(Received for publication, October 7, 1994)
From the
To examine whether the surface redistribution of the insulin receptor from microvilli, where it sits in its unoccupied form, to the nonvillous domain, where it is internalized through clathrin-coated pits, is an active movement or a passive redistribution linked to the release of a restraint maintaining it on microvilli, we have generated a mutated insulin receptor with a truncation of exons 17-22 and tracked it biochemically and morphologically. Biochemical analysis indicates that this mutated receptor is constitutively internalized and recycled even in the absence of ligand. Quantitative electron microscope autoradiography analysis reveals that it does not preferentially associate with microvilli in its unoccupied form but is normally segregated in clathrin-coated pits through the preserved signal sequence(s) of exon 16. We conclude that (a) insulin receptor internalization is initiated through receptor kinase activation and autophosphorylation, which free the receptor from constraints maintaining it on microvilli; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors; (c) these sequences are not uncovered by kinase activation; and (d) the ``code'' maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.
Most plasma membrane receptors and their ligands undergo receptor-mediated endocytosis and are taken up by cells through the formation of clathrin-coated vesicles(1, 2, 3, 4, 5) . But, while transport protein receptors such as the receptors for low density lipoproteins or transferrin are rapidly and constitutively internalized, signaling receptors such as those for insulin and epidermal growth factor internalize at rapid rates only when ligand is bound(1, 2, 3) . These two classes of receptors differ not only on the basis of their function (delivering of nutrients to the cells versus intracellular signaling) and of the ligand dependence or independence of the internalization process but also in their topography. Class I receptors, typified by the low density lipoprotein and the transferrin receptors, are present in clathrin-coated pits in their unoccupied form while class II receptors, like the insulin receptor, are preferentially found associated with surface microvilli in their unbound state(3, 6, 7) . Following entry into the cells, the intracellular pathway followed by receptors of these two classes is similar; the ligand-receptor complex is cleaved at the acidic pH of endosomes and its two components are targeted in different directions: the ligand is generally routed to lysosomes while the receptor is classically recycled back to the cell surface where it can be reused(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The signals responsible for targeting receptors for endocytosis are
being defined. Receptor-mediated endocytosis requires specific amino
acid sequences found in the cytoplasmic tail (frequently the
submembranous domains) of receptors. They have in common a propensity
to form a tight turn exposing an aromatic residue (preferentially
a
tyrosine)(2, 5, 10, 11, 12, 13, 14, 15, 16, 17) .
These sequences are recognized by proteins (probably the adaptors) that
link the receptors to clathrin-coated pits (18, 19, 20, 21, 22) .
Similar sequences in the juxtamembrane cytoplasmic domain of the
insulin receptor (GPLY and NPEY) have been found to be necessary for
endocytosis(23, 24, 25, 26, 27) .
In the case of the insulin receptor, however, these sequences are not
sufficient for endocytosis; rapid internalization also requires
activation of the receptor tyrosine kinase, probably accounting for the
observed ligand dependence of insulin receptor
endocytosis(27, 28, 29, 30, 31) .
The tyrosine kinase of the epidermal growth factor receptor has also
been shown to be required for its
internalization(32, 33) . Tyrosine kinase activation
is involved in the surface redistribution of the insulin receptor from
microvilli to the nonvillous surface of the
cell(27, 28) , but how the receptor tyrosine kinase
initiates this initial step of endocytosis is not clear. Tyrosine
kinase activation could play an active role and initiate a controlled
redistribution of the receptor in the direction of the clathrin-coated
pits of the nonvillous domain of the cell surface. Conversely, tyrosine
kinase activation could simply act to release receptors from
microvilli, whereupon localization to clathrin-coated pits and
endocytosis occurs without further kinase-dependent signaling.
In the present study, we have addressed this question by examining in detail the endocytotic itinerary of an insulin receptor deleted of the cytoplasmic tyrosine kinase domain and COOH terminus, thus leaving only the juxtamembrane domain intact. Biochemical and ultrastructural analyses indicate that the truncated insulin receptor does not preferentially associate with microvilli and undergoes efficient constitutive endocytosis. We conclude that the signal allowing the anchoring of the unoccupied insulin receptor on microvilli is contained within the regulatory domain of the insulin receptor and that the tyrosine kinase activation of the insulin receptor initiates insulin receptor internalization through the release of a brake maintaining it on microvilli. Moreover, the submembranous endocytotic motifs of the insulin receptor, once available to the endocytotic machinery, appear sufficient for endocytosis.
Insulin internalization was measured as
described(30, 35) . Briefly, cells in 6-well dishes
(10
cells/dish) were equilibrated at 37 °C in
Krebs-Ringer phosphate, 10 mM HEPES, 0.2% bovine serum
albumin. The cells were then exposed to 10 pM
I-insulin for various periods of time, after which
the cells were rinsed rapidly three times in ice-cold saline (pH 7.6)
and then exposed to 1 ml of incubation medium at pH 4.0 for 6 min on
ice to remove surface-bound insulin. This plus a further 1 ml of rinse
(pH 4.0) were combined, and radioactivity corresponding to
surface-bound insulin was determined in a
counter. The rinsed
cells were solubilized in detergent, and radioactivity corresponding to
internalized insulin was also determined. Counts bound and internalized
in the presence of excess (300 nM) unlabeled insulin were less
than 10% of the counts observed with labeled insulin only and were
subtracted.
Trypsinization of intact cells and the determination of insulin binding activity in solubilized extracts of untreated or trypsinized cells in order to determine the relative proportions of intracellular and cell surface receptors were performed as described(35) .
We first
examined the internalization of wild-type (WThIR) or truncated
(hIRex17-22) receptors expressed in Rat-1 fibroblasts and
labeled with an insulin photoaffinity probe. The cells were initially
exposed to ligand in the cold so only the surface receptors would be
labeled. The cells were then exposed to UV light to cross-link the
ligand to the receptor and warmed at 37 °C for various periods of
time. At each time point studied, cells were trypsinized to degrade any
receptors still at the cell surface. As the receptors internalized they
became trypsin-resistant such that internalized receptors remained
intact while the receptors remaining at the plasma membrane were
degraded by trypsin. Intact labeled
-subunits of the receptor were
then quantified after reducing gel electrophoresis and autoradiography.
As can be seen in Fig. 1, before warming (time 0)
greater than 95% of either normal or truncated hIR were at the cell
surface and susceptible to trypsin degradation. With time at 37 °C,
normal insulin receptors so labeled undergo internalization such that
after 40 min, 52.6% of the receptors were trypsin-resistant. The
truncated receptors did internalize more slowly, but after 40 min a
significant number of them (22.2%) were intracellular. By comparison,
the intracellular fraction of a noninternalizing kinase-defective
receptor did not exceed 8-9% even after 60 min(30) .
Labeling of receptors using this technique was performed at relatively
high but not saturating insulin concentrations (
2 nM)
where roughly 50% receptor occupancy would be expected. Thus, the
proportion of labeled receptors could not exceed 50% even assuming only
a minimal degree of inefficiency of covalent coupling of the
photoprobe. If nonligand-occupied hIR
ex17-22 were
internalizing as well as the receptors labeled by the ligand, the total
internalization rate of the hIR
ex17-22 could be comparable
with that of the WThIR. We therefore examined whether unoccupied
hIR
ex17-22 might also be undergoing constitutive
endocytosis.
Figure 1:
Internalization of wild-type and
hIRex17-22 receptors labeled with
I-NAPA-DP
insulin.
I-NAPA-DP insulin was bound to cells expressing
wild-type (WT) hIR or the truncated mutant hIR
(hIR
ex17-22) and the cells photoderivatized as described.
After various times at 37 °C the cells were trypsinized and
analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A, autoradiogram showing intact hIR
-subunits in treated
cells. Receptors from cells not treated with trypsin are on the left (total), indicating the total number of labeled
receptors. In cells trypsinized before warming (0) less than
5% of the receptors remain intact, illustrating that the labeled
receptors are initially exclusively at the cell surface and that the
trypsinization procedure is effective in degrading nearly all of the
surface receptors. As a function of time at 37 °C, more receptors
escape trypsin sensitivity as they internalize. B,
quantitation of internalized receptors as a function of time at 37
°C. Two experiments such as the one illustrated above were
performed, and the labeled bands visualized by autoradiography were
excised and radioactivity determined in a
counter. The counts in
untrypsinized cells (A, total) were taken as 100%, and the
percent of that total that were trypsin-resistant is
plotted.
Figure 2:
A,
distribution of wild-type and hIRex17-22 receptors in
unstimulated cells. Cells expressing either WThIR (solid bar)
or hIR
ex17-22 (dotted bar) were solubilized in the
absence of insulin and total receptor number determined in solution as
described(30) . Cells were also trypsinized to degrade cell
surface receptors and the number of trypsin-resistant (intracellular)
receptors determined in solubilized extracts. The percentages of
intracellular receptors (trypsin resistant/total) are plotted and are
the means of three experiments (±S.E.), each performed in
triplicate. Insulin binding assays were performed as binding
competition curves; these revealed no significant difference in
affinity between the receptors from cells trypsinized or not, so the
plotted results are of tracer (10 pM) insulin binding. B, change in intracellular receptor number after insulin
treatment. Cells were treated for 60 min at 37 °C with near
saturating insulin (30 nM), and the cell receptor number
(intracellular and total) was determined as above. Results are plotted
as the percent increase in intracellular receptors shown in panel A and are the means of three experiments assayed in
duplicate.
Receptor distribution
after treatment of cells with the lysosomotropic drug chloroquine that
blocks receptor recycling was also investigated and was also consistent
with the truncated hIRex17-22 constitutively undergoing an
endocytotic itinerary. As shown in Fig. 3, after exposure to
chloroquine but no insulin for 24 h, hIR
ex17-22 cells had
lost 20.3 ± 5.4% of their surface insulin binding compared with
a negligible loss (1.0 ± 3.6%) of binding in cells expressing
normal receptors. There was a concomitant increase in the fraction of
intracellular trypsin-resistant receptors after chloroquine treatment
of the truncated but not normal receptors, comparable with what was
seen with cells expressing normal receptors but after insulin treatment (Fig. 3).
Figure 3:
Effect of chloroquine treatment on insulin
receptor distribution in the absence of insulin. Left, cells
expressing WThIR (solid bar) or hIRex17-22 (dotted bar) were exposed to 100 µM chloroquine
for 1 h. Cell surface insulin binding was then assayed and compared
with that of control cells incubated in the absence of chloroquine.
Results are the means of two independent experiments, each assayed in
triplicate. Right, cells treated with or without chloroquine
were either trypsinized or not, and the percentage of intracellular
(trypsin-resistant) receptors was determined in solubilized extracts of
cells as described under ``Experimental Procedures.'' Results
are the means of two experiments, each assayed in
quintuplicate.
Figure 4:
I-insulin internalization in
Rat-1 fibroblasts transfected with WThIR or hIR
es17-22.
Results presented are the average of the analysis of four different
Epon blocks. For each time point and each cell line =
600-800 autoradiographic grains were quantitated. Results are
expressed as a percent of the total number of grains associated with
the cells whose centers were >250 nm from the plasma
membrane.
As previously observed in isolated
rodent hepatocytes, human monocytes and various cultured
cells(38, 39, 40, 41) , I-insulin preferentially associated with microvilli on
the surface of wild-type cells at 4 °C, demonstrating that the
unoccupied insulin receptor was preferentially localized on these
surface domains in cultured Rat-1 fibroblasts (Fig. 5). Warming
of the incubation medium to 37 °C resulted in a redistribution of
the radioactive hormone-hIR complex in the direction of the nonvillous
domain of WT cells (Fig. 5). By contrast, in
hIR
ex17-22 cells,
I-insulin did not
preferentially associate with microvilli at 4 °C (Fig. 5),
and at 37 °C, the labeled material remained localized on the
nonvillous regions of the cell surface at all time points studied (Fig. 5).
Figure 5:
Surface redistribution of I-insulin in Rat-1 fibroblasts transfected with WThIR or
hIR
ex17-22. Results presented are the average of the
analysis of four different Epon blocks. For each time point and each
cell line, 600-800 autoradiographic grains were quantitated.
Results are expressed as a percent of the total number of grains
associated with the cell surface (±250 nm from the plasma
membrane) whose centers were within 250 nm of
microvilli.
The redistribution of I-insulin on
the surface of WT cells was accompanied by a progressive sequestration
of the radioactively labeled material in clathrin-coated pits. By 2 h
at 4 °C less than 4% were associated with these surface
differentiations while by 30 min this value reached 14.5% (Fig. 6A). By contrast, in hIR
ex17-22 cells,
by 2 h of incubation at 4 °C and before warming, 13.7% of
surface-bound
I-insulin was already present in
clathrin-coated pits, and this association of the radiolabeled ligand
with clathrin-coated pits remained practically constant with time at 37
°C. When the quantification was restricted to autoradiographic
grains present on the nonvillous domain of the cell surface and the
grains present on this surface domain at all time points were pooled,
the propensity of occupied WThIR and hIR
ex17-22 insulin
receptors to anchor to clathrin-coated pits was identical (Fig. 6B).
Figure 6:
Association of I-insulin
with clathrin-coated pits. A, percent of autoradiographic
grains present on the total surface of Rat-1 fibroblasts transfected
with WThIR or hIR
ex17-22 that are associated with
clathrin-coated pits. B, percent of the autoradiographic
grains present on the nonvillous surface of Rat-1 fibroblasts
transfected with WThIR or hIR
ex17-22 that are associated
with clathrin-coated pits. Results presented are the average of the
analysis of four different Epon blocks. Results are expressed as a
percent of the total number of grains associated with the cell surface
(±250 nm from the plasma membrane) whose centers were within 250
nm from a clathrin-coated pit. In B, values are the mean of
the values obtained at the 5, 15, and 30 min time points ± S.E. (n = 3).
The requirement of receptor tyrosine kinase activation and autophosphorylation for tyrosine kinase receptor internalization is widely accepted(28, 29, 30, 31, 32, 33, 42, 43, 44) . In the case of the insulin receptor, this activation governs the first step of receptor internalization: the ligand-dependent induction of receptor redistribution from microvilli to the nonvillous domain of the cell surface(27, 28) . A question is whether this process is active or passive. The ``active'' hypothesis would involve activation of enzyme(s) (probably via phosphorylation reactions) inducing a directed redistribution of the receptors on the cell surface and controlling their segregation in the internalization gates: the clathrin-coated pits. The ``passive'' process would imply that a brake is keeping the receptors out of the endocytotic machinery. In this case, ligand binding would result, via kinase activation and receptor autophosphorylation, in the release of this block to internalization. In the case of this passive hypothesis, two further possibilities exist: either the process is mediated via phosphorylation of a specific substrate(s) that mediates internalization or autophosphorylation of the receptor molecule itself constitutes the signal(s) for internalization through unmasking of endocytotic signal sequences. The present study addresses these questions. Data presented demonstrate that (a) the process is occurring through the release of a brake: receptor kinase activation and autophosphorylation free the receptor from constraints maintaining it on microvilli of the cell surface; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors, which have access to the nonvillous domain of the cell surface; (c) these sequences are not uncovered by kinase activation; and (d) the ``code'' maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.
In its
unoccupied state, the insulin receptor preferentially localizes on
microvilli(27, 28, 38, 39, 40, 41) .
This may facilitate its optimal interaction with circulating insulin.
What maintains the receptor on these thin digitations of the cell
surface remains unknown, but an interaction with cytoskeleton elements
particularly rich in the submembrane domain of these regions is highly
probable. Taken together with previous observations, present data
indicate that the receptor site involved in this potential interaction
is contained neither in the juxtamembrane domain nor in the
COOH-terminal tail (27, 28) but in the regulatory
domain of the cytoplasmic tail of the receptor and that it is abolished
by kinase activation. The regulatory domain contains dileucine motifs
that have been shown to control endocytosis of several receptors
including the insulin receptor(45, 46) . ()Studies are in progress to determine their exact role in
the initial stages of insulin receptor internalization and especially
in the anchoring of the unoccupied receptor on microvilli.
We
observed that the truncated receptor with a cytoplasmic tail containing
only the juxtamembrane domain of the receptor but no kinase domain did
not preferentially associate with microvilli in its unoccupied form.
Rather, the unoccupied truncated receptor showed the same propensity as
an activated normal hIR to associate with the nonvillous domain of the
cell surface, arguing against an active insulin-induced surface
redistribution of the receptor mediated by kinase activation and
receptor autophosphorylation. Moreover, taken together with previous
observations that kinase-inactive or autophosphorylation-deficient
insulin receptors preserve their capacity to associate with
clathrin-coated pits(27, 47) , present data showing
that hIRex17-22 receptors exhibit full capacity to be
segregated in clathrin-coated pits strengthen the argument that kinase
activation is not required for clathrin-coated pit association and that
the signal sequences allowing insulin receptor anchoring in
clathrin-coated pits are not unmasked by kinase activation.
Whether
the activated tyrosine kinase phosphorylates a specific substrate(s)
that mediates the release from microvilli or whether
autophosphorylation of the receptor molecule itself constitutes the
signal for this release remains an open question. In support of the
first possibility are the recent observations that ligand-induced
internalization of two tyrosine kinase receptors (platelet-derived
growth factor receptor and fibroblast growth factor receptor) depends
on the recruitment, at the autophosphorylation site of the receptor, of
specific regulatory proteins implicated in the initiation of a
phosphorylation cascade (phosphatidylinositol 3-kinase and
phospholipase C, respectively)(42, 43) . In the
case of the insulin receptor, neither IRS-1 nor phosphatidylinositol
3-kinase, two substrates that play key roles in the early stages of
postreceptor insulin signal transduction, seem involved in mediating
insulin receptor
internalization(24, 27, 48) . On the other
hand, the presence in the cytosol of unidentified specific factors
required for kinase-dependent internalization of insulin receptors was
recently proposed(44) . Thus, a dichotomy in the signals
initiated by kinase activation that mediate biological action and
internalization is highly probable. Strengthening this argument is our
recent observation that insulin receptor internalization is not
required for transmission of insulin's biological
actions(49, 50) .
When insulin receptors have
access to the nonvillous domain of the cell, their internalization
becomes relatively nonspecific and similar to that of most receptors
that are constitutively internalized whether a ligand is bound or not (i.e. transferrin receptor, low density lipoprotein receptor,
. . . ). In these conditions, the internalization sequence(s)
identified in the juxtamembrane domain of the insulin receptor and
which is (are) analogous to the ones present in most of these receptors
(tight turn exposing an aromatic amino acid) is (are) sufficient
to promote a rapid and efficient internalization of the receptor. Based
on the slow internalization of insulin mediated by the
hIR
ex17-22 receptor, we had earlier concluded that the
receptor did not undergo endocytosis at a rate equivalent to the
complete receptor(25) . In these experiments, however, it is
difficult to compare ligand-independent or -constitutive endocytosis
with ligand-triggered endocytosis because the measure of endocytosis is
the ligand itself. If endocytosis is constitutive, ligand-occupied
receptors will not be preferentially internalized, so the
internalization rate will appear lower than ligand-dependent
endocytosis because those unoccupied receptors that are concomitantly
internalized are not being scored. Biochemical comparisons of the
resting distribution of hIR
ex17-22 and WThIR between the
surface and the inside of the cells and of the influence of chloroquine
and insulin on these distributions confirm this interpretation of the
data. (a) A larger fraction of receptors was internal in
hIR
ex17-22 cells than in hIR cells, indicating a significant
continuous turnover of receptors in hIR
ex17-22 cells but not
in hIR cells; (b) a blockade of receptor recycling by
chloroquine increased the intracellular fraction of
hIR
ex17-22 receptor but had no effect on unoccupied hIR
receptors, which is also in favor of a constitutive internalization
recycling of hIR
ex17-22 receptors; and (c) insulin
had no effect on the distribution of hIR
ex17-22 receptors
while it led to an increase in the proportion of internal hIR receptors
confirming that insulin receptor internalization was essentially
ligand-dependent in hIR cells.
Our observations disagree with those
recently published by Smith et al.(51, 52) who claimed that insulin-induced kinase
activation and autophosphorylation did not control the surface
redistribution of the insulin receptor but were required for the
receptor to concentrate in clathrin-coated pits. Since in both cases
the mutations studied were very similar and Rat-1 fibroblasts were used
as the host cells, the most likely explanation for the differing
conclusions is in the morphological probe used. Large electron-dense
probes (i.e. ferritin or colloidal gold) have frequently
raised suspicions about the reliability of results generated due to the
fact that these large molecules could perturb the subcellular
distribution of the bound molecule and to the difficulty in producing a
conjugate that preserved the full biological properties of the native
ligand. In the present work as well as in previous ones (for review see (6) and (7) ), we have tracked, by quantitative
electron microscope autoradiography, monoiodinated I-human insulin, a ligand with full biochemical and
biological activation and whose use is widely accepted as valid.
In conclusion, taken together with previous observations(26, 27, 28) , data presented allow us to propose the following ordered sequence of events, including ligand-dependent and ligand-independent steps, leading to insulin receptor internalization in target cells.
1) In its unoccupied and unstimulated state, the insulin receptor preferentially associates with microvilli on the cell surface. This preferential association is dependent on the integrity of the cytoplasmic tail of the receptor, but neither the juxtamembrane domain, nor the COOH-terminal domain, nor kinase activation and receptor autophosphorylation play a role in this association.
2) Insulin binding releases the constraint maintaining the receptor on microvilli and does so via receptor kinase activation and autophosphorylation of the three tyrosine residues present in the regulatory domain.
3) The insulin-receptor complex, freely mobile on the cell surface, next associates with the internalization gates, the clathrin-coated pits, via signal sequences contained in the juxtamembrane domain of the cytoplasmic tail of the receptor. These sequences, which are constitutively unmasked (neither kinase activation nor specific autophosphorylation of the three tyrosines of the kinase domain is required to uncover them), are sufficient to promote rapid and efficient internalization of the receptor.
4) The subsequent invagination, budding, and pinching off to form clathrin-coated vesicles would then proceed as with all classes of receptors employing this pathway(53) .