 0.1 M Tris, pH 8.0, 0.1 M NaCl, 10 mM EDTA, Triton X-100 (1% vol/vol), sodium deoxycholate (0.1% wt/vol), aprotinin (1% vol/vol), antipain (4 µg/ml), benzamidine (20 µg/ml), and 2 µg/ml each of leupeptin, chymostatin, and pepstatin. The total amount of protein in the resulting cell lysate was determined using the Bradford protein assay as recommended by the manufacturer (Bio-Rad). The resulting cell lysates were subjected to quantitative Western blot analysis as described previously (51), except that, after incubation with the 86f7 monoclonal antibody, the membranes were probed with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce) and the proteins were visualized using enhanced chemiluminescence (ECL) as recommended by the manufacturer (Pierce). Bands were quantified using an Ambis radioanalytical imaging system.
Surface binding and internalization of the monoclonal antibody
86f7.
86f7 hybridoma media was adjusted to 50% ammonium sulfate, and after
centrifugation, the concentrated antibody was dialyzed against PBS.
86f7 antibody was iodinated using IODO-GEN (Pierce) to a specific
activity of 5-13 µCi/µg. Transfected Caco-2 cells were grown
on 12- or 24-mm Costar Transwell filters for 10-14 days. Cells
were rinsed with binding buffer (PBS containing 0.1 mM
CaCl2, 1.0 mM MgCl2, and 0.5% BSA), and
iodinated 86f7 was then added to the apical or basolateral surface at a
concentration of 0.5-1.0 µg/ml in serum-free DMEM containing 1% BSA
and incubated for 2 h at 37°C. After cells were chilled to 4°C
in an ice-water bath, media was removed from both surfaces and counted
to confirm the integrity of the monolayer. Media was then combined and
an aliquot was precipitated with 10% trichloroacetic acid and compared with a control for nonprecipitable counts. Cells were then washed three
times each with ice-cold binding buffer and wash buffer (binding buffer
without BSA). Surface-bound 86f7 was removed by washing in ice-cold low
pH buffer (50 mM glycine, pH 2.8, 150 mM NaCl), rinsed with wash
buffer, and cells were removed from the filters by treatment with 0.1 N
NaOH. Total protein was determined from the cell lysate by the Lowry
method (Bio-Rad DC protein assay). Radioactivity in the trichloroacetic
acid-soluble media, low pH wash, and cell lysates were counted in a
-counter and normalized to total protein to give degraded, surface,
and internalized values, respectively. Nontransfected Caco-2 cells were
used as a control for nonspecific binding. An internalization index was
determined by the sum of internalized and degraded values divided by
the surface-bound values and serves to normalize for variations in the
level of receptor expression among cell lines.
Surface binding and internalization of -glucuronidase.
To determine surface ratios and extent of internalization of the
endogenous human IGF-II/MPR, similar experiments as described above
were performed on nontransfected Caco-2 cells except that the iodinated
lysosomal enzyme
-glucuronidase (2.0 nM) was used in place of the
monoclonal antibody 86f7.
-glucuronidase was purified and
iodinated as described previously (9). Nonspecific binding
was determined by incubation in the presence of 10 mM Man-6-P. To
determine the amount of surface-bound ligand, cells were washed with 10 mM Glc-6-P (nonspecific ligand) followed by 10 mM Man-6-P (specific ligand).
Steady-state surface distribution of the IGF-II/MPR.
Transfected Caco-2 cells were grown in 12- or 24-well tissue culture
plates to postconfluence. On days 7 and 8 of
growth, cells were washed and chilled to 4°C with binding buffer and
incubated with 0.5-0.65 µg 125I-86f7 in binding
buffer for 3 h at 4°C to measure the amount of receptor on the
cell surface. To determine total IGF-II/MPR levels, parallel wells were
incubated with 125I-86f7 in the presence of 0.1%
saponin. Cells were washed three times each with ice-cold binding
buffer and wash buffer and then harvested in 0.1N NaOH. Radioactivity
was measured in a -counter and normalized to total protein. The
amount of cell surface receptors (measured in the absence of saponin)
was calculated as a percentage of the total receptors (measured in the
presence of saponin). Nontransfected cells were used as a control for
nonspecific binding.
Confocal microscopy. Transfected Caco-2 cells were grown on 12-mm Costar Transwell polycarbonate filters (pore size, 0.4 mm) for 10 days before immunostaining. Cells were fixed as described previously (13) in a 1:9 methanol/acetone solution followed by rehydration with PBS. A 1:200 dilution of the monoclonal antibody 86f7 (specific for the bovine IGF-II/MPR) was added to both the apical and basolateral surfaces for 1 h at room temperature. After washing with PBS, both surfaces were incubated with 1:100 dilution of a fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Pierce) for 1 h at room temperature. Both antibodies were diluted in PBS containing 3% goat serum. Nontransfected cells were incubated with the polyclonal antibody B2.5 (raised against bovine liver IGF-II/MPR) followed by fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Pierce). After washing, the filters were removed and mounted in VECTASHIELD (Vector Laboratories). Images were obtained with a Bio-Rad MRC 600 laser scanning confocal imaging system mounted on a Nikon Optiphot microscope using a 100× objective. Data analysis was accomplished using MetaMorph software.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Expression of the bovine IGF-II/MPR in human Caco-2 cells.
To distinguish recombinant bovine IGF-II/MPRs expressed in Caco-2 cells
from the endogenous human receptor, species-specific antibodies were
identified. We obtained four monoclonal antibodies generated against
the bovine IGF-II/MPR. To determine whether these antibodies recognize
the human IGF-II/MPR produced by Caco-2 cells, Caco-2 cells were
metabolically labeled with [35S]methionine and the
IGF-II/MPR was purified by pentamannosyl phosphate-agarose
affinity chromatography. [35S]methionine-labeled protein
encompassing domains 1-13 of the extracytoplasmic
region of the bovine IGF-II/MPR was used as a positive control
(10). Equal aliquots of either the purified Caco-2
IGF-II/MPR or the purified bovine IGF-II/MPR (domains
1-13) were incubated with various antibodies plus protein
A-Sepharose. Figure 1A shows
that the four monoclonal antibodies (32g, 56f, 70h, and 86f7)
efficiently recognized the purified bovine IGF-II/MPR encompassing
domains 1-13 but were unable to
immunoprecipitate the purified human Caco-2 IGF-II/MPR. In contrast,
the polyclonal antibody B2.5 efficiently recognized both the human
Caco-2 and bovine IGF-II/MPRs. As a control, the supernatants from
samples in Fig. 1A lanes 1, 3-7, 9, and 15 were
reimmunoprecipitated with the polyclonal antibody B2.5 to demonstrate
that intact Caco-2 or bovine IGF-II/MPR were, in fact, present in these
samples (Fig. 1B). These results demonstrated that the
monoclonal antibodies 32g, 56f, 70h, and 86f7 exhibit
species-specificity in that they recognize the bovine, but not the
human, IGF-II/MPR. In addition, the observation that these monoclonal
antibodies precipitate a construct that lacks the cytoplasmic region of
the IGF-II/MPR (construct encodes domains 1-13 of the
extracytoplasmic region, see Fig. 1) demonstrates that mutations placed
in the cytoplasmic region of the receptor will not inhibit antibody
recognition. We have previously mapped the epitope of the 32g, 56f, and
86f7 monoclonal antibodies to domain 5 of the extracytoplasmic region of the IGF-II/MPR (3). Thus
the use of these monoclonal antibodies allowed for further specific
analyses of recombinant bovine IGF-II/MPRs expressed in Caco-2 cells.
|
|
|
Cell surface expression of the IGF-II/MPRs.
The steady-state cell surface distribution of the recombinant
IGF-II/MPRs was measured. The percentage of each construct on the cell
surface (representing apical plus basolateral surfaces) was determined
by incubating the stably transfected Caco-2 cells with iodinated
86f7 monoclonal antibody in the absence or presence of
saponin. In the absence of saponin, only the receptors on the cell
surface will be recognized in the intact cells, whereas in the presence
of saponin, which partially permeabilizes membranes, the total receptor
population is available for antibody recognition. Table
1 shows that the percentage of the
Leu75, Arg124, and YAYA receptors on the cell
surface is similar to that of the wild-type receptor. In contrast,
Val8 and Asn21 constructs express a
significantly higher percentage (62 and 47%, respectively) of receptor
on the cell surface at steady state than does the wild-type receptor,
which is consistent with the loss of the endocytosis motif
(Y24KYSKV29) in these constructs (see Fig. 2).
|
Polarized surface distribution and internalization.
To determine whether the mutant receptors maintained their polarized
distribution, the iodinated 86f7 monoclonal antibody was added
to either the apical or basolateral medium of Caco-2 cells grown on
filter inserts, and incubations were carried out at 37°C to measure
the ability of the receptor to undergo endocytosis from the cell
surface followed by a 4°C incubation to measure surface expression of
the receptor. The results demonstrate that the recombinant wild-type
bovine receptor exhibits a similar polarized surface distribution and
internalization to the endogenous IGF-II/MPR (Table 1).
Leu75, Arg124, and YAYA constructs are similar
to the wild-type receptor in their basolateral enrichment on cell
surface. In addition, Leu75 and Arg124
constructs internalize antibody from the basolateral surface to a
similar extent as the wild-type receptor. In contrast, the YAYA
construct exhibits a decreased level of internalization from the
basolateral surface, consistent with alteration of the endocytosis signal in this mutant (see Fig. 2). Additional truncations of the
cytoplasmic domain (Val8 and Asn21) result in a
dramatic change in the distribution of the receptor on the cell surface
compared with that of the wild-type receptor: both Val8 and
Asn21 constructs are expressed predominantly at the apical
cell surface, and limited internalization is observed (Table 1), which
is again consistent with the loss of the endocytosis signal in these
mutants (Fig. 2). These results were confirmed using the
Man-6-P-containing ligand, -glucuronidase: Val8 and
Asn21 constructs differed dramatically from the wild-type
receptor in that little [125I]
-glucuronidase was
internalized from the basolateral surface (data not shown).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IGF-II/MPR mediates the lysosomal targeting of Man-6-P-containing soluble acid hydrolases and IGF-II. Although much information is available concerning the sorting pathways traversed by the IGF-II/MPR in the targeting of lysosomal enzymes to the lysosome in nonpolarized cells (34) and in the uptake of IGF-II, which has been shown to function as an autocrine growth factor in intestinal epithelial cells (49, 50, 52), very little is known about the trafficking of the receptor and its ligands in polarized epithelial cells. Our recent studies have shown that the IGF-II/MPR exhibits a polarized plasma membrane distribution with a threefold enrichment on the basolateral surface of Caco-2 cells (9). In addition, we have found that the IGF-II/MPRs expressed on the two cell surfaces are functionally distinct: unlike the receptor on basolateral membranes, the IGF-II/MPR on the apical surface cannot endocytose lysosomal enzymes (9). Furthermore, we have shown that the secretion-recapture pathway in which the IGF-II/MPR internalizes secreted lysosomal enzymes from the basolateral surface plays a critical role in establishing the steady-state polarized distribution of phosphorylated lysosomal enzymes (enrichment of secretion into the apical medium) in intestinal epithelial cells (47). To begin to understand the mechanism underlying this functional difference, we have investigated the sorting of the IGF-II/MPR in polarized Caco-2 cells by expressing mutant forms of the receptor in stably transfected cells and monitoring their steady-state surface distribution and cell surface internalization. Our observation that the bovine IGF-II/MPR when expressed in human Caco-2 cells exhibits a similar trafficking pattern to the endogenous receptor, coupled with our ability to distinguish the recombinant MPRs from the endogenous receptor, has made this approach a viable system to evaluate the effects that a specific region of the IGF-II/MPR has on its polarized distribution in intestinal epithelial cells.
Numerous studies have illustrated that sorting of membrane proteins to the basolateral plasma membrane is determined by the presence of specific residues in the cytoplasmic domain that serve as sorting signals (12, 13, 16, 41, 42). These cytoplasmic basolateral targeting signals are typically found to be dominant over apical sorting determinants. Evidence for this conclusion comes from the observation that mutant forms of basolateral membrane proteins lacking their cytosolic signals are sorted predominantly to the apical surface (19, 32, 39). We have observed a similar finding with the IGF-II/MPR: deletion of all but 7 (Val8 construct), 20 (Asn21 construct), or 35 (Asp36 construct) residues of the receptor's 163 residue cytosolic region resulted in a redistribution from the basolateral surface to nearly exclusive expression on the apical surface (Table 1 and Figs. 5 and 6). A comparison of the Asp36, Ala51, Leu75, and Arg124 constructs revealed that the removal of 128, but not 113, 89, or 40, amino acids from the COOH terminus of the receptor resulted in the loss of basolateral expression and the predominant polarized distribution of the receptor at the apical cell surface (Table 1 and Figs. 5 and 6). These results demonstrate that amino acids contained within the region of the cytoplasmic domain encompassing residues 36-50 are essential for entry of the IGF-II/MPR into the basolateral sorting pathway. The cytosolic tail of the IGF-II/MPR has been shown to be phosphorylated at Ser85 and Ser156 (30) and contains a conserved casein kinase II site followed by a dileucine motif (D154DSDEDLLHV163) at the COOH terminus as well as an aromatic-based sequence (Y24KYSKV29) located 24-29 residues from the transmembrane region that has been shown to be important for rapid internalization of the receptor (27) (see Fig. 2). Analysis of a mutant in which the tyrosine residues at positions 24 and 26 were replaced with alanine residues (YAYA construct) revealed no significant alteration in the steady-state polarized surface distribution of the receptor (Table 1 and Fig. 4). These results demonstrate that the IGF-II/MPR basolateral sorting signal differs from the class of known basolateral sorting signals that are tyrosine based, because Tyr24 and Tyr26 are the sole tyrosine residues in the cytosolic region of the bovine IGF-II/MPR (25). In addition, our preliminary data (D. A. Wick and N. M. Dahms, unpublished data) show that Val29, but not Asn30, is critical for internalization of the receptor in Caco-2 cells, which is in agreement with previous studies (4). Analysis of the Asp36 mutant revealed that Val29 and N30 are not involved in the basolateral targeting signal (Table 1 and Fig. 6). Together, IGF-II/MPR contains a basolateral sorting signal that is not colinear with its phosphorylation sites, Tyr-based internalization signal, or dileucine motif known to be required for lysosomal enzyme targeting via endosomal compartments (5, 6, 21, 27), and thus IGF-II/MPR is a member of a growing number of proteins (2, 16, 24, 35, 37) that contain basolateral signals that differ from Tyr- and Leu-based internalization motifs.
The sequence D36ENETEWLMEEIQPP50 located within the cytoplasmic domain of the IGF-II/MPR, which this study has identified as being critical for the basolateral surface expression of the receptor, is highly conserved; a comparison of the bovine, human, rat, mouse, and chicken sequences reveals changes only at positions 36 (Asn in chicken), 48 (Ala in chicken), and 49 (not conserved). On analysis of basolateral targeting sequences found in other proteins, this 15-residue region of the IGF-II/MPR bears a striking similarity to the basolateral targeting motif recently identified for the transmembrane growth factor, stem cell factor (SCF) (46). Within the 36-residue cytoplasmic domain of SCF, a single leucine residue is required for basolateral targeting of SCF, and the presence of an acidic cluster NH2 terminus to the leucine residue enhances the efficiency of basolateral sorting [K1KKQSSLTRAVENIQINEEDNEISMLQQKEREFQEV36 (critical residues underlined)]. Unlike the invariant chain that uses Met-Leu as a dihydrophobic basolateral sorting signal (36), substitution of the methionine residue adjacent to the critical leucine residue did not affect basolateral targeting of SCF (46). Thus the SCF basolateral targeting signal represents a novel motif in that a monomeric, rather than a dimeric, leucine residue mediates basolateral sorting. Additional studies will be required to determine whether the IGF-II/MPR utilizes a basolateral targeting motif similar to that of SCF or the invariant chain.
A recent study by Distel et al. (12) has shown that the cation-dependent MPR (CD-MPR) contains a basolateral sorting determinant in its cytoplasmic region that differs from the signals known to be involved in its internalization from the cell surface and in its sorting of lysosomal enzymes to the lysosome. The authors identified the 12 amino acids (QRLVVGAKGMEQ) juxtaposed to the transmembrane region as being essential for basolateral targeting of the CD-MPR in MDCK cells. The authors hypothesized that the RXXV sequence within this region of the CD-MPR is of particular importance, because a similar motif has been shown to be important for basolateral delivery of a truncated version of the poly-Ig receptor (1). In addition, the authors state that a similar motif is present near the transmembrane region of the IGF-II/MPR (REMV located 5-8 residues from the transmembrane region). Our results clearly demonstrate that the basolateral sorting signal of the IGF-II/MPR in Caco-2 cells does not reside in the 35 residues adjacent to the transmembrane region of the receptor and thus the IGF-II/MPR utilizes a different basolateral sorting signal than the CD-MPR. However, it will be necessary to determine whether the CD-MPR utilizes the same sorting signal in Caco-2 cells as in MDCK cells, because it has been reported that sorting signals can be interpreted differently by different polarized cells (42).
In summary, we have shown that the IGF-II/MPR utilizes a structural determinant in its cytoplasmic region for basolateral delivery that differs from that used for rapid internalization from the cell surface and for sorting of lysosomal enzymes in endosomal compartments. We have shown that this basolateral sorting signal is located 36-50 residues from the transmembrane region and contains a dihydrophobic sequence (Leu-Met) within a cluster of acidic residues. However, additional studies will be required to identify the specific residue(s) in this region that are essential for entry of the IGF-II/MPR into the basolateral sorting pathway. In nonpolarized cells, it has been demonstrated that the cytoplasmic region of the IGF-II/MPR is recognized by the clathrin adaptor molecules AP-1 and AP-2 (15, 31) as well as by the newly identified protein TIP47 (11, 38) that function to facilitate the intracellular trafficking of the receptor. Future studies will be directed toward identifying the components of the cellular machinery of polarized epithelial cells that mediate the basolateral sorting of the IGF-II/MPR.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Stuart Kornfeld and Dr. Peter Lobel for their generous gift of mutant IGF-II/MPR plasmids and Dr. Donald Messner for providing the bovine IGF-II/MPR-specific monoclonal antibodies.
![]() |
FOOTNOTES |
---|
First published October 3, 2001;10.1152/ajpgi.00028.2001
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases DK-44200. This work was done during the tenure of an Established Investigatorship from the American Heart Association (to N. M. Dahms).
Address for reprint requests and other correspondence: N. M. Dahms, Medical College of Wisconsin, Dept. of Biochemistry, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: ndahms{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 18 January 2001; accepted in final form 27 September 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aroeti, B,
Kosen PA,
Kuntz ID,
Cohen FE,
and
Mostov KE.
Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor.
J Cell Biol
123:
1149-1160,
1993[Abstract].
2.
Beau, I,
Groyer-Picard MT,
Le Bivic A,
Vannier B,
Loosfelt H,
Milgrom E,
and
Misrahi M.
The basolateral localization signal of the follicle-stimulating hormone receptor.
J Biol Chem
273:
18610-18616,
1998
3.
Brzycki-Wessell, M,
Hill D,
Alvarez J,
Messner D,
and
Dahms NM.
Characterization of monoclonal antibodies specific for the mannose 6-phosphate/insulin-like growth factor II receptor (Abstract).
Mol Biol Cell
5:
435a,
1994.
4.
Canfield, WM,
Johnson KF,
Ye RD,
Gregory W,
and
Kornfeld S.
Localization of the signal for rapid internalization of the bovine cation-independent mannose 6-phosphate/insulin-like growth factor-II receptor to amino acids 24-29 of the cytoplasmic tail.
J Biol Chem
266:
5682-5688,
1991
5.
Chen, HJ,
Remmler J,
Delaney JC,
Messner DJ,
and
Lobel P.
Mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor. A consensus casein kinase II site followed by 2 leucines near the carboxyl terminus is important for intracellular targeting of lysosomal enzymes.
J Biol Chem
268:
22338-22346,
1993
6.
Chen, HJ,
Yuan J,
and
Lobel P.
Systematic mutational analysis of the cation-independent mannose 6-phosphate/insulin-like growth factor II receptor cytoplasmic domain. An acidic cluster containing a key aspartate is important for function in lysosomal enzyme sorting.
J Biol Chem
272:
7003-7012,
1997
7.
Dahms, NM,
Lobel P,
and
Kornfeld S.
Mannose 6-phosphate receptors and lysosomal enzyme targeting.
J Biol Chem
264:
12115-12118,
1989
8.
Dahms, NM,
Rose PA,
Molkentin JD,
Zhang Y,
and
Brzycki MA.
The bovine mannose 6-phosphate/insulin-like growth factor II receptor. The role of arginine residues in mannose 6-phosphate binding.
J Biol Chem
268:
5457-5463,
1993
9.
Dahms, NM,
Seetharam B,
and
Wick DA.
Expression of insulin-like growth factor (IGF)-I receptors, IGF-II/cation-independent mannose 6-phosphate receptors (CI-MPRs), and cation-dependent MPRs in polarized human intestinal Caco-2 cells.
Biochim Biophys Acta
1279:
84-92,
1996[ISI][Medline].
10.
Dahms, NM,
Wick DA,
and
Brzycki-Wessell MA.
The bovine mannose 6-phosphate/insulin-like growth factor II receptor. Localization of the insulin-like growth factor II binding site to domains 5-11.
J Biol Chem
269:
3802-3809,
1994
11.
Diaz, E,
and
Pfeffer SR.
TIP47: a cargo selection device for mannose 6-phosphate receptor trafficking.
Cell
93:
433-443,
1998[ISI][Medline].
12.
Distel, B,
Bauer U,
Le Borgne R,
and
Hoflack B.
Basolateral sorting of the cation-dependent mannose 6-phosphate receptor in Madin-Darby canine kidney cells. Identification of a basolateral determinant unrelated to clathrin-coated pit localization signals.
J Biol Chem
273:
186-193,
1998
13.
El Nemer, W,
Colin Y,
Bauvy C,
Codogno P,
Fraser RH,
Cartron JP,
and
Le Van Kim CL.
Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.
J Biol Chem
274:
31903-31908,
1999
14.
Fuhlbrigge, RC,
Fine SM,
Unanue ER,
and
Chaplin DD.
Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA.
Proc Natl Acad Sci USA
85:
5649-5653,
1988[Abstract].
15.
Glickman, JN,
Conibear E,
and
Pearse BM.
Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.
EMBO J
8:
1041-1047,
1989[Abstract].
16.
Hobert, ME,
Kil SJ,
Medof ME,
and
Carlin CR.
The cytoplasmic juxtamembrane domain of the epidermal growth factor receptor contains a novel autonomous basolateral sorting determinant.
J Biol Chem
272:
32901-32909,
1997
17.
Hoflack, B,
and
Kornfeld S.
Purification and characterization of a cation-dependent mannose 6-phosphate receptor from murine P388D1 macrophages and bovine liver.
J Biol Chem
260:
12008-12014,
1985
18.
Hunziker, W,
and
Fumey C.
A di-leucine motif mediates endocytosis and basolateral sorting of macrophage IgG Fc receptors in MDCK cells.
EMBO J
13:
2963-2967,
1994[Abstract].
19.
Hunziker, W,
Harter C,
Matter K,
and
Mellman I.
Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant.
Cell
66:
907-920,
1991[ISI][Medline].
20.
Jadot, M,
Canfield WM,
Gregory W,
and
Kornfeld S.
Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.
J Biol Chem
267:
11069-11077,
1992
21.
Johnson, KF,
and
Kornfeld S.
The cytoplasmic tail of the mannose 6-phosphate/insulin-like growth factor-II receptor has two signals for lysosomal enzyme sorting in the Golgi.
J Cell Biol
119:
249-257,
1992[Abstract].
22.
Keller, P,
and
Simons K.
Post-Golgi biosynthetic trafficking.
J Cell Sci
110:
3001-3009,
1997
23.
Kornfeld, S.
Structure and function of the mannose 6-phosphate/insulinlike growth factor II receptors.
Annu Rev Biochem
61:
307-330,
1992[ISI][Medline].
24.
Le Gall, AH,
Powell SK,
Yeaman CA,
and
Rodriguez-Boulan E.
The neural cell adhesion molecule expresses a tyrosine-independent basolateral sorting signal.
J Biol Chem
272:
4559-4567,
1997
25.
Lobel, P,
Dahms NM,
Breitmeyer J,
Chirgwin JM,
and
Kornfeld S.
Cloning of the bovine 215-kDa cation-independent mannose 6-phosphate receptor.
Proc Natl Acad Sci USA
84:
2233-2237,
1987[Abstract].
26.
Lobel, P,
Dahms NM,
and
Kornfeld S.
Cloning and sequence analysis of the cation-independent mannose 6-phosphate receptor.
J Biol Chem
263:
2563-2570,
1988
27.
Lobel, P,
Fujimoto K,
Ye RD,
Griffiths G,
and
Kornfeld S.
Mutations in the cytoplasmic domain of the 275 kd mannose 6-phosphate receptor differentially alter lysosomal enzyme sorting and endocytosis.
Cell
57:
787-796,
1989[ISI][Medline].
28.
Matter, K,
Hunziker W,
and
Mellman I.
Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants.
Cell
71:
741-753,
1992[ISI][Medline].
29.
Matter, K,
Yamamoto EM,
and
Mellman I.
Structural requirements and sequence motifs for polarized sorting and endocytosis of LDL and Fc receptors in MDCK cells.
J Cell Biol
126:
991-1004,
1994[Abstract].
30.
Meresse, S,
Ludwig T,
Frank R,
and
Hoflack B.
Phosphorylation of the cytoplasmic domain of the bovine cation-independent mannose 6-phosphate receptor. Serines 2421 and 2492 are the targets of a casein kinase II associated to the Golgi-derived HAI adaptor complex.
J Biol Chem
265:
18833-18842,
1990
31.
Meyer, C,
Zizioli D,
Lausmann S,
Eskelinen EL,
Hamann J,
Saftig P,
von Figura K,
and
Schu P.
µ1A-Adaptin-deficient mice: lethality, loss of AP-1 binding and rerouting of mannose 6-phosphate receptors.
EMBO J
19:
2193-2203,
2000
32.
Mostov, KE,
de Bruyn Kops A,
and
Deitcher DL.
Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis.
Cell
47:
359-364,
1986[ISI][Medline].
33.
Munier-Lehmann, H,
Mauxion F,
and
Hoflack B.
Function of the two mannose 6-phosphate receptors in lysosomal enzyme transport.
Biochem Soc Trans
24:
133-136,
1996[ISI][Medline].
34.
Neufeld, EF.
Lysosomal storage diseases.
Annu Rev Biochem
60:
257-280,
1991[ISI][Medline].
35.
Odorizzi, G,
and
Trowbridge IS.
Structural requirements for basolateral sorting of the human transferrin receptor in the biosynthetic and endocytic pathways of Madin-Darby canine kidney cells.
J Cell Biol
137:
1255-1264,
1997
36.
Odorizzi, G,
and
Trowbridge IS.
Structural requirements for major histocompatibility complex class II invariant chain trafficking in polarized Madin-Darby canine kidney cells.
J Biol Chem
272:
11757-11762,
1997
37.
Okamoto, CT,
Song W,
Bomsel M,
and
Mostov KE.
Rapid internalization of the polymeric immunoglobulin receptor requires phosphorylated serine 726.
J Biol Chem
269:
15676-15682,
1994
38.
Orsel, JG,
Sincock PM,
Krise JP,
and
Pfeffer SR.
Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein.
Proc Natl Acad Sci USA
97:
9047-9051,
2000
39.
Prill, V,
Lehmann L,
von Figura K,
and
Peters C.
The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells.
EMBO J
12:
2181-2193,
1993[Abstract].
40.
Prydz, K,
Brandli AW,
Bomsel M,
and
Simons K.
Surface distribution of the mannose 6-phosphate receptors in epithelial Madin-Darby canine kidney cells.
J Biol Chem
265:
12629-12635,
1990
41.
Rodionov, DG,
Nordeng TW,
Kongsvik TL,
and
Bakke O.
The cytoplasmic tail of CD1d contains two overlapping basolateral sorting signals.
J Biol Chem
275:
8279-8282,
2000
42.
Roush, DL,
Gottardi CJ,
Naim HY,
Roth MG,
and
Caplan MJ.
Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.
J Biol Chem
273:
26862-26869,
1998
43.
Simonsen, A,
Stang E,
Bremnes B,
Roe M,
Prydz K,
and
Bakke O.
Sorting of MHC class II molecules and the associated invariant chain (Ii) in polarized MDCK cells.
J Cell Sci
110:
597-609,
1997
44.
Trowbridge, IS,
Collawn JF,
and
Hopkins CR.
Signal-dependent membrane protein trafficking in the endocytic pathway.
Annu Rev Cell Biol
9:
129-161,
1993[ISI].
45.
von Figura, K,
and
Hasilik A.
Lysosomal enzymes and their receptors.
Annu Rev Biochem
55:
167-193,
1986[ISI][Medline].
46.
Wehrle-Haller, B,
and
Imhof BA.
Stem cell factor presentation to c-Kit. Identification of a basolateral targeting domain.
J Biol Chem
276:
12667-12674,
2001
47.
Wick, DA,
Seetharam B,
and
Dahms NM.
Biosynthesis and secretion of the mannose 6-phosphate receptor and its ligands in polarized Caco-2 cells.
Am J Physiol Gastrointest Liver Physiol
277:
G506-G514,
1999
48.
Yeaman, C,
Grindstaff KK,
and
Nelson WJ.
New perspectives on mechanisms involved in generating epithelial cell polarity.
Physiol Rev
79:
73-98,
1999
49.
Zarrilli, R,
Pignata S,
Romano M,
Gravina A,
Casola S,
Bruni CB,
and
Acquaviva AM.
Expression of insulin-like growth factor (IGF)-II and IGF-I receptor during proliferation and differentiation of CaCo-2 human colon carcinoma cells.
Cell Growth Differ
5:
1085-1091,
1994[Abstract].
50.
Zarrilli, R,
Romano M,
Pignata S,
Casola S,
Bruni CB,
and
Acquaviva AM.
Constitutive insulin-like growth factor-II expression interferes with the enterocyte-like differentiation of CaCo-2 cells.
J Biol Chem
271:
8108-8114,
1996
51.
Zhang, Y,
Wick DA,
Haas AL,
Seetharam B,
and
Dahms NM.
Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells.
Biochim Biophys Acta
1267:
15-24,
1995[ISI][Medline].
52.
Zhang, Y,
Wick DA,
Seetharam B,
and
Dahms NM.
Expression of IGF-II and IGF binding proteins in differentiating human intestinal Caco-2 cells.
Am J Physiol Endocrinol Metab
269:
E804-E813,
1995
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |