(Received for publication, April 14, 1997)
From the Department of Morphology, University of
Geneva, 1211 Geneva, Switzerland and the § Diabetes
Branch, NIDDK, National Institutes of Health,
Bethesda, Maryland 20892
Two leucines (Leu986 and
Leu987) have recently been shown to take part in the
control of human insulin receptor (HIR) internalization (Renfrew-Haft,
C., Klausner, R. D., and Taylor, S. I. (1994) J. Biol. Chem. 269, 26286-26294). The aim of the present study was to further investigate the exact mechanism of this control process. Constitutive and insulin-induced HIR internalizations were studied biochemically and morphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986-987) mutant HIR (HIR
AA1) or HIR truncated at either amino acid residue 981 (HIR 981) or
1000 (HIR
1000). Data collected indicate that: (a) the three mutant HIR show a reduced association with microvilli as compared
with HIR wild-type; (b) the two receptors containing the
dileucine motif (HIR WT and HIR
1000) show the highest propensity to
associate with clathrin-coated pits, independently of kinase activation; (c) the two receptors lacking the dileucine
motif but containing two tyrosine-based motifs, previously described as
participating in clathrin-coated pit segregation, associate with these
surface domains with a lower affinity than the two others,
(d) in the presence of the kinase domain, an unmasking of
the tyrosine-based motifs mediated by kinase activation is required.
These results indicate that the dileucine motif is not sufficient by itself, but participates in anchoring HIR on microvilli and that another sequence, located downstream from position 1000 is crucial for this event. This dileucine motif also plays a role in HIR segregation in clathrin-coated pits. This latter function is additive with that of the tyrosine-based motifs but the role of the dileucine motif predominates. Eventually, the clathrin-coated pit anchoring function of the dileucine motif is independent of receptor kinase activation in contrast to the tyrosine-based motifs.
The internalization of many signaling receptors (i.e.
insulin, epidermal growth factor receptors, CD4 ... ) is a
ligand-dependent process which requires the ligand-mediated
activation of a tyrosine kinase, intrinsic to or associated with the
cytoplasmic domain of the receptor (1-5). In the case of the insulin
receptor, activation of this tyrosine kinase releases a constraint
maintaining the unoccupied receptor on cytoskeleton-rich microvilli (1,
2, 6). The activated receptor then moves in the plane of the plasma membrane, and gains access to the non-villous domains of the cell surface, where it is internalized via the clathrin-coated pits. Very
little information is available regarding the receptor domain(s) involved in the retention of unoccupied receptors on microvilli or
about the cellular component(s) that anchor(s) the insulin receptor on
microvilli. The mechanism(s) allowing kinase activation to induce the
release of the insulin receptor to migrate away from microvilli are
also unresolved. In contrast, some light was shed on the subsequent
surface steps. The speed of the surface shift has been shown to depend
at least in part on the structure of the receptor transmembrane domain
(7-9) similar to what was observed in the case of other receptors
(10). Furthermore, the last surface event, the association with
clathrin-coated pits, has been shown to require the integrity of two
tyrosine-based signals present in the juxtamembrane cytoplasmic domain
of the insulin receptor (GPLY and NPEY) (11-14). Similar motifs
forming a turn and exposing an aromatic amino acid (preferentially
a tyrosine) have been described as participating in the internalization of various receptors including receptors for which internalization is
ligand-independent (3, 15-19).
Recently, a dileucine motif present within exon 17 (Leu986 and Leu987) has been shown to participate in the control of insulin receptor internalization and sorting inside the cell (20). Mutation of these two leucines for alanines indeed results in a decreased insulin-induced internalization of the mutant receptor. In addition, the direct fusion of this dileucine motif (together with four neighboring amino acids) to the carboxyl terminus of Tac to form a chimeric molecule identified this motif as a lysosomal sorting sequence (20). The present work was designed to further investigate the role of this dileucine motif encoded by exon 17. To that end, two truncated receptors (at positions 981 and 1000, respectively) were prepared in addition to the dileucine mutant receptor previously described. Constitutive as well as insulin-induced internalization processes were analyzed both biochemically and morphologically at the electron microscopic level. Results collected indicate that the sequence anchoring the unoccupied insulin receptors on microvilli is located downstream from Glu1000 and that the dileucine motif at positions 986-987 participates in the control of this anchoring. In addition, our observations reveal that this dileucine motif plays a role in the segregation of the insulin receptor in clathrin-coated pits. This latter function of the dileucine motif is additive with that of the tyrosine-based motifs present in exon 16 but the role of the dileucine motif predominates. Receptor tyrosine kinase is not required for this function of the dileucine motif. In contrast, the function of tyrosine-based motifs to segregate receptors in clathrin-coated pits requires kinase activation.
The plasmids containing either the mutant insulin
receptor with 2 alanines substituted for the Leu-Leu pair at positions
986-987 or the insulin receptor truncated at position 981 were
previously described (20-22). The receptor truncated at position 1000 was constructed as follows. A fragment containing nucleotides
2289-3265 of the human insulin receptor cDNA (23) was amplified by
polymerase chain reaction using the full-length insulin receptor
cDNA cloned into pSP64 vector (Promega Corp., Madison, WI) as
template and the following oligonucleotide primers:
5-GCCGAGGACCCTAGGCCATCTCGGA-3
(nucleotides 2289-2313
of the sense strand with an AvrII restriction site
(underlined)) and
5
-AAGACTTAAGCTAATACACCATGCC-3
(nucleotides
3138-3162 of the antisense strand with a BfrI restriction site (underlined) and a stop codon (bold)). The polymerase chain reaction product was digested with AvrII and BfrI
to yield a 858-base pair fragment. This fragment was substituted for
the AvrII/BfrI segment (2298-4326) of the
wild-type insulin receptor cDNA. This construction was verified by
nucleotide sequencing and subcloned into a bovine papilloma virus-based
expression vector pBPV (Pharmacia Biotech Inc.), in which insulin
receptor cDNA expression was driven by the murine metallothionin
promoter.
NIH 3T3 cells
(~750,000 cells) in Petri dishes (10 cm in diameter) were transfected
using 40 µl of Lipofectin (Life Sciences Technologies Inc.) and a
mixture of 1 µg of pRSV-Neo (a plasmid encoding neomycin resistance)
and 5 µg of pBPV-HIR 1000. After selection for resistance to the
antibiotic G418 (800 µg/ml) (Life Technologies, Inc.), stable
transfectants were isolated by FACS (fluorescence-activated cell
sorter). Cells were recovered by EDTA treatment and incubated for 30 min with 500 µl of biotinylated anti-HIR1 antibody (1:500
in phosphate-buffered saline, 1% bovine serum albumin) at 4 °C,
After three washes, 100 µl of streptavidin R-phycoerytherin conjugate
(1:20) (Caltag Laboratories Inc., San Franscico, CA) were added for 30 min in the dark. Flow cytometry was performed on a Becton Dickinson
FACScan. Cell surface expression was verified by
125I-insulin binding.
Confluent monolayers of NIH 3T3 cells expressing
either the wild-type or mutant receptors were first incubated for
2 h at 4 °C in the presence of 5 pM
125I-mAb 83-14 (constitutive internalization) or 50 pM 125I-insulin (insulin-induced
internalization) then transferred to 37 °C for various periods of
time. At each time point studied, the unbound 125I-ligand
was removed and cells were washed three times with cold phosphate-buffered saline. Cells were then subjected to three 5-min
washes with cold phosphate-buffered saline at pH 1.5 to release the
125I-ligand bound at the cell surface. Finally, cells were
lysed in 1 N NaOH and all samples (acid washes and lysates)
were counted in a -counter to determine the internalized
radioactivity versus total radioactivity.
Confluent
monolayers of NIH 3T3 cells were grown up in Petri dishes (10 cm in
diameter). Cell surface proteins were biotinylated as described in Ref.
24. Briefly, cells were incubated in phosphate-buffered saline
containing 0.5 mg/ml N-hydroxysuccinimide long chain biotin (Pierce) for 30 min at 4 °C. Cells were then incubated in 5 ml of
binding buffer in the absence or presence of 107
M insulin for 15 min at 37 °C. Cell surface proteins
were subjected to Pronase digestion (2.5 mg/ml, Boehringer Mannheim)
for 1 h at 4 °C. Thereafter, cells were washed and solubilized
in 500 µl of lysis buffer containing protease inhibitors. Insulin
receptors were immunoprecipitated with a mixture of rabbit antibodies
(B7 and B10) or with a monoclonal antibody (83-14) directed against the
-subunits of the human insulin receptor. After SDS-polyacrylamide (6.5%) gel electrophoresis and electroblotting, nitrocellulose sheets
were probed with horseradish peroxidase-linked streptavidin (Amersham)
at a dilution of 1:500. After extensive washes, enhanced chemiluminescence (ECL) detection was performed according to the manufacturer's instructions (Amersham). Signals were analyzed by
densitometry (Molecular Dynamics).
After
incubation at 37 °C in the presence of tracer amount
of 125I-insulin (1011 M), cells
were fixed, dehydrated, and quantitated as described previously (1).
For each time point studied, three Epon blocks were prepared and
sectioned. About 450-600 grains were analyzed from morphologically
intact cells. Grains within a distance of 1 ± 250 nm from the
plasma membrane were considered associated with cell surface; grains
inside the cytoplasm and >250 nm from the plasma membrane were
considered internalized. Grains present at the plasma membrane fell
into the following classes: microvilli, clathrin-coated pits,
non-villous nonclathrin-coated pit segments, and uninterpretable.
Grains were considered associated with microvilli or clathrin-coated
pits if the center was <250 nm from the surface domains.
NIH 3T3 fibroblast cell
lines were transfected with an expression vector encoding wild-type or
one of three mutant insulin receptors: HIR with a pair of alanines in
place of the dileucines normally present at positions 986 and 987 (AA1)
(20), HIR truncated at position 981 (981) or truncated at position
1000 (
1000) (Fig. 1). HIR AA1
exhibited close to normal
-subunit phosphorylation as well as
insulin-induced phosphorylation of insulin receptor substrate-1 (20),
while, as expected, HIR
981 and HIR
1000, were not
autophosphorylated (data not shown).
Constitutive and Insulin-stimulated Internalizations of Insulin Receptors in NIH 3T3 Fibroblasts Expressing Normal or Mutant Insulin Receptors
HIR cytoplasmic domain contains several dileucine
motifs, one of which (leucines 986-987) has been proposed to play a
role in HIR internalization (20). In an attempt to clarify the exact function of this dileucine motif in the internalization process, the
internalization previously reported defect was studied by comparing
constitutive and insulin-induced insulin receptor internalizations in
HIR AA1 and HIR WT cells by various methodological approaches. First,
comparison of the constitutive internalization (i.e. in the
absence of insulin) of the two receptors was carried out by using a
monoclonal anti-human insulin receptor antibody (83-14) coupled to
125I as a ligand (25). Under these conditions, constitutive
HIR internalization observed in AA1 cells and in WT cells were
identical (Fig. 2). Second,
insulin-induced internalization was compared in WT and AA1 cells by
tracking 125I-insulin internalization both by the acid wash
technique and by quantitative EM autoradiography. Both techniques
confirmed an inhibition of the internalization rate and a decrease in
the total amount of radioactivity recovered inside AA1 cells at each time point studied (Fig. 2 and data not shown). Third, we compared constitutive and insulin-induced internalization in the same experiment by analyzing internalization of biotinylated receptors. According to
this procedure, cell surface proteins were labeled with biotin, and
cells further incubated at 37 °C in the presence or absence of
insulin (107 M) for 15 min to promote
receptor internalization. At the end of this incubation, biotinylated
receptors at the cell surface were digested with Pronase. Internalized
insulin receptors were protected from digestion. After cells were
solubilized, biotinylated receptors were immunoprecipitated with
anti-insulin receptor antibody (mAb 83-14). Biotinylated receptors were
quantitated in a blotting experiment using horseradish
peroxidase-labeled streptavidin (Figs. 3,
A and B). Under these circumstances, the slow
constitutive internalization of AA1 receptors was accelerated by
insulin, but this stimulation was smaller in magnitude than that
observed in WT cells, where more than 70% of biotinylated receptors
became resistant to Pronase digestion by 15 min of incubation at
37 °C (Fig. 3). Taken together, these internalization studies
demonstrate that insulin-stimulated HIR internalization is inhibited
when the two leucines present at positions 986 and 987 are replaced by
alanines, while constitutive internalization is relatively normal in
this mutant receptor.
To collect further information on the role of the dileucine per
se, internalization studies analogous to the one described above,
were carried out with two truncated receptors. These two receptors (HIR
981 and HIR
1000) have in common the absence of the kinase domain
but they differ by the presence (HIR
1000) or absence (HIR
981)
of the dileucine motif. Following biotinylation of the cell surface,
constitutive internalization of HIR
981 and HIR
1000 was more
rapid than in the case of HIR WT (Fig. 3). Moreover, insulin did not
accelerate internalization of either truncated receptors, confirming
that these two mutant receptors were internalized in a constitutive way
(Fig. 3). Based on these observations, further studies of these two
truncated receptors were performed by measuring receptor-mediated
endocytosis of 125I-mAb 83-14.
In both cases, the internalization rate as well as the maximal amount
of radioactive material internalized were significantly higher than
those recorded in WT cells (Fig. 4). The
highest values were obtained in 1000 cells, where >55% of total
cell associated radioactivity was found inside the cells by 15 min of
incubation at 37 °C (Fig. 4). In the case of
981 cells, both the
internalization rate and the maximal amount of radioactivity
internalized were intermediate between those obtained in
1000 cells
and in WT cells (Fig. 4). Thus, HIR
1000 contains, in its native and
unoccupied state, all the elements allowing the optimal internalization
of HIR while HIR
981 appears to lack one or more of these
elements.
125I-mAb 83-14 and 125I-Insulin Initial Localization and Redistribution on the Surface of NIH 3T3 Fibroblasts Expressing Normal and Mutant Insulin Receptors
To further understand why the two truncated receptors differ in terms of their internalization capacities and in an attempt to shed some light on how the 986-987 dileucine motif participates in HIR internalization, a morphological ultrastructural analysis of the surface distribution of the above described mutants was carried out.
As determined by quantitative EM autoradiography analysis, in
conditions revealing the localization of unoccupied receptors (2 h of
incubation at 4 °C in the presence of 125I-mAb 83-14),
HIR WT preferentially associated with microvilli on the surface of NIH
3T3 cells (Fig. 5). In contrast, in the case of receptors truncated at position 981 and 1000 and constitutively internalized, such preferential initial localization was not observed (Fig. 5). These observations confirm and extend the previously described lack of association with microvilli of a receptor truncated at position 965 (6). HIR AA1 behaved similarly to the truncated receptors: at the end of a 4 °C incubation in the presence of either
125I-mAb 83-14 or 125I-insulin, it did not
preferentially associate with microvilli (Figs. 5 and
6).
As a function of incubation time at 37 °C, 125I-insulin
moved in the plane of the plasma membrane of WT cells so that by 30 min of incubation at 37 °C, 70% of the autoradiographic grains were scored as associated with non-villous domains of the cell surface, indicating a preferential association with these surface regions (Fig.
6A). In the case of AA1 cells, although HIR AA1 did not preferentially associate with microvilli (see above), a surface redistribution of 125I-insulin was observed, attesting to a
preferential association of the mutant receptor with the non-villous
domains of the cell surface (Fig. 6A). In contrast, the two
receptors containing an active kinase (HIR WT and HIR AA1), did not
redistribute in the sole presence of 125I-mAb 83-14 (Fig.
6) although these two receptors did undergo internalization in response
to insulin binding. In the case of the two truncated receptors tagged
with 125I-mAb 83-14, a redistribution reflecting a
progressive concentration of the receptors in the non-villous domains
of the cell surface was noted (Fig. 6B).
Taken together with the biochemical observations (see above), these morphological data demonstrate that the receptor signal sequence responsible for HIR anchoring on microvilli is located downstream from amino acid 1000. The dileucine motif present at positions 986-987 also participates in this anchoring process although it is not sufficient to maintain the unoccupied receptor on microvilli.
125I-mAb 83-14 and 125I-Insulin Association with Clathrin-coated Pits on the Non-villous Surface of NIH 3T3 Fibroblasts Expressing Normal and Mutant Insulin ReceptorsIn the
presence of insulin, normal insulin receptors are classically
segregated in the internalization gates present on the non-villous
surface domains: the clathrin-coated pits (26-28). A rapid
concentration of HIR WT in clathrin-coated pits was similarly observed
in NIH 3T3 fibroblasts in the presence of insulin (Fig. 7B). As described previously
(25), even in the absence of insulin (125I-mAb 83-14 experiments) a progressively increasing proportion of HIR WT was
associated with clathrin-coated pits (Fig. 7A). However,
association with clathrin-coated pits remained significantly lower than
that observed when 125I-insulin was used as ligand (Fig. 7,
A and B). Under similar experimental conditions
(125I-mAb 83-14 experiments), HIR AA1 association with
clathrin-coated pits remained low at all time points studied (Fig.
7A). In the presence of 125I-insulin, HIR AA1
progressively associated with clathrin-coated pits but with a lower
affinity for these surface domains than HIR WT under the same
conditions (Fig. 7B). HIR 981, which undergoes ligand-independent internalization (see above and Fig. 3), also associated with clathrin-coated pits but with a low affinity, similar
to that of HIR AA1 in the presence of insulin (Fig. 7B). In
contrast, HIR
1000, which also undergoes constitutive
internalization but at a higher rate than HIR
981 (see above and
Fig. 3), showed the highest association with clathrin-coated pits at
all time points studied. By 2 h of incubation at 4 °C, 13% of
the radioactive ligand was already present within these surface domains
(in contrast to
5% in all other cell lines), and this value reached
a maximum of 23% by 30 min of incubation at 37 °C) (Fig.
7A).
To better discriminate the propensity of the various HIR to associate
with clathrin-coated pits independently of their capacity to anchor to
microvilli, the morphological quantitative analysis was limited to the
autoradiographic grains present on the non-villous domains of the cell
surface. In this analysis, both HIR WT (+insulin) and HIR 1000
showed the highest affinity for clathrin-coated pits (Fig.
8). In contrast, the two mutant receptors
which lack the 986-987 dileucine motif (HIR AA1 (+insulin) and HIR
981), showed a comparable propensity to associate with these surface invagination gates but their affinity for clathrin-coated pits was half
that noted for the two first receptors (Fig. 8). Based on these
observations, it can be concluded that the dileucine motif is involved
in HIR anchoring on clathrin-coated pits. Moreover, since a significant
residual association with clathrin-coated pits is detected in both HIR
AA1 (+insulin) and HIR
981, it is evident that (an)other motif(s)
is(are) additive to the dileucine motif in this function. The GPLY and
NPEY motifs, present in the juxtamembrane domain of HIR and retained in
both HIR AA1 and HIR
981, have been demonstrated previously to play
such a role (11-14). Present data indicate that this second set of
motifs is masked in the absence of insulin since in the presence of
125I-mAb 83-14, which does not activate HIR, the
association of HIR AA1 with clathrin-coated pits is dramatically
reduced as compared with that observed when 125I-insulin is
used as a ligand (Fig. 8). In contrast, such unmasking mediated by
insulin binding does not seem required in the case of the dileucine
motif since in its inactivated form (i.e. tagged with
125I-mAb 83-14), HIR WT is able to efficiently associate
with clathrin-coated pits despite the masking of the juxtamembrane
domain (Fig. 8).
Cell surface receptors taken up by clathrin-coated pits can be subdivided in two categories. Class I receptors, including transport protein receptors (i.e. low density lipoprotein, transferrin, and asialoglycoprotein receptors) are spontaneously segregated in clathrin-coated pits and are continuously internalized and recycled even in the absence of ligand (29). In contrast to these nutrient receptors, signaling receptors (class II receptors, i.e. insulin, epidermal growth factor receptors) must first bind their respective ligand to have access to clathrin-coated pits which mediate their internalization (29). Present data demonstrate that the truncation of the HIR cytoplasmic tail, either at position 981 or at position 1000, transforms this typical class II receptor into a class I receptor. Indeed, as demonstrated either by tagging the receptor with an anti-insulin receptor antibody (that does not activate the receptor tyrosine kinase (25)) or through biotinylation of the cell surface proteins, internalization of these two truncated receptors shows the characteristics of constitutive internalization. It occurs at a high rate, independent of insulin binding. These observations are similar to the one obtained with a HIR truncated at position 965 which, as determined by different techniques, appeared also to be constitutively internalized (6). They extend, however, these former observations by eliminating 35 amino acids (amino acids 965-999) from the domain which is the candidate to determine the ligand-specificity of HIR internalization. Taken together with previous observations that the last 95 amino acids of the COOH-terminal tail of HIR are not required for insulin-induced HIR internalization (1), it can thus be concluded that the HIR domain(s) governing insulin-induced HIR internalization is (are) contained between amino acids 1000 and 1248.
In apparent contradiction with these conclusions are the results
obtained with the mutant insulin receptor in which the two leucines at
positions 986-987 have been replaced by alanines. Indeed, although the
AA1-mutant insulin receptor retains the normal amino acid sequence
downstream from amino acid 1000, the unoccupied receptor exhibits a
decreased association with microvilli, suggesting that the dileucine
motif plays a role in this association. However, the presence of this
dileucine motif within 1000 receptors does not improve their ability
to associate with microvilli as compared with
981 receptors,
indicating that the insulin receptor domain centered on leucines
986-987 is not sufficient to allow HIR anchoring on microvilli. It
must rather act in conjunction with another major domain present
downstream from position 1000.
To maintain unoccupied normal HIR on microvilli requires not only a specific anchoring sequence present in the cytoplasmic domain of HIR ("receptor side" component discussed above) but also a "cellular side" partner with which the receptor should interact. No information is available regarding this "cellular side" component of the brake, but the association of unoccupied HIR with microvilli suggests an implication of cytoskeleton elements which are particularly enriched in these regions. Various surface proteins including epidermal growth factor receptors and L-selectin interact with cytoskeleton elements (30-32), but, to our knowledge, the mechanism responsible for their positioning on microvilli remains unknown. Members of the ERM (ezrin, radixin, moesin) family of cytoskeletal proteins, abundant in microvilli and becoming tyrosine-phosphorylated in response to the formation of various ligand-receptor complexes (i.e. epidermal growth factor receptors and CD4), represent good candidates for such a function (33, 34).
Despite the fact that the unoccupied AA1-mutant HIR was not retained
efficiently on microvilli, the constitutive internalization of this
mutant receptor was not increased. On the other hand, the
insulin-stimulated internalization of HIR AA1 remained lower than that
of HIR WT. Thus, both constitutive and insulin-stimulated HIR AA1
internalizations are relatively less efficient than corresponding HIR
WT internalization processes. Present data demonstrate that defects in
internalization of HIR AA1 were caused by a decreased propensity of the
mutant receptor to associate with clathrin-coated pits. This is the
first direct demonstration of the involvement of dileucine motifs in
the segregation of HIR in clathrin-coated pits. It provides the clue to
the inhibition of internalization previously described for a series of
receptors with a mutation of dileucine motif(s) present in their
cytoplasmic tail, including CD4, CD3 ( and
chains),
interleukin-6 receptor, IgG Fc receptor, insulin receptor, Iip 31 invariant chain which associates with major histocompatibility complex
II molecules, and interferon-
receptor (20, 35-38). It remains an
open question how dileucine motifs mediate receptor segregation in
clathrin-coated pits. Recent studies making use of a yeast two-hybrid
system have failed to demonstrate a direct interaction with the µ chains of the AP-complexes similar to the one observed in the case of
tyrosine-based internalization motifs (i.e. a short
four-amino acid sequence containing a tyrosine at position 1 followed
by two basic amino acids and ending with a hydrophobic residue (39,
40)). However, these results do not rule out the possibility of either
an interaction of dileucine motifs with another chain of the AP-complex
or the participation of a connecting molecule, intermediate between the
dileucine motif and the AP-complex, as recently suggested in the case
of CD4 (41).2
Two tyrosine-based motifs present in the juxtamembrane domain of the
cytoplasmic tail of HIR (GPLY and NPEY) have previously been
demonstrated to participate in HIR segregation in clathrin-coated pits
(1, 2, 6, 13, 14). Although different from the 4-amino acid stretches
that interact directly with AP-2, similar motifs forming a turn and
exposing an aromatic amino acid (preferentially a tyrosine) have been
described as participating in the internalization of various receptors
via clathrin-coated pits (43-46). The high constitutive
internalization rate of HIR
981, which includes the two
tyrosine-based motifs but does not contain the dileucine motif, and the
ability of this receptor to preferentially associate with
clathrin-coated pits confirm these observations. The comparison of the
behavior of this truncated HIR with that of HIR
1000 reveals, however, that the optimal anchoring is obtained when, in addition, the
dileucine-based motif of exon 17 is present. Thus, at least three
signals are required in the cytoplasmic domain of HIR for maximally
efficient association with clathrin-coated pits: the two tyrosine-based
motifs encoded by exon 16 and a dileucine-based motif encoded by exon
17.
Both AA1 and 981 receptors contain the two juxtamembrane
tyrosine-based motifs participating in anchoring in clathrin-coated pits, and lack the dileucine-based motif. Nevertheless, in the absence
of kinase activation, HIR AA1 differs from HIR
981 in its ability to
anchor on these internalization gates since: (a) constitutive internalization of insulin receptors occurs at a higher
rate in
981 cells than in AA1 cells and (b)
981
receptors show a higher propensity to associate with clathrin-coated
pits than AA1 receptors, in the course of constitutive internalization. However, in the presence of insulin, AA1 receptors increase their affinity for clathrin-coated pits so that it becomes comparable to that
of the
981 truncated receptors (Fig. 7). These observations suggest
that the tyrosine-based signal sequences present in the juxtamembrane
domain are mostly inaccessible in the inactivated receptor and that
these motifs are unmasked by kinase activation as previously suggested
(13). In contrast, the dileucine motif does not seem to be masked in
the inactivated receptors. Indeed, as described previously in Chinese
hamster ovary cells (10), kinase-inactive insulin receptors (with
masked tyrosine-based motifs, see above), present on the non-villous
surface domains of NIH 3T3 fibroblasts, show a high propensity to
associate with clathrin-coated pits. In these conditions, where the
kinase is inactive, the implication of the dileucine motif is evidenced by the reduced ability of HIR AA1 receptors to associate with these
surface domains as compared with that of HIR WT receptors.
Although two motifs have been implicated in the anchoring of the
insulin receptor on clathrin-coated pits, the dileucine motif appears
to play the most important role. Indeed, when association with
clathrin-coated pits was quantitated exclusively in terms of the
radioactive ligands present on the non-villous domains of the cell
surface (Fig. 8), several lines of evidence suggested the major role of
the dileucine motif in this anchoring function: (a)
inactivated HIR WT (acting via the dileucine motif) showed a higher
propensity than activated HIR AA1 (acting through the tyrosine motifs)
to associate with clathrin-coated pits; (b) for both HIR AA1
and HIR WT, only small differences were noted between the incubations
carried out in the presence or absence of insulin (which reflected the
role of the unmasking of tyrosine domains), (c) major
differences (reflecting the role of the dileucine motif) separated HIR
981 and HIR
1000 receptors, (d) in the presence of
insulin, significative differences distinguished HIR WT receptors (tyrosine-based and dileucine motifs exposed) and HIR AA1 receptors (only tyrosine-based motifs exposed), and (e) similarly, in
the absence of insulin, HIR WT receptors (dileucine motif present) showed a significantly higher propensity to associate with
clathrin-coated pits than HIR AA1 receptors (dileucine motif absent).
This major role of the dileucine motif in HIR association with
clathrin-coated pits could help to resolve the dilemma regarding the
potential dual function of the juxtamembrane tyrosine-based motifs
involved not only in association with clathrin-coated pits but also
representing the site of insulin receptor substrate-1 and Shc binding
to the insulin receptor (42, 47, 48).
In conclusion, the dileucine-based motif present at positions 986-987
of the cytoplasmic domain of the -subunit of the insulin receptor
plays a dual role in the control of HIR internalization. The first
function consists in participating in the anchoring of the unoccupied
receptor on microvilli. Its second role resides in the anchoring of the
receptor (occupied or not) on the internalization gates: the
clathrin-coated pits. This latter function is independent of receptor
kinase activation in contrast with the tyrosine-based motif
participation in this same function.
We thank G. Porcheron-Berthet for skilled technical assistance.