(Received for publication, August 31, 1995; and in revised form, December 4, 1995)
From the
The interleukin-6 (IL-6) receptor complex is composed of two different subunits, the IL-6 binding protein (IL-6R, gp80) and the signal transducing component gp130. Our previous studies revealed that the 10-amino acid sequence TQPLLDSEER within the intracellular domain of gp130 is crucial for the efficient internalization of IL-6. Since this sequence contains a putative di-leucine internalization motif, we further analyzed this region by constructing two additional deletions and a series of point mutants. Analyses of these mutants showed that the di-leucine pair (Leu-145 and Leu-146) is essential for ligand internalization, with leucine 145 being less resilient to exchanges. Furthermore, when a chimeric protein (Tac-STQPLL) composed of the Tac antigen fused to the hexapeptide STQPLL of gp130 was studied, we found that this sequence is sufficient to mediate endocytosis and lysosomal targeting of the chimera. Mutational analysis of three serine residues upstream of the di-leucine motif revealed that mutation of serine 139 to an alanine reduces the initial internalization rate by 50%. This finding suggests that a serine phosphorylation may be important for rapid endocytosis.
Interleukin-6 (IL-6) ()displays a broad range of
biologic activities (for review, see Kishimoto and Hirano (1988), Van
Snick(1990), and Heinrich et al.(1990)) including the
stimulation of immunoglobulin production by activated B cells (Hirano et al., 1985), the regulation of myeloma/plasmacytoma (Kawano et al., 1988; Schwab et al., 1991) and hybridoma
proliferation (Nordan and Potter, 1986; Van Damme et al.,
1987), and the induction of acute phase protein synthesis in liver
cells (Gauldie et al., 1987; Andus et al., 1987,
1988; Castell et al., 1990). Binding of IL-6 to its receptor
(IL-6R) induces homodimerization of two gp130 molecules, thereby
transducing the signal into the cell (Murakami et al., 1993).
Monomeric gp130 is phosphorylated on serine and threonine, but not on
tyrosine (Murakami et al., 1993). In contrast, dimerized gp130
is also phosphorylated on tyrosine and exhibits an increased
phosphorylation on serine and threonine residues (Murakami et
al., 1993). Whereas the identity of gp130-associated
serine-threonine kinases remains to be elucidated, it was recently
shown that IL-6 stimulation triggers the activation of tyrosine kinases
of the JAK family already bound to the cytoplasmic domain
(Lütticken et al., 1994; Stahl et
al., 1994). Moreover, the signal transducers and activators of
transcription (STAT) APRF/STAT3 and STAT1
become
tyrosine-phosphorylated in response to IL-6
(Lütticken et al., 1994; Akira et
al., 1994). They homo- or heterodimerize and translocate to the
nucleus, where they bind to responsive elements of IL-6 target genes
(Wegenka et al., 1994). Before nuclear translocation,
APRF/STAT3 and STAT1
undergo an additional serine phosphorylation
(Lütticken et al., 1995; Wen et
al., 1995).
After binding to its receptor, IL-6 is rapidly internalized and degraded by rat hepatocytes and human hepatoma cells HepG2 (Nesbitt and Fuller, 1992; Zohlnhoefer et al., 1992). In HepG2 cells, this internalization leads to a net loss of IL-6 binding sites at the cell surface, indicating that the IL-6R is down-regulated by its ligand (Zohlnhoefer et al., 1992). The down-regulation might play an important role as a protection against overstimulation. Recently, we showed by co-expression of wild-type and mutant forms of both, the IL-6 receptor and gp130, in transiently transfected COS-7 cells that gp130 is essential for the efficient endocytosis of IL-6 and receptor down-regulation (Dittrich et al., 1994). Whereas the cytoplasmic domain of the IL-6 receptor was not significantly involved in the internalization process, deletion of the corresponding domain of gp130 resulted in an almost complete impairment of IL-6 internalization. Mutants with different truncations within the intracellular domain of gp130 led to the identification of the 10 amino acid sequence TQPLLDSEER that is crucial for efficient endocytosis (Dittrich et al., 1994).
During the last few years, it has
become clear that efficient internalization of transmembrane receptor
proteins requires a signal sequence in the cytoplasmic tail of the
protein (Vaux, 1992). There are at least two different types of
internalization signal sequences: tyrosine- and di-leucine-based
internalization motifs. Tyrosine motifs have been identified in a
variety of receptor molecules including the low density lipoprotein
receptor, the transferrin receptor, and the asialoglycoprotein receptor
(for review, see Trowbridge et al.(1993)). Substitution of
single amino acids within such motifs and structural predictions
suggested that an important characteristic of these sequences is the
formation of a ``tight -turn'' (Collawn et al.,
1990; Bansal and Gierasch, 1991). This structure might be recognized by
adaptor proteins, which mediate the accumulation of these receptors in
clathrin-coated pits (Collawn et al., 1990). Recently,
leucine-leucine or leucine-isoleucine sequence motifs, important for
internalization and/or lysosomal targeting, were found in the
intracellular domains of the
- and
-chain of the T cell
receptor complex, CD4, the cation-dependent mannose 6-phosphate
receptor, the cation-independent mannose 6-phosphate/insulin-like
growth factor II receptor, and the interferon
-receptor
(Letourneur and Klausner, 1992; Shin et al., 1991; Johnson and
Kornfeld, 1992; Chen et al., 1993; Farrar and Schreiber,
1993). It is not known whether the secondary structure of these motifs
is related to a tight
-turn or not. It is the current hypothesis
that these motifs reside within a random coil structure (Sandoval et al., 1994).
In this study, we demonstrate that the di-leucine motif (STQPLL, positions 141-146) in the cytoplasmic tail of gp130 is essential for the efficient endocytosis of IL-6. Furthermore, serine 139 located six amino acids upstream of the two leucine residues was found to be important for rapid internalization. We suggest that it may become phosphorylated after IL-6 stimulation, which in turn leads to an exposure of the internalization signal to the endocytotic machinery.
N-Succinimidyl-3-(4-hydroxy-5-[I]iodophenyl)propionate
(Bolton-Hunter reagent; 74 TBq/mmol) was purchased from Amersham Corp.
A frequently used buffer was PBS (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na
HPO
, 1.76 mM KH
PO
, pH 7.2).
The cloning vector pBluescriptIIKS(+) was purchased from Stratagene (Heidelberg, Germany).
Figure 1:
Schematic representation of the point
mutations and deletions in the cytoplasmic tail of gp130. The sequence
is shown in the single-letter code and the mutated residues Ser-137,
Ser-139, Ser-141, Leu-145, and Leu-146 in the wild-type sequence are
written in boldface. The wild-type and mutated forms of gp130,
respectively, were transiently expressed in COS-7 cells together with
the wild-type IL-6 receptor (COS-WT, -L145A, -L146A, -L145/146A,
-L145I, -L146I, -S137A, -S139A, -S141A, -S139/141A, -131, and
-
133).
Figure 2:
Internalization of surface-bound I-IL-6 by IL-6 receptor complexes composed of wild-type
IL-6 receptor and wild-type (WT) or mutant gp130. Three days
after transfection, cells were washed twice with ice-cold binding
medium. After incubation with 1 nM of
I-IL-6 for
2 h at 4 °C, cells were warmed to 37 °C for the indicated times
without removing unbound ligand. Cells were then set on ice for 2 h and
washed 3 times with PBS to eliminate unbound ligand. Surface-bound
I-IL-6 (open symbols) was determined after
subjecting cells to 0.5 M NaCl/HCl, pH 1, for 3 min.
Internalized
I-IL-6 (closed symbols) was
measured in the
-counter after lysis of the cells in 1 M NaOH. Data are expressed as a percentage of the amount of IL-6
bound at time point 0. The sum of surface-bound and internalized IL-6
at later time points adds up to more than 100%, indicating that an
intracellular pool of cycling receptors is loaded with IL-6 during the
6-h incubation time. Values are the mean of two to four independent
experiments. CO, untransfected cells (triangles).
Figure 3:
Competition of binding of I-IL-6 to the IL-6 receptor complex in the presence of
increasing concentrations of unlabeled IL-6. Three days after
transfection, cells were washed twice with ice-cold binding medium.
Then, cells were incubated with 100 pM
I-IL-6
for 4 h at 4 °C in the presence of increasing amounts of unlabeled
IL-6 (0.1-1000 nM). After 4 h, medium was removed, and
cells were washed 3 times with ice-cold PBS in order to remove all
unbound ligand. Finally, cells were lysed in 1 M NaOH, and
cell-associated radioactivity was measured in the
-counter. Data
are expressed as a percentage of control incubations containing only
labeled ligand. Values are the mean of two independent
experiments.
To determine if there are additional
internalization motifs downstream of the di-leucine motif, we mutated
both leucines to alanines in the full-length gp130 protein
(L145A/L146A; Fig. 1). When transfected into COS-7 cells, this
mutant bound IL-6IL-6R complexes with an affinity comparable with
that of wild-type gp130 (Fig. 3B) but internalized only 16% of initially surface-bound ligand within the first 30 min. In addition
the number of surface-binding sites was reduced only by 13% within 6 h (Fig. 2D). This finding corroborates the crucial function
of leucine residues 145 and 146.
Figure 4:
Internalization of surface bound I-IL-6 by IL-6 receptor complexes composed of wild-type (WT) IL-6 receptor and mutant gp130. Three days after
transfection, cells were washed twice with ice-cold binding medium.
After incubation with 1 nM
I-IL-6 for 2 h at 4
°C, cells were warmed to 37 °C for the indicated times without
removing unbound ligand. Cells were then placed on ice for 2 h and
washed 3 times with PBS to eliminate unbound ligand. Surface bound
I-IL-6 (open symbols) was determined after
subjecting cells to 0.5 M NaCl/HCl, pH 1, for 3 min.
Internalized
I-IL-6 (closed symbols) was
measured in the
-counter after lysis of the cells in 1 M NaOH. Data are expressed as described in Fig. 2. Values are
the mean of two to four independent
experiments.
Figure 5: Confocal immunofluorescence microscopy of COS-7 cells expressing Tac-gp130 and Tac-insulin receptor (IR) chimeras, respectively. 48 h after transfection, COS-7 cells were incubated with anti-Tac antibody for 2 h at 4 °C. After the prebound antibody was internalized for 30 min at 37 °C, cells were fixed, permeabilized, and stained with an rhodamine-conjugated anti-mouse antibody. Representative medial optical cuts are shown. In each panel, the hexapeptide sequence fused to the Tac antigen is indicated, and the source of the peptide is identified in parentheses. Bar, 25 and 50 µm, respectively.
Figure 6: Localization of Tac-STQPLL (A-D) and Tac-STQPAA (E-H) relative to transferrin (TF) and CD63, respectively. 48 h after transfection, HeLa cells were incubated with anti-Tac antibody for 2 h at 4 °C. After the prebound antibody was internalized at 37 °C for 15 min (A, B, E, F in the presence of 10 ng/ml iron-loaded transferrin) and 90 min (C, D, G, H), respectively, cells were fixed, permeabilized, and stained with anti-transferrin (A, B, E, F) or anti-CD63 (C, D, G, H) antibody. Antibodies bound to their specific antigen were detected using a 1/200 dilution of rhodamine or fluorescein isothiocyanate-conjugated second antibodies.
Figure 7:
IL-6-stimulated degradation of wild-type (WT) gp130 and the di-leucine mutant (L145A/L146A). Confluent
monolayers of COS-7 cells, transfected with wild-type or mutant gp130
together with wild-type IL-6 receptor, were incubated for 1 h in DMEM
containing [S]methionine and
[
S]cysteine. Thereafter, the cells were
transferred to medium containing 1 nM IL-6 and unlabeled
methionine and cysteine for 0-8 h. The cells were then
solubilized, and the wild-type and mutant forms of gp130 were
immunoprecipitated with an specific monoclonal antibody (GPX-7). Finally, the immunoprecipitates were analyzed by
SDS-PAGE followed by fluorography. The x-ray film was exposed for 2
days. The amount of wild-type and mutant protein present at each time
point was quantitated by densitometry as described under
``Experimental Procedures.'' Half-lives were determined by
curve fitting using an exponential
equation.
Upstream
of the di-leucine motif of gp130, three serine residues exist, Ser-137,
Ser-139, and Ser-141 (Fig. 1). In order to study whether any of
these are required for efficient endocytosis, we mutated each of the
serine residues to alanine (Fig. 1). Each mutant cDNA was
transfected together with the wild-type IL-6 receptor cDNA into COS-7
cells. All mutant receptor complexes were expressed equally and bound
IL-6 with a comparable affinity (Fig. 3). Since we observed the
strongest effect during the initial internalization, we measured the
specific internalization rates (k) as outlined in
(Lund et al., 1990, Backer et al., 1991). Fig. 8A shows the ratio of intracellular to cell-surface
I-IL-6 as a function of time for the mutant and wild-type
gp130 proteins. Over a 12-min time frame, in which receptor recycling
and degradation is negligible, cells expressing wild-type gp130
internalized IL-6 with a rate constant of k
= 0.100 ± 0.029 min
(Fig. 8B). For mutant S139A we measured a rate
constant that was reduced by 50% (k
= 0.050
± 0.010 min
). Mutants S137A and S141A showed
a somewhat reduced internalization rate, however this effect was not
statistically significant (k
= 0.088
± 0.006 min
; k
= 0.072 ± 0.028 min
). The double mutant
S139A/S141A underwent an even slower rate of internalization than
mutant S139A (k
= 0.037 ± 0.004
min
). The lowest rate, however, was found with the
di-leucine mutant L145A/L146A (k
= 0.016
± 0.010 min
). These results suggest that in
addition to the di-leucine motif serine 139 is important for rapid
endocytosis.
Figure 8:
125I-IL-6 internalization by COS-7 cells
expressing wild-type IL-6 receptor plus wild-type (WT) or
mutant gp130. COS-7 cells transiently expressing either wild-type or
mutant gp130 together with the wild-type IL-6 receptor were exposed to
1 nMI-IL-6 for 2-12 min at 37 °C. At
the indicated times after IL-6 addition, surface-bound and internalized
I-IL-6 were determined as described under
``Experimental Procedures.'' Panel A, the ratio of
the intracellular IL-6 to surface-bound IL-6 is plotted as a function
of time for COS-7 cells expressing either wild-type or mutant gp130.
The results are the means ± the S.E. of three experiments, each
performed in duplicate. Panel B, the internalization rate
constants (k
) for the different IL-6
receptor complexes in each cell line were calculated as described under
``Experimental Procedures.''
In this study, we identified a di-leucine internalization motif (STQPLL, amino acids 141-146) within the cytoplasmic tail of gp130. There is also a putative tyrosine-containing internalization sequence (YSTV) from amino acid 118 to 121 in the intracellular domain. However, this sequence by itself is not sufficient for mediating efficient endocytosis (Dittrich et al., 1994). As with the tyrosine-based motifs in which the tyrosine often can be replaced by a phenylalanine, the leucines can tolerate conservative changes. Thus the second leucine (Leu-146) can be changed to an alanine or to an isoleucine, resulting in a slightly diminished but persistent internalization. The first leucine (Leu-145), however, appears less resilient to change, showing a diminished function when changed to an isoleucine and a complete loss of function when replaced by an alanine.
While the di-leucine motif first manifested itself as an endocytotic signal, we could show that the addition of the hexapeptide STQPLL to the cytoplasmic tail of the Tac antigen enables this molecule not only to become internalized but also to be targeted to the lysosomes. This observation is supported by the results of the site-directed mutagenesis studies in the intact gp130 molecule. In the full-length gp130 the di-leucine motif appears to be essential for both ligand-induced endocytosis and increased receptor degradation. However, the effect of the mutated leucine pair (L145A/L146A) on receptor half-life was less pronounced than on IL-6 internalization and receptor down-regulation. One possibility may be that there exist additional signals in the cytoplasmic tail of gp130 that code for the delivery of the signal transducer to the degradative pathway independent of ligand binding. This is in agreement with the short half-life of this protein in HepG2, Madin-Darby canine kidney (Gerhartz et al., 1994), and COS-7 cells (this study).
By introducing a point mutation at
serine residue 139, we demonstrated that this serine is important for
rapid internalization of IL-6. Currently we do not know whether this
serine is phosphorylated upon IL-6 stimulation. However, we suggest
that it may become phosphorylated for three reasons. First, gp130
exhibits an increased phosphorylation on serine residues after IL-6
stimulation (Murakami et al., 1993). Second, the position of
serine 139, six amino acids upstream of the di-leucine motif, aligns
very well with the positions of the phosphoserines identified for the
CD3- chain, CD4 and the cation-independent mannose 6-phosphate
receptor (Dietrich et al., 1994; Shin et al., 1990,
1991; Chen et al., 1993). Third, down-stream of serine 139 we
find a negative charge at position +1 and two putative negative
charges at positions +2 (Ser-141) and +3 (Thr-142). If the
latter would be phosphorylated, this would make serine 139 a possible
target of casein kinase II (Meggio et al., 1994). However,
future studies have to address this question in more detail.
For the
CD3- chain and CD4, it has been shown that the PMA-induced
down-regulation is impaired after mutation of the identified serines to
valine or alanine. In the mannose 6-phosphate receptor system, the
transport from the TGN, trans-Golgi network to the lysosomes was
impaired in such a serine mutant. Until now it is not known which
kinase(s) phosphorylate(s) these serine residues. The serines
identified reside either within a consensus sequence for
phosphorylation by protein kinase C (CD3-
, CD4) or by casein
kinase II (CD3-
, CI-M6PR). Interestingly, Hoflack and co-workers
detected a casein kinase II-like activity that copurifies with the HAI
adaptor complex and is associated to its 47-kDa subunit (Meresse et
al., 1990). In vitro this enzyme phosphorylates the
cation-independent mannose 6-phosphate receptor cytoplasmic domain on
serine 2492 upstream of the di-leucine motif.
The functional role of
the ligand-induced serine phosphorylation however, is still
speculative. For CD4 it was shown that serine phosphorylation causes
dissociation of the protein tyrosine kinase p56 from its
cytoplasmic domain, thus allowing interaction of CD4 with the
endocytotic machinery to occur (Pelchen-Matthews et al.,
1992). Probably, ligand-induced serine phosphorylation is analogous to
tyrosine autophosphorylation observed for the epidermal growth factor
receptor in which this phosphorylation enhances the interaction with
the endocytotic machinery, i.e. the plasma membrane associated
-adaptin (Sorkin and Carpenter, 1993). For the CD3-
chain, it
has been suggested that receptor-mediated cell activation leads to an
increase in kinase activity and thereby to a serine phosphorylation.
This subsequently induces a conformational change that results in an
increased accessibility of phosphoserine-dependent di-leucine-based
motifs to adaptors and leads to a down-regulation of the receptor
(Dietrich et al., 1994). Since the mutation of serine 139 to
an alanine does not inhibit IL-6 internalization completely, but
reduces the initial internalization rate by 50%, we envision a similar
mechanism for the IL-6 induced down-regulation of its receptor complex.