(Received for publication, June 3, 1994; and in revised form, October 24, 1994)
From the
Insulin causes rapid insulin receptor autophosphorylation, receptor endocytosis, and phosphorylation of its principle substrate (IRS-1). Using rat adipocytes, we studied the dynamics of receptor autophosphorylation, the kinase activity, and the IRS-1 phosphorylation state relative to the subcellular localization of these proteins. After 2 min of insulin exposure, the specific phosphotyrosine content of the insulin receptor in the internal membranes (IM) peaks at a level 5-6-fold higher than the plasma membrane (PM) receptor and then declines after 5-8 min to a level similar to the PM receptor. The exogenous kinase activity of these receptors exactly mirrored their phosphotyrosine content. The distribution of IRS-1 is 80% cytosolic, 20% IM-associated, and essentially undetectable in the PM. The phosphorylation state of IRS-1 in the IM parallels that of the insulin receptor, but cytosolic IRS-1 phosphorylation remains constant. Insulin-dependent GLUT4 translocation to the PM occurs after the peak of IRS-1 phosphorylation. The data are consistent with the hypothesis that insulin action may be mediated by receptor internalization and interaction with its substrate(s) associated with internal membranes. A small fraction of phosphorylated insulin receptors is sufficient for signal transduction. The dephosphorylation of the insulin receptor and IRS-1 in the IM appears to be a concerted process, possibly mediated by the same enzyme.
The biological effects of insulin are mediated by binding of the
hormone to its cell surface receptor, which results in subunit
autophosphorylation and activation of its exogenous tyrosine kinase
activity (reviewed in (1, 2, 3, 4) ). This kinase activity
is requisite for the many biological effects of insulin, including the
critically important activation of glucose transport(5) .
Concomitant with or shortly after receptor activation, the insulin
receptor undergoes endocytosis into an acidic compartment where
dissociation of the ligand occurs(6) , and then the receptors
cycle back to the cell surface(7, 8) . Internalization
has been shown to be dependent on receptor autophosphorylation and
kinase activity(9) , as well as on an intact juxtamembrane
region of the insulin receptor(10) . Previous studies have
shown that internalized insulin receptors are phosphorylated and active
toward exogenous substrates(11, 12, 13) ,
thus suggesting a possible physiological role for internalization in
the propagation of the insulin signal. Another consequence of
internalization is the termination of the insulin signal by two
processes. Dissociation and degradation of the ligand is required to
prevent continual stimulation of autophosphorylation, and then
dephosphorylation leads to deactivation of the receptor's
exogenous kinase activity. Studies in rat hepatoma cells have shown
that the phosphorylation of internalized receptors persists even after
the dissociation of insulin but that receptors are dephosphorylated
prior to recycling back to the cell surface(12) . A number of
studies have examined the dynamics of internalization of the insulin
receptor(14, 15, 16, 17) , and other
studies have focused on the intrinsic kinetic properties of insulin
receptor autophosphorylation and kinase activity in
vitro(18) . However, the physiological interrelationship
among autophosphorylation, exogenous kinase activity, and endocytosis
has not been systematically investigated. Also, only limited studies of
insulin receptor dephosphorylation have been performed(19) ,
and the physiologically relevant phosphotyrosine phosphatase(s) has yet
to be identified.
Insulin produces a range of pleiotropic effects in
target cells that include acute metabolic changes and longer term
growth promoting effects. The metabolic effects include the
translocation of a pool of insulin-sensitive glucose transporters from
internal vesicles to the cell surface, thereby promoting the uptake of
glucose in insulin-sensitive tissues such as fat and
muscle(20) . The cascade that leads to the activation of these
processes is not clearly defined but is likely to include the
phosphorylation of the principal insulin receptor substrate
(IRS-1)()(21) . Phosphorylated IRS-1 has been shown
to interact with several Src homology 2 (SH2) domain-containing
proteins(22, 23, 24, 25, 26) .
Phosphatidylinositol 3-kinase (PI-3-kinase) is one such SH2
domain-containing protein whose activity is stimulated by insulin (27, 28, 29) via binding of phosphorylated
IRS-1. This activation appears to be restricted to the microsomal
fraction of fat cells(30) . The relationship of these effectors
to downstream targets of insulin action such as glucose transport are
as yet unclear, although recent data using fat cells have shown that
Wortmannin, an inhibitor of PI-3-kinase, blocks glucose transport
activation by insulin at similar concentrations to those required for
PI-3-kinase inhibition(31) .
The fact that the activation of
PI-3-kinase takes place in the microsomal membranes in fat cells (30) raises the issue of how this might be occurring. Previous
studies from our lab showed that a microsomal membrane-associated
protein of M 160,000 was rapidly phosphorylated on
tyrosine residues in response to insulin(32) , although we do
not know if this protein corresponds to IRS-1. As a result of this
study, we raised the possibility that there might be direct contact
between activated insulin receptor at the cell surface and internal
membrane-associated pp160 (possibly IRS-1) or that internalization of
the insulin receptor in an activated state results in substrate
phosphorylation in internal membranes. In light of the recent data
linking activation of microsomal PI-3-kinase to glucose transport
activation, we decided to examine the dynamics of insulin receptor
activation and endocytosis relating to IRS-1 phosphorylation and GLUT4
translocation in rat adipocytes. In the present study, we use a
physiological concentration of insulin to study the phosphorylation,
activation, and subsequent dephosphorylation and deactivation of the
insulin receptor and IRS-1 as a function of time and subcellular
compartmentalization.
Figure 1:
The
internalized insulin receptor is initially highly phosphorylated when
compared with cell surface receptor. Fat cells isolated from rat
epididymal fat pads were treated with insulin and, at the indicated
times, were homogenized. Internal vesicles and plasma membranes were
isolated by differential centrifugation (see ``Experimental
Procedures''). Samples from each time point were separated by
SDS-PAGE using a 3-10% gradient gel in the absence of reducing
agents and electrotransferred to PVDF Immobilon membranes. Western blot
analysis was performed (A) using an anti-insulin receptor
antibody R1064 (upperpanel) and an
anti-phosphotyrosine antibody 4G10 (lowerpanel).
Shown is the holoreceptor of M
350,000. R1064 blots were developed using
I-protein A, and 4G10 blots were developed using goat
anti-mouse coupled to horseradish peroxidase using chemiluminescence.
The data were scanned using a computing densitometer, manipulated to
reflect total receptor amounts, and graphically displayed as arbitrary
units (A.U.). B shows the time course of
internalization of the insulin receptor from the plasma membrane (closedcircles) to the internal membranes (opencircles). C shows the level of phosphotyrosine
in the two fractions as above. The immunoblots presented are
representative of three experiments.
Figure 4: The time course of phosphorylation of the insulin receptor in the internal membrane fraction is mirrored by its exogenous kinase activity and by the time course of IRS-1 phosphorylation in the same fraction. The immunoblots from Fig. 1and Fig. 3were scanned and expressed as phosphotyrosine content per insulin receptor or IRS-1, respectively, by normalizing to the amount of each protein as determined by Western blotting. The exogenous kinase activity data from Fig. 2is also normalized to the amount of insulin receptor and plotted as a time course. All of the data are in arbitrary units (A.U.). The upperpanel shows the time course of specific phosphorylation of insulin receptor in the internal membranes. The middlepanel shows the time course of specific exogenous kinase activity of insulin receptors in the internal membranes. The lowerpanel shows the time course of specific phosphorylation of IRS-1 in the internal membranes.
Figure 3:
IRS-1 is phosphorylated at the internal
membrane with a time course that immediately follows that of the
exogenous kinase activity of the insulin receptor. Fat cells were
treated with insulin for various times, and IM, PM, and cytosol were
separated as described above. Plasma membranes (400 µg) from each
time point were separated by SDS-PAGE and immunoblotted with antibodies
to IRS-1 (A). Concentrated cytosolic protein (200 µg, 4%
of total cytosolic) and solubilized internal membrane protein (200
µg, 18% of total internal) from various time points were
immunoprecipitated using anti-IRS-1 antibodies as described under
``Experimental Procedures.'' The immunoprecipitates were
separated by SDS-PAGE using a 7% gel and immunoblotted (B)
using anti-IRS-1 (upperpanel) or
anti-phosphotyrosine antibodies (lowerpanel). IRS-1
blots were developed using I-protein A, and
phosphotyrosine blots were developed using goat anti-mouse coupled to
horseradish peroxidase using chemiluminescence. The immunoblots shown
are representative of two experiments. The increase seen in the amount
of IRS-1 in the cytosol with time is an artifact of the process of
concentrating the cytosol since the total amount of IRS-1 in the
cytosol does not change with time.
Figure 2: The exogenous kinase activity of the insulin receptor in the internal membranes is higher than that at the plasma membranes. Membranes extracted from fat cells treated with insulin for various times, as in Fig. 1above, were solubilized in Triton X-100, and insulin receptors were partially purified using wheat germ-agarose beads. These partially purified receptor fractions were used to assess exogenous kinase activity against poly Glu/Tyr without further treatment with insulin, as described under ``Experimental Procedures.'' Equal aliquots of each fraction were separated by SDS-PAGE and immunoblotted using anti-insulin receptor antibody, and the amount of receptor in each fraction was quantified by scanning. The data are expressed as arbitrary units (A.U.) of kinase activity normalized to the amount of insulin receptor. The shadedbars represent the internal membranes, and the openbars represent the plasma membranes. The assay was performed in triplicate, and the standard errors are shown. These data are representative of three experiments.
Fig. 4shows the following time course experiments: 1) the specific phosphotyrosine content of insulin receptors in the internal membranes, 2) the specific exogenous kinase activity of these insulin receptors, and 3) the specific phosphotyrosine content of IRS-1 in the same fraction. These data indicate that there is a tight correlation between receptor autophosphorylation, exogenous kinase activity, and IRS-1 phosphorylation in the internal membranes.
Figure 5: Insulin-induced GLUT4 translocation to the plasma membrane takes place in a time frame that follows IRS-1 phosphorylation. Fat cells were treated with insulin for various times, and plasma membranes and internal membranes were isolated as described under ``Experimental Procedures.'' After samples from each time point were separated by SDS-PAGE using a 10% gel and electro-transferred to PVDF Immobilon membranes, Western blot analysis was performed using 1F8(37) , an antibody to GLUT4 (upperpanel). 1F8 blots were developed using goat anti-mouse coupled to horseradish peroxidase using chemiluminescence. The data were scanned and expressed as fraction of total cellular GLUT4 (PM + IM) as a function of time (lowerpanel). The immunoblot shown is representative of two experiments.
Figure 6:
A small fraction of the insulin receptors
in the internal membranes and plasma membrane is phosphorylated. Fat
cells were treated with insulin for 2 or 16 min. PM and IM fractions
were isolated as described above, WGA-purified, and immunoprecipitated
using 4G10, an anti-phosphotyrosine antibody. The supernatants (SUP) and pellets were separated by SDS-PAGE using a
3-10% gradient gel in the absence of reducing agents and
immunoblotted using anti-phosphotyrosine antibody (upperpanel) and anti-insulin receptor antibody (lowerpanel). Shown is the holoreceptor of M
350,000. R1064 blots were
developed using
I-protein A, and 4G10 blots were
developed using goat anti-mouse coupled to horseradish peroxidase using
chemiluminescence. The immunoblots shown are representative of three
experiments.
Rat adipocytes are most probably the best system for studying the biochemistry and cell biology of the important physiological response to insulin, namely stimulation of glucose transport. This is because adipocytes respond very well to insulin after isolation, they can be obtained in large quantities, and they are easily fractionated into well characterized subcellular compartments(33, 34) . Thus, it is largely from studies with these cells that it has been determined that insulin-activated glucose transport involves the recruitment of a specific glucose transporter isoform, GLUT4, from an intracellular vesicular storage pool to the cell surface(20) . However, the steps after receptor activation that result in communication with these vesicles and that allow their movement to the plasma membrane remain largely obscure. Recently, the major substrate for the insulin receptor, IRS-1, was cloned, and the predicted sequence was that of a large soluble protein with numerous possible tyrosine phosphorylation sites(38, 39, 40) . These sites can interact with the SH2 domains of effector proteins, and it has been shown in rat adipocytes that insulin stimulates PI-3-kinase activity via the interaction of IRS-1 with PI-3-kinase and that this occurs exclusively in the low density microsomal membranes in adipocytes(30) . Moreover, Wortmannin, an inhibitor of PI-3-kinase, inhibits insulin-stimulated glucose transport in adipocytes with the same concentration dependence as it inhibits the kinase(31) . Thus, in light of these studies, it appeared to us that activation of IRS-1 and PI-3-kinase must be occurring in microsomal membranes (defined as IMs in the present study) and that the interaction of IRS-1 and the insulin receptor might be occurring as a result of receptor internalization. Our data are consistent with this notion.
We show
that insulin receptors are rapidly internalized within 2 min of insulin
treatment, reaching a steady state level after 8 min. Our results are
in agreement with other work done in adipocytes showing that insulin
receptors internalize with a t of 2-3 min
and reach a steady state of endocytosis and recycling after 6-8
min(15, 16, 17) . We used a physiological
concentration of insulin in our study that would be non-saturating with
respect to receptor occupancy to more closely mimic conditions in
vivo. Previous studies in fat cells have shown that the
internalization of insulin receptors is insulin concentration-dependent (16, 17) and that at saturating concentrations of
insulin, internalization is accelerated and receptors remain highly
activated, thus possibly masking the deactivation processes involved in
insulin signaling(11, 13) . We show that internal
membrane insulin receptors rapidly reach a level of specific
phosphorylation that is 5-6-fold that of insulin receptors
located in the plasma membrane and that this subsequently falls to
steady state levels in an equally rapid time frame. This may be due to
preferential internalization of phosphorylated receptors or to a lower
phosphatase activity in the IM relative to the PM at early time points.
When we examined the exogenous kinase activity of insulin receptors
activated in cells, we found that the time course of exogenous kinase
activity exactly mirrors the time course of phosphorylation of the
insulin receptor isolated from both PM and IM. The finding that the
insulin receptor's exogenous kinase activity is at its highest
level in the IM and only within a short time frame after insulin
stimulation, along with the strong correlation with IRS-1
phosphorylation in that fraction, suggests that internalized insulin
receptors as opposed to plasma membrane receptors may be the
functionally active species with respect to substrate (IRS-1)
phosphorylation. This conclusion is further supported by the fact that
we detect essentially no IRS-1 at the cell surface. Therefore, our data
are consistent with the hypothesis that insulin action may be mediated
as a consequence of receptor internalization.
Studies of insulin receptor endocytosis performed in rat liver also showed that endosomal insulin receptors exhibit greater autophosphorylation and exogenous kinase activities when compared with plasma membrane receptor. These studies showed that the time course of autophosphorylation activity and exogenous kinase activity in both fractions peaked between 1 and 3 min after insulin stimulation followed by an equally rapid drop to steady state levels(36) . However, our data, which show a direct correlation between the phosphotyrosine levels and the exogenous kinase activities at all time points in both fractions, are in contradiction with the data of Burgess et al.(13) . From numerous in vitro studies, it would be expected that autophosphorylation and exogenous kinase activity would correlate completely(1) . However, Burgess et al.(13) showed that at their peak, the endosomal receptors from liver are 2-3-fold less phosphorylated than PM receptors but that they are 30% more active at saturating concentrations of insulin. Even at lower insulin concentrations, they demonstrated a lack of correlation between phosphotyrosine level and exogenous kinase activity. These differences may be due to the existence of different regulatory systems in fat and liver coincident with their differing functions. Differences between insulin receptors from different tissues have been previously demonstrated at the level of their intrinsic kinase activity(18) , and differences may be expected at more complex regulatory steps. Our data are consistent with that of Klein et al.(11, 41) , who also used isolated rat adipocytes. They show that when exposed to saturating concentrations of insulin, internalized receptors are fully active with a specific kinase activity equal to plasma membrane receptors. However, at lower non-saturating concentrations of insulin, the specific kinase activity of internalized receptors was shown to be higher than that of plasma membrane receptors(11) , as we also show here at early time points of insulin exposure.
Since immunoblotting assesses the average phosphorylation state of a population of receptors, we used anti-phosphotyrosine antibodies to immunoprecipitate phosphorylated insulin receptors to differentiate phosphorylated and unphosphorylated receptors and to examine their relative amounts in the two fractions. A surprising result from this experiment was that only a small fraction of internalized receptor (<10%) was immunoprecipitable with anti-phosphotyrosine antibody. This finding suggests either that a considerable amount of non-phosphorylated receptors are internalized as bystanders without being phosphorylated or that dephosphorylation occurs rapidly and concomitantly with internalization. We cannot distinguish between these possibilities at present, although the bystander theory has some indirect support from studies showing that this phenomenon occurs for epidermal growth factor receptors(42) .
Our data show that microsome-associated IRS-1 undergoes a peak of phosphorylation and then is rapidly dephosphorylated, whereas cytosolic IRS-1 quickly reaches a steady phosphorylation state that does not change over the time of our experiments (Fig. 3). This difference may represent a differential regulation of the two IRS-1 pools underlying different pathways of insulin action, although the function of the cytosolic pool remains unclear at this time. As previously noted, Kelly and Ruderman (30) showed that low density microsomes (IM)-associated IRS-1 accounts for essentially all of the insulin-stimulated PI-3-kinase activity, and they found no cytosolic PI-3-kinase activity associated with IRS-1 as determined by immunoprecipitation with anti-phosphotyrosine antibodies. However, it is possible that phosphorylated cytosolic IRS-1 may function as a docking protein for the other SH2 domain-containing proteins such as Syp and Grb-2(22, 23, 25) , and we are currently addressing this possibility.
The data of Fig. 3also support the notion that the phosphotyrosine phosphatase activity or activities, which dephosphorylate IRS-1 and the insulin receptor, are present in the internal membrane fraction. For the insulin receptor, it is unclear whether this activity or activities also exist at the plasma membrane. From in vitro observations (data not shown), we detect the presence of a stronger phosphotyrosine phosphatase activity at the PM than at the IM. However, since phosphotyrosine phosphatases have been shown to be promiscuous in vitro(43, 44, 45, 46) , it is unclear whether this observation has any physiological significance. The similarity in the time course of dephosphorylation of the insulin receptor and IRS-1 in the internal membrane fraction suggests the presence of one or more phosphotyrosine phosphatases whose activity is similarly regulated to shut off the insulin signal in a concerted manner.
In Fig. 7, we present a model describing the possible interpretations of our data in the framework of related studies from other workers. As previously discussed, we favor the hypothesis that internalized insulin receptors are the important population with regard to IRS-1 phosphorylation (Fig. 7, pathway1). Alternatively, receptors activated at the plasma membrane could be in physical proximity to intracellular vesicles containing IRS-1 such that substrate can be phosphorylated (pathway2). A third possibility is that either plasma membrane or internalized receptor could phosphorylate IRS-1 in the cytosol, and the phosphorylated protein would then become membrane associated (pathway3). However, we see no change in microsomal IRS-1 at 1-4 min after insulin exposure (Fig. 3) when its phosphotyrosine content is changing dramatically ( Fig. 3and Fig. 4), although we cannot rule out a very rapid exchange of phosphorylated cytosolic IRS-1 for unphosphorylated microsomal IRS-1. The possibility that IRS-1 and the insulin receptor are in the same membrane compartment seems unlikely because Kelly and Ruderman (30) showed that activated insulin receptors on the one hand, and PI-3-kinase-associated IRS-1, therefore tyrosine phosphorylated IRS-1 on the other hand, are located in different fractions of sucrose gradients from fat cell microsomal membranes. Moreover, GLUT4-containing vesicles contain no IRS-1, insulin receptor, or PI-3-kinase(30, 47) . Thus, the working model of Fig. 7postulates that after phosphorylation of IRS-1, a signal is propagated by an unknown number of steps, possibly including PI-3-kinase activation(31) , such that GLUT4 translocation occurs. We are in the process of conducting further experiments to refine this model. Interestingly, Di Guglielmo et al.(48) have proposed a somewhat similar model for epidermal growth factor receptor-mediated signal transduction in the liver.
Figure 7: Possible mechanisms by which the insulin receptor can phosphorylate IRS-1. The numbers (1-3) indicate possible pathways of this process as discussed in the text.
In summary, we demonstrated that internalized insulin receptors are more phosphorylated and more active than plasma membrane insulin receptors and remain highly phosphorylated and active for a short time that is coincident with the time frame of phosphorylation of IRS-1. The maximal activation of the receptor and IRS-1 precede that of GLUT4 translocation. Both the insulin receptor and IRS-1 are subsequently dephosphorylated in a similar time frame, suggesting the existence of a concerted mechanism for dephosphorylation, which may be insulin regulated. Additionally, we show that the phosphorylated receptors in the IM fraction during the phosphorylation peak are only a small fraction of the total internalized, suggesting that non-phosphorylated receptors can internalize as bystanders.