©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Dynamics of Signaling during Insulin-stimulated Endocytosis of Its Receptor in Adipocytes (*)

(Received for publication, June 3, 1994; and in revised form, October 24, 1994)

Bassil Kublaoui Jongsoon Lee Paul F. Pilch (§)

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The biological effects of insulin are mediated by binding of the hormone to its cell surface receptor, which results in beta 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)(^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(r) 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.


EXPERIMENTAL PROCEDURES

Materials

Collagenase for digestion of fat pads was purchased from Worthington Chemical Corp. Immobilon-P PVDF was purchased from Millipore. I-protein A and [-P]ATP were from DuPont NEN. Wheat germ agglutinin-agarose was obtained from EY laboratories. Poly Glu/Tyr 4:1 and horseradish peroxidase-linked goat anti-mouse antibody were obtained from Sigma. Enhanced chemiluminescence reagents were from Dupont NEN. The x-ray film used was Hyperfilm-MP from Amersham Corp. Prestained molecular weight markers were from Sigma, Pharmacia Biotech Inc., and Life Technologies, Inc. Monoclonal anti-phosphotyrosine antibodies were purchased from Upstate Biotechnologies and Leinco Technologies Inc. Anti-IRS-1 antibodies were a generous gift from Dr. Michael Gibbs (Dept. of Cardiovascular and Metabolic Diseases, Pfizer Inc., Groton, CT).

Cell Fractionation

Fat cells were isolated from rat epididymal fat pads from 200-250-g male Sprague-Dawley rats as previously described(33, 34) . Briefly, fat pads were dissected and placed in modified Krebs Ringer phosphate bovine serum albumin buffer containing 12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO(4), 0.6 mM Na(2)HPO(4), 0.4 mM NaH(2)PO(4), 2.5 mMD-glucose, 2% bovine serum albumin at 37 °C. The fat pads were cut into small pieces and digested with 1.75 mg/ml crude collagenase for 45 min to 1 h at 37 °C. Fat cells were isolated by filtration through nylon mesh and, after this procedure, by centrifugation. Adipocytes were washed three times with Krebs Ringer buffer and were allowed to equilibrate at 37 °C for 30 min prior to insulin treatment. Equal volumes of adipocytes were then treated with insulin or not to a final concentration of 10 nM. At the indicated times, the cells were washed twice with the following homogenization buffer (HES) at 12 °C: 20 mM HEPES, 5 mM EDTA, 250 mM sucrose, 5 mM benzamidine, 1 µM each of pepstatin, leupeptin, aprotinin, 1 mM phenylmethylsulfonyl fluoride, 100 mM NaF, 1 mM Na(3)VO(4), 20 mM tetrasodium pyrophosphate, and 1 mM molybdate. Cells were then homogenized using a 30-ml Potter-Elvehjem Teflon pestle, and subcellular fractions were isolated as described by Simpson et al.(33, 34) . Plasma membranes, light and heavy microsomes (LM and HM), mitochondria and nuclei, and cytosol were isolated by differential centrifugation and were suspended in HES with the following concentrations of phosphatase inhibitors: 100 mM NaF, 1 mM Na(3)VO(4), 1 mM molybdate, and 20 mM pyrophosphate. The tyrosine phosphatase inhibitors used were sufficient to inhibit dephosphorylation of the insulin receptor and IRS-1 as determined by an incubation of the various fractions at 4 °C overnight in the presence or absence of inhibitors. For most experiments, an internal membrane (IM) fraction consisting of the combine LM and HM fractions was harvested by centrifugation of the supernatant from the plasma membrane isolation at 200,000 times g for 1 h.

Gel Electrophoresis and Immunoblotting

Protein concentration was determined on the suspended membrane fractions or on SDS-solubilized fractions using the BCA protein assay system (Pierce). SDS-PAGE was performed as described by Laemmli(35) . We used 7, 10, or 3-10% acrylamide gels as indicated. After electrophoresis, gels were transferred onto PVDF Immobilon membrane at 1200 mA-h, and the membranes were used for Western blotting. Membranes were immunoblotted with R1064, a polyclonal antibody to the C terminus of the insulin receptor, 4G10, a monoclonal anti-phosphotyrosine antibody, PY20, a monoclonal anti-phosphotyrosine antibody, or R2656, a polyclonal antibody to IRS-1. Polyclonal antibody blots were visualized using I-protein A followed by autoradiography while monoclonal antibody blots were performed using goat anti-mouse antibody coupled to horseradish peroxidase followed by chemiluminescence. For quantitative analysis, membranes were either cut and counted or the films were scanned using a computing densitometer from Molecular Dynamics.

Exogenous Kinase Activity Assays

Membrane fractions were suspended in HES with the following concentrations of phosphatase inhibitors: 100 mM NaF, 1 mM Na(3)VO(4), 1 mM molybdate, and 20 mM pyrophosphate. They were solubilized using 1% Triton X-100 for 1 h at 4 °C and incubated with wheat germ agglutinin-agarose (WGA) beads. The WGA beads were washed twice with buffer A (30 mM HEPES, 0.1% Triton X-100, 0.01% sodium azide, with protease and phosphatase inhibitors as above), then twice with buffer B (without phosphatase inhibitors), and then eluted with 0.3 MN-acetyl glucosamine in buffer B. Aliquots were then used for the exogenous kinase assay without further treatment with insulin in the presence of 10 mM MgCl(2), 8 mM MnCl(2), 50 µM ATP, 0.5 mg/ml poly Glu/Tyr 4:1, and 10 µCi of [-P]ATP per sample in a final volume of 100 µl. The reactions were stopped after 30 min by the addition of EDTA to a final concentration of 67 mM. The assay mixtures were spotted on 3 times 3-cm 3MM Whatman paper and placed in a 10% trichloroacetic acid, 10 mM pyrophosphate solution and washed three times. The filters were then dried and subjected to Cerenkov counting.

Immunoprecipitation

IRS-1 immunoprecipitations were performed as follows. Cytosolic samples were concentrated 6-10-fold using 20-ml Centricon concentrators. Internal membranes and plasma membranes were solubilized in 1% Triton X-100. The fractions were simultaneously incubated with anti-IRS-1 antibody and protein A trisacryl overnight at 4 °C, washed, and eluted with 1 times Laemmli sample buffer. Immunoprecipitation with anti-phosphotyrosine antibodies was performed as for IRS-1 except that the membrane fractions were first subjected to WGA purification as described above, and the immunoprecipitation was performed for 3 h.


RESULTS

The Temporal Relationship between the Autophosphorylation and Endocytosis of the Insulin Receptor

To examine the dynamics of insulin receptor endocytosis with respect to receptor phosphorylation state, isolated adipocytes were treated with 10 nM insulin at 37 °C, and at the indicated times, they were washed with 12 °C homogenization buffer and subsequently homogenized. The homogenate was fractionated by differential centrifugation to separate internal membranes from plasma membranes, and equal amounts of protein from each fraction were used for electrophoresis. The IM fraction consists of the combined HM and LM fractions (see ``Experimental Procedures''), and we used this because we determined that the proportion of the total receptor that internalized into the HM (30%) behaved indistinguishably from the proportion that internalized into the LM (70%) (data not shown). The insulin receptor and its phosphotyrosine content were examined by Western blot analysis as a function of time after insulin treatment (Fig. 1A). As expected(15, 16, 17) , there is essentially no receptor present in internal membranes in the absence of insulin, but 1 min after insulin exposure, phosphorylated receptor is detected in microsomal membranes (Fig. 1A). Fig. 1, B and C, shows densitometric scans of these data. Internal insulin receptor reaches steady state levels by 8-10 min after insulin treatment in agreement with the prior studies of Marshall (15) who only monitored insulin binding. The phosphotyrosine content of the pool of internalized receptors also rapidly reaches a steady state level as does the phosphotyrosine content of the plasma membrane receptors (Fig. 1C). Since the amount of receptor in the internal membranes is low at early times after insulin exposure (but the phosphotyrosine content is high), the specific phosphotyrosine content/internalized receptor rises to 5-6-fold that of PM receptors after 2 min of insulin treatment and subsequently drops to a steady state level (see Fig. 4). Internalized insulin receptors are derived from the plasma membrane, and therefore these data suggest either that there is a preferential internalization of phosphorylated receptors or that at early time points there is less dephosphorylation of internalized receptors relative to PM receptors. At later time points, insulin receptors are being rapidly dephosphorylated in a time frame that precedes that of recycling to the plasma membrane.


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 alpha(2)beta(2) holoreceptor of M(r) 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.



The Time Course of Exogenous Kinase Activity of Insulin Receptors Derived from Internal Membranes and Plasma Membranes of Adipocytes Treated with Insulin

The exogenous kinase activity of insulin receptors activated in cells was assayed after various times of insulin exposure using poly Glu/Tyr as a substrate. A volume of WGA eluate equal to that used in the kinase assay was subjected to SDS-PAGE, and the relative amount of insulin receptor in each fraction was assessed by Western blotting with anti-insulin receptor antibody and anti-phosphotyrosine antibody. We established that the phosphotyrosine content before and after the WGA purification was identical (data not shown). Fig. 2shows that at 1 and 2 min after cellular insulin exposure, the specific exogenous kinase activity of IM versus PM receptors is 5-6-fold higher for the internalized receptors, after which it drops to a steady state level equal in the two fractions. The time course of exogenous kinase activity normalized to the amount of insulin receptor mirrors the phosphotyrosine content of the insulin receptors at each time point (see Fig. 4). These results show that insulin receptors are most active in the internal membranes and only for a brief interval. These results also indicate that in fat cells, unlike the case for similar experiments conducted in rat liver(13, 36) , the phosphotyrosine content of insulin receptors correlates well with their exogenous kinase activity.

The Time Course of Phosphorylation of IRS-1

The data of Fig. 1and Fig. 2show that insulin receptors are rapidly internalized in a kinase-active form, demonstrating that they are able to phosphorylate potential substrates at cellular locations other than the plasma membrane. Hence, we examined the location and phosphorylation state of IRS-1 at various times after treating adipocytes with insulin, as shown in Fig. 3. The plasma membrane fraction contains very little IRS-1 and does not show a detectable phosphotyrosine signal for IRS-1. The IRS-1 associated with the internal membrane is unphosphorylated in the basal state and undergoes rapid phosphorylation within 2 min, followed by dephosphorylation in a time frame similar to that of insulin receptor exogenous kinase activity (see Fig. 4). Based on Western blotting analysis, we determined that roughly 20% of total cellular IRS-1 is associated with the internal membrane fraction and that the remainder is cytosolic. 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. Unlike the internal membrane-associated IRS-1, the cytosolic IRS-1 appears to escape rapid dephosphorylation as seen in Fig. 3, suggesting the possible presence of active phosphotyrosine phosphatases at the internal membranes.

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.

The Time Course of Translocation of GLUT4

We examined GLUT4 translocation, which is a major and important consequence of insulin action in fat cells, to determine its relationship to IRS-1 phosphorylation. Cells treated with insulin for various times were fractionated, and the plasma membranes were subjected to SDS-PAGE and immunoblotting using the anti-GLUT4 antibody 1F8(37) . Fig. 5shows that GLUT4 appears at the plasma membrane as early as 1 min after insulin treatment but that it peaks after 8 min. In contrast, the maximal degree of receptor activation and IRS-1 phosphorylation precedes that of GLUT4 translocation.


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.



Immunoprecipitation of Insulin Receptors from Plasma Membranes and Internal Membranes Using Anti-phosphotyrosine to Examine the Ratio of Phosphorylated to Unphosphorylated Receptors in the Two Compartments

One possible explanation for the results from Fig. 1and Fig. 2is that phosphorylated insulin receptors are preferentially internalized over unphosphorylated receptors. However, since there is obviously a mixture of phosphorylated and unphosphorylated receptors, particularly at the cell surface, we wished to determine how much of the total population in a given membrane fraction was phosphorylated. To do this, we used anti-phosphotyrosine antibodies for immunoprecipitation and immunoblotted using both anti-insulin receptor antibodies and anti-phosphotyrosine antibodies. Fig. 6shows that all of the tyrosine-phosphorylated receptors were brought down with antibody to this epitope, but that over 90% of the insulin receptors remained in the supernatant in all conditions. This is a surprising result in the case of the internalized receptor in that such a small proportion of these receptors is actually phosphorylated and is responsible for the high level of exogenous kinase activity seen above. At relatively short times after exposure to 10 nM insulin, it would be expected that only a small fraction of the plasma membrane insulin receptors would be occupied with ligand and therefore would be phosphorylated, and this is what was observed.


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 alpha(2)beta(2) holoreceptor of M(r) 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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK-36424 and DK-30425 and the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118.

(^1)
The abbreviations used are: IRS-1, insulin receptor substrate 1; PI-3-kinase, phosphatidylinositol 3-kinase; PAGE, polyacrylamide gel electrophoresis; IM, internal membranes; PM, plasma membrane; LM and HM, light and heavy microsomes; PVDF, polyvinylidene difluoride; WGA, wheat germ agglutinin-agarose.


ACKNOWLEDGEMENTS

We thank Dr. Lise Coderre, Dr. Konstantin Kandror, and Galini Thoidis for helpful discussion.


REFERENCES

  1. Lee, J., and Pilch, P. F. (1994) Am. J. Physiol. 266, C319-C334
  2. White, M. F., and Kahn, C. R. (1994) J. Biol. Chem. 269, 1-4 [Free Full Text]
  3. Tavaré, J. M., and Siddle, K. (1993) Biochim. Biophys. Acta 1178, 21-39 [Medline] [Order article via Infotrieve]
  4. Taylor, S. I., Cama, A., Accili, D., Barbetti, F., Quon, M. J., De la Luz Sierra, M., Suzuki, Y., Koller, E., Levy-Toledano, R., Wertheimer, E., Moncada, V. Y., Kadowaki, H., and Kadowaki, T. (1992) Endocr. Rev. 13, 566-595 [Medline] [Order article via Infotrieve]
  5. Morgan, D. O., and Roth, R. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 41-45 [Abstract]
  6. Marshall, S., and Olefsky, J. M. (1979) J. Biol. Chem. 254, 10153-10160 [Medline] [Order article via Infotrieve]
  7. Fehlmann, M., Carpentier, J. L., Van Obberghen, E., Freychet, P., Thamm, P., Saunders, D., Brandenburg, D., and Orci, Lelio (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 5921-5925 [Abstract]
  8. Marshall, S., Green, A., and Olefsky, J. M. (1981) J. Biol. Chem. 256, 11464-11470 [Free Full Text]
  9. McClain, D. A., Maegawa, H., Lee, J., Dull, T. J., Ullrich, A., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 14663-14671 [Abstract/Free Full Text]
  10. Backer, J. M., Shoelson, S. E., Haring, E., and White, M. F. (1991) J. Cell Biol. 115, 1535-1545 [Abstract]
  11. Klein, H. H., Freidenberg, G. R., Matthaei, S., and Olefsky, J. M. (1987) J. Biol. Chem. 262, 10557-10564 [Abstract/Free Full Text]
  12. Backer, J. M., Kahn, C. R., and White, M. F. (1989) J. Biol. Chem. 264, 1694-1701 [Abstract/Free Full Text]
  13. Burgess, J. W., Wada, I., Ling, N., Khan, M. N., Bergeron, J. J. M., and Posner, B. I. (1992) J. Biol. Chem. 267, 10077-10086 [Abstract/Free Full Text]
  14. Huecksteadt, T., Olefsky, J. M., Brandenberg, D., and Heidenreich, K. A. (1986) J. Biol. Chem. 261, 8655-8659 [Abstract/Free Full Text]
  15. Marshall, S. (1985) J. Biol. Chem. 260, 4136-4144 [Abstract]
  16. Sonne, O., and Simpson, I. A. (1984) Biochim. Biophys. Acta 804, 404-413 [Medline] [Order article via Infotrieve]
  17. Wang, C., Sonne, O., Hedo, J. A., Cushman, S. W., and Simpson, I. A. (1983) J. Biol. Chem. 258, 5129-5134 [Abstract/Free Full Text]
  18. O'Hare, T., and Pilch, P. F. (1989) J. Biol. Chem. 264, 602-610 [Abstract/Free Full Text]
  19. Mooney, R. A., and Anderson, D. L. (1989) J. Biol. Chem. 264, 6850-6857 [Abstract/Free Full Text]
  20. Birnbaum, M. J. (1992) Int. Rev. Cytol. 137, 239-297 [Medline] [Order article via Infotrieve]
  21. White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186 [Medline] [Order article via Infotrieve]
  22. Baltensperger, K., Kozma, L. M., Cherniack, A. D., Klarlund, J. K., Chawla, A., Banerjee, U., and Czech, M. P. (1993) Science 260, 1950-1952 [Medline] [Order article via Infotrieve]
  23. Skolnik, E. Y., Batzer, A., Li, N., Lee, C.-H., Lowenstein, E., Mohammadi, M., Margolis, B., and Schlessinger, J. (1993) Science 260, 1953-1955 [Medline] [Order article via Infotrieve]
  24. Skolnik, E. Y., Lee, C.-H., Batzer, A., Vicentini, L. M., Zhou, M., Daly, R., Myers, M. J., Jr., Backer, J. M., Ullrich, A., White, M. F., and Schlessinger, J. (1993) EMBO J. 12, 1929-1936 [Abstract]
  25. Kuhné, M. R., Pawson, T., Lienhard, G. E., and Feng, G.-S. (1993) J. Biol. Chem. 268, 11479-11481 [Abstract/Free Full Text]
  26. Myers, M. G., Backer, J. M., Sun, X. J., Shoelson, S., Hu, P., Schlessinger, J., Yoakim, M., Schaffhausen, B., and White, M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10350-10354 [Abstract]
  27. Ruderman, N. B., Kapeller, R., White, M. F., and Cantley, L. C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1411-1415 [Abstract]
  28. Endemann, G., Yonezawa, K., and Roth, R. A. (1990) J. Biol. Chem. 265, 396-400 [Abstract/Free Full Text]
  29. Kelly, K. L., Ruderman, N. B., and Chen, K. S. (1992) J. Biol. Chem. 267, 3423-3428 [Abstract/Free Full Text]
  30. Kelly, K. L., and Ruderman, N. B. (1993) J. Biol. Chem. 268, 4391-4398 [Abstract/Free Full Text]
  31. Okada, T., Kawano, Y., Sakakibara, T., Hazeki, O., and Ui, M. (1994) J. Biol. Chem. 269, 3568-3573 [Abstract/Free Full Text]
  32. Del Vecchio, R. L., and Pilch, P. F. (1989) Biochim. Biophys. Acta 986, 41-46 [Medline] [Order article via Infotrieve]
  33. Simpson, I. A., Yver, D. R., Hissin, P. J., Wardzala, L. J., Karnieli, E., Salans, L. B., and Cushman, S. W. (1983) Biochim. Biophys. Acta 763, 393-407 [Medline] [Order article via Infotrieve]
  34. Simpson, I. A., and Cushman, S. W. (1986) Annu. Rev. Biochem. 55, 1059-1089 [CrossRef][Medline] [Order article via Infotrieve]
  35. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  36. Khan, M. N., Baquiran, G., Brule, C., Burgess, J., Barbara, F., Bergeron, J. J. M., and Posner, B. I. (1989) J. Biol. Chem. 264, 12931-12940 [Abstract/Free Full Text]
  37. James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185 [CrossRef][Medline] [Order article via Infotrieve]
  38. Sun, X. J., Rothenberg, P., Kahn, C. R., Backer, J. M., Araki, E., Wilden, P. A., Cahill, D. A., Goldstein, B. J., and White, M. F. (1991) Nature 352, 73-77 [CrossRef][Medline] [Order article via Infotrieve]
  39. Keller, S. R., Aebersold, R., Garner, C. W., and Lienhard, G. E. (1993) Biochim. Biophys. Acta 1172, 323-326 [Medline] [Order article via Infotrieve]
  40. Sun, X. J., Crimmins, D. L., Myers, M. G., Jr., Miralpeix, M., and White, M. F. (1993) Mol. Cell. Biol. 13, 7418-7428 [Abstract]
  41. Klein, H. H., Freidenberg, G. R., Kladde, M., and Olefsky, J. M. (1986) J. Biol. Chem. 261, 4691-4697 [Abstract/Free Full Text]
  42. Wiley, H. S., Walsh, B. J., and Lund, K. A. (1989) J. Biol. Chem. 264, 18912-18920 [Abstract/Free Full Text]
  43. Walton, K. M., and Dixon, J. E. (1993) Annu. Rev. Biochem. 62, 101-120 [CrossRef][Medline] [Order article via Infotrieve]
  44. Tonks, N. K., Yang, Q., Flint, A. J., Gebbink, M. F. B. G., Franza, B. R., Jr., Hill, D. E., Sun, H., and Brady-Kalnay, S. (1992) Cold Spring Harbor Symp. Quant. Biol. 57, 87-94 [Medline] [Order article via Infotrieve]
  45. Hashimoto, N., Feener, E. P., Zhang, W.-R., and Goldstein, B. J. (1992) J. Biol. Chem. 267, 13811-13814 [Abstract/Free Full Text]
  46. Brautigan, D. L. (1992) Biochim. Biophys. Acta 1114, 63-77 [CrossRef][Medline] [Order article via Infotrieve]
  47. Del Vecchio, R. L., and Pilch, P. F. (1991) J. Biol. Chem. 266, 13278-13283 [Abstract/Free Full Text]
  48. Di Guglielmo, G. M., Baass, P. C., Ou, W., Posner, B. I., and Bergeron, J. J. M. (1994) EMBO J. 13, 4269-4277 [Abstract]

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