©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction between the Insulin Receptor and Its Downstream Effectors
USE OF INDIVIDUALLY EXPRESSED RECEPTOR DOMAINS FOR STRUCTURE/FUNCTION ANALYSIS (*)

(Received for publication, August 10, 1995; and in revised form, December 4, 1995)

Keren Paz (1) Hedva Voliovitch (1) Yaron R. Hadari (1) Charles T. Roberts Jr. (2)(§) Derek LeRoith (2) Yehiel Zick (1)(¶)

From the  (1)Department of Chemical Immunology, the Weizmann Institute of Science, Rehovot 76100, Israel and the (2)Diabetes Branch, National Institutes of Health, Bethesda, Maryland 20982

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) beta subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1331) of IR were expressed in bacteria as (His)(6)-fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four ``blocks'' of the PH domain (Pro^5-Pro) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions.


INTRODUCTION

The insulin receptor (IR) (^1)is an heterotetrameric transmembrane glycoprotein composed of two extracellular alpha subunits and two transmembrane beta subunits linked by disulfide bonds. The alpha subunits contain the insulin-binding domain, while the transmembrane beta subunits function as a tyrosine-specific protein kinase that undergoes autophosphorylation following insulin binding (for reviews, see (1) and (2) ). Autophosphorylation activates the insulin receptor kinase (3) and enables it to phosphorylate endogenous proteins, including insulin receptor substrate-1 (IRS-1) (4) , IRS-2(5) , and Shc(6) .

Several structural regions have been defined within the intracellular part of the beta subunit. These are the juxtamembrane (JM) region, the kinase region, and the carboxyl-terminal (CT) region(7, 8) . The JM region (Arg-Leu) contains at least one autophosphorylation site (Tyr), which resides in an LX(4)NPXYXSXSD motif (numbering of IR amino acids is according to Ullrich et al.(7) ). Replacement of Tyr with Phe or Ala impairs receptor signal transmission and abolishes both the metabolic and growth-promoting effects of insulin, even though autophosphorylation in other regions is normal and the kinase is fully active in vitro(9, 10) . This appears to be due to an inability of the mutant receptors to mediate the phosphorylation of endogenous receptor substrates, including IRS-1. Indeed, overexpression of IRS-1 can rescue certain biological effects in cells overexpressing the mutated receptor(1) . Similarly, the IGF-1 and the IL-4 receptors, which contain LX(4)NPXYXS motifs, also phosphorylate IRS-1(11, 12) . These findings strongly suggest that the JM region of IR is involved in the interactions with IRS-1.

The CT region (Leu-Ser) possesses only limited (44%) homology with the related IGF-1 receptor or with other protein tyrosine kinases(13) . It contains two autophosphorylation sites at Tyr and Tyr whose role in receptor signaling is still unresolved(14) . Mutations of these residues augments insulin-dependent activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase, suggesting that these tyrosine residues negatively regulate the growth-promoting effects of insulin(15) . Similarly, cells expressing a receptor mutant in which the carboxyl-terminal 43 amino acids (including these Tyr residues) were deleted (IR) exhibit impaired metabolic effects (16, 17) and impaired induction of c-fos(18) but have augmented mitogenic signaling(16, 17) .

The best studied IR substrates are IRS-1 and Shc. IRS-1 undergoes phosphorylation on multiple Tyr residues(1) , which serve as docking sites for SH2-containing proteins like the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, GRB2, Nck, and the protein tyrosine phosphatase SH-PTP2. IRS-1 contains a pleckstrin-homology (PH) domain at its extreme amino-terminal region(19, 20) , whose deletion markedly impairs Tyr phosphorylation of IRS-1 in vivo and the capability of IRS-1 to associate with, and activate, phosphatidylinositol 3-kinase(21, 22) . A phosphotyrosine binding (PTB) domain resides at the carboxyl-terminal end of the PH region of IRS-1. This domain, which is present in several signaling molecules, interacts with NPXY motifs of Tyr-phosphorylated growth factor receptors(23, 24, 25, 26) .

Another substrate of the insulin receptor kinase is Shc(6) , which has three different isoforms of 46, 52, and 66 kDa. Tyr-phosphorylated Shc forms specific complexes with the SH2 domain of Grb2 to further propagate the insulin signal by activation of mSOS/Ras/mitogen-activated protein kinase signaling pathway(27, 28, 29) . Yonezawa et al.(30) suggested that the CT region of the IR might mediate the interactions between IR and Shc. In contrast, the presence of a PTB domain at the amino-terminal region of Shc (23, 24, 25, 26, 31) suggests that Shc has the potential to interact either with the JM or the CT region of the IR.

To better characterize the interactions between the IR and its downstream effectors, we examined the ability of individually expressed receptor domains to interact with IRS-1 and Shc. Our findings suggest that the IR can directly interact with its downstream effectors through distinct receptor regions and that autophosphorylation of Tyr residues located in the CT domain can modulate these interactions.


EXPERIMENTAL PROCEDURES

Materials

Insulin (I5500), and wheat germ agglutinin (WGA) resins (L1882) were purchased from Sigma. ProBond Ni beads were obtained from Invitrogen (San Diego, CA). Monoclonal anti-phosphotyrosine PY-20 and polyclonal anti-Shc (S14630) were obtained from Transduction Labs (Lexington, KY). Monoclonal anti-T7bulletTag antibodies were from Novagene. Polyclonal anti-IRS-1 antibodies (anti-YR-1) were prepared as described previously(32) . Polyclonal anti-IR antibodies (ST-50) were kindly provided by Simeon I. Taylor (National Institutes of Health, Bethesda, MD).

Generation of Plasmids Expressing Epitope-tagged Receptor Regions

A plasmid containing the full-length IR cDNA was used as a template to PCR-amplify 124- and 260-base pair fragments corresponding to the JM and CT regions of the IR. Two primers, 5`-GGGCCCGGATCCGGACTTTGGAATGACCAGAGAC-3` (^2)and 5`-GGGCCCAAGCTTCGGACCACATGTCAGAAGAAGT-3`, were used to amplify the sequence encoding amino acids 943-984 of the JM region of the IR, while primers 5`-GGGCCCGGATCCGCTGGAGATTGTCAACCTGCTC-3` and 5`-GGGCCCAAGCTTCGTTTTTCTTGCCTCCGTTCAT-3` were used to amplify the sequence encoding amino acids 1245-1331 of the CT region of the IR. The PCR products were digested with BamHI and HindIII, gel-purified, and ligated into the pTrcHis-B bacterial expression vector (Invitrogen, San Diego CA), which contains (His)(6) tag sequence upstream of the multiple cloning site. The constructs were then transformed into Escherichia coli Top-10, and sequencing of both expression plasmids (denoted pTrcHis-JM and pTrcHis-CT) was carried out to ensure proper, in-frame, ligation of the inserts.

Peptide Expression and Purification

Cultures of E. coli Top-10 containing the pTrcHisB-JM or pTrcHisB-CT constructs were grown at 37 °C in 1 liter of LB medium containing 100 µg/ml ampicillin. When the A reached 0.5, IPTG was added to a final concentration of 2 mM, and incubation was continued for an additional 6 h at 37 °C. Cells were centrifuged for 10 min at 2000 times g, and the pellet was resuspended in 20 ml of buffer A (20 mM sodium phosphate, 500 mM NaCl, pH 7.8). The solution was sonicated 3 times for 1 min and centrifuged for 40 min at 40,000 times g. The supernatant was collected, and Triton X-100 was added to a final concentration of 1%. The supernatant was then loaded onto an affinity column containing 2 ml of ProBond Ni beads (Invitrogen). The column was washed with 20 ml of buffer A containing 1% Triton X-100, 60 ml of buffer A supplemented with 0.5 M NaCl, and 20 ml of buffer B (20 mM sodium phosphate, 500 mM NaCl, pH 6.3). Peptides were eluted from the resin with 0-0.6 M imidazole gradient in buffer B. Eluted peptides were extensively dialyzed against phosphate-buffered saline and stored at -20 °C.

Purification of Rat Liver IR on WGA Columns

Insulin receptors were purified from liver plasma membranes of 10-week-old rats. The preparation of membranes, solubilization in Triton X-100, and affinity chromatography of IR on WGA coupled to Sepharose beads were carried out as described previously(3) .

Peptide Phosphorylation

20-µl aliquots of WGA-purified IR were incubated with 10 µl of insulin (10M) at 22 °C for 10 min. Purified peptide samples (10 µl, 1 mg/ml) of either (His)(6)-JM or (His)(6)-CT, were added, and incubation was carried out for an additional 20 min. At the end of this incubation, 40 µl of a reaction mix (50 mM Hepes, 10 mM magnesium acetate, 4 mM manganese acetate, and 1 mM ATP, pH 7.4) was added, and incubation was continued for another 15 min. The phosphorylated peptides were bound to ProBond Ni beads, washed with buffer B, and used as affinity matrixes as described below.

Precipitation of Cytosolic Proteins Derived from NIH-3T3 O(2) CHO Cells with IR Peptides

Cytosolic extracts were prepared either from NIH-3T3 or CHO cells overexpressing the IR (33) or from CHO cells overexpressing IRS-1(22) . When indicated, we made use of CHO cells expressing a mutated form of IRS-1 (IRS-1), in which 60 amino acids from the amino-terminal region (Pro^5-Pro) were deleted (22) . Cells, in 150-mm dishes, were cultured in 20 ml of Dulbecco's modified Eagle's medium or Ham's F-12 medium, respectively containing 10% fetal calf serum. Cells were starved in 20 ml of serum-free medium for 16 h and then incubated with or without insulin (10M) for 1 min at 37 °C. Cells were washed 3 times with phosphate-buffered saline and harvested in 800 µl of buffer C (25 mM Tris-HCl, 2 mM sodium orthovanadate, 0.5 mM EGTA, 10 mM NaF, 10 mM sodium pyrophosphate, 80 mM beta-glycerophosphate, 25 mM NaCl, 10 mM MgCl(2), 10 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride, pH 7.4). After 3 freeze-thaw cycles, the cells were microcentrifuged at 12,000 times g for 30 min at 4 °C, and the supernatant was collected. From the supernatant, 300-µg (300 µl) aliquots were incubated for 2 h with 10 µg of purified JM or CT peptides at 4 °C. Incubation was continued for an additional hour after the addition of 50 µl of ProBond Ni beads prewashed in buffer C. The beads were washed 4 times with buffer C containing 200 mM imidazole and 0.1% Nonidet P-40, 2 times with buffer C, and boiled in 60 µl of Laemmli ``sample buffer''(34) . Samples were resolved by means of SDS-PAGE and immunoblotted with the appropriate antibodies.


RESULTS

Expression and Purification of Epitope-tagged Peptides Corresponding to the JM and CT Region of IR

Polypeptides of 41, and 86 amino acid long, corresponding to the Arg-Lys (JM) and Leu-Asn (CT) domains of the IR, respectively, were produced as epitope-tagged fusion peptides that contain at their amino terminus a (His)(6) sequence, fused in-frame to an epitope for the T7bulletTag antibody (Novagen). Transcription was induced with IPTG, cell extracts were made, and formation of the (His)(6)-JM and (His)(6)-CT fusion peptides was detected by immunoblotting with T7bulletTag antibodies. The expressed peptides were purified over ProBond Ni resin (Invitrogen) and eluted with imidazole. (His)(6)-JM (JM) (Fig. 1, top) and (His)-CT (CT) (not shown) fusion peptides, having the expected M, were synthesized in an IPTG-dependent manner. Up to 6 and 3 mg of JM and CT, respectively, were purified in such a manner from 1 liter of culture medium. Purified peptides (e.g. JM (Fig. 1, bottom) were extensively dialyzed against phosphate-buffered saline (to remove excess imidazole), aliquoted, and stored at -20 °C.


Figure 1: Purification of epitope-tagged JM. Top panel, the pTrcHis-JM vector was expressed in E. coli Top-10 cells. After IPTG induction, the bacteria were lysed, and supernatants were prepared as described under ``Experimental Procedures.'' 10 ml of supernatant was applied to a ProBond Ni column and eluted with 0-0.6 M imidazole gradient. From the total, effluent, and each eluted fraction, 50 µl were run on 12% SDS-polyacrylamide gel, transferred to nitrocellulose and immunoblotted with T7bulletTag antibodies. Bottom panel, fractions 5-8, eluted from the column, were pooled and dialyzed, and samples (10 µg) were subjected to 12% SDS-PAGE. Samples were either Coomasie-stained (left) or were transferred to nitrocellulose and immunoblotted with T7bulletTag antibodies (right). This is a representative of an experiment carried out at least three times.



Shc Specifically Interacts with the CT Peptide

Incubation of (His)(6)-JM and (His)(6)-CT peptides with extracts from CHO cells, overexpressing the IR, resulted in preferential association of the three Shc isoforms with the immobilized CT peptide (Fig. 2, a and c). Shc also bound, albeit to a lesser extent, to the JM peptide. The interactions of Shc with the CT peptide were not affected when Shc was derived from insulin-treated cells (Fig. 2e); however, Shc binding was significantly enhanced (about 2-fold) when the CT peptide was subjected to in vitro Tyr phosphorylation by a partially purified insulin receptor kinase prior to the incubation with the cell extracts (Fig. 2, b and d). These results suggest that the CT region of IR is sufficient to bind Shc, and that Tyr-phosphorylation of the CT region potentiates these interactions.


Figure 2: Interactions of JM and CT-peptides with Shc. A, cytosolic extracts were prepared from CHO cells overexpressing the IR. Samples (300 µg) were incubated with 10 µg of purified JM, CT, or buffer alone. Ni beads were added, and after extensive washes, the beads were boiled in 60 µl of Laemmli sample buffer. Samples (including a 30-µg aliquot of the original total extract) were resolved by means of 10% SDS-PAGE and immunoblotted with anti-Shc antibodies. B, same as in A save for the fact that the extracts were incubated either with native (CT) or Tyr-phosphorylated CT (pCT). C, quantitation of the intensity of the bands, corresponding to the three Shc isoforms shown in A. Results of three independent experiments are presented as mean ± S.D. D, quantitation of the intensity of the bands, corresponding to the three Shc isoforms shown in B. Results of five independent experiments are presented as mean ± S.D. E, same as in A save for the fact that extracts from control or insulin-treated cells were incubated with CT.



IRS-1 Specifically Interacts with the JM Polypeptide

In a similar set of experiments, the interaction of IRS-1 with receptor peptides was studied. Incubation of (His)(6)-JM and (His)(6)-CT peptides with extracts of CHO cells, overexpressing the insulin receptor, resulted in specific association of IRS-1 with the immobilized JM peptide, as detected by blotting with anti-IRS-1 antibodies (Fig. 3, a and b). No difference was seen between IRS-1 derived from control or insulin-treated cells (Fig. 3c). In contrast, IRS-1 failed to associate with the CT-peptide either when derived from control or insulin-treated cells. Significant binding of IRS-1 (60% of the total present in 300 µg cell extracts) was observed at peptide concentrations of 1 µM. These results suggest that the JM region is sufficient to mediate high affinity interactions between the IR and IRS-1. To study the effects of Tyr phosphorylation of the JM region on its interactions with Shc and IRS-1, attempts to phosphorylate this peptide have been made. Although several experimental conditions were applied, we were unable to obtain any significant phosphorylation of this peptide.


Figure 3: Interactions of JM and CT with IRS-1. A, cytosolic extracts were prepared from CHO cells overexpressing the IR. Samples (300 µg) were incubated with 10 µg of purified JM, CT, or buffer alone. Ni beads were added, and after extensive washes, the beads were boiled in 60 µl of Laemmli sample buffer. Samples (including a 30-µg aliquot of the original total extract) were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 antibodies. B, quantitation of the intensity of the bands, corresponding to IRS-1 shown in A. Results of three independent experiments are presented as mean ± S.D. C, same as in A save for the fact that extracts were derived from control or insulin-treated cells.



Interaction of Tyr-phosphorylated IRS-1 with the JM Region

Although insulin treatment of intact cells did not alter the fraction of IRS-1 retained by immobilized JM peptide, we wished to compare the binding characteristic of IRS-1 with those of Tyr-phosphorylated IRS-1. Immunoblotting with either anti-IRS-1 or anti P-Tyr antibodies revealed that insulin-induced Tyr-phosphorylation of IRS-1 decreased its interactions with the JM peptide (Fig. 4). While 60% of the total IRS-1 remained associated with the JM peptide, only 25% of the total Tyr-phosphorylated IRS-1 still formed complexes with this domain. Thus, Tyr-phosphorylated IRS-1 tends to dissociate more readily from the JM peptide.


Figure 4: Binding of Tyr-phosphorylated IRS-1 to JM. A, cytosolic extracts were prepared from insulin-treated CHO cells overexpressing the IR. The extracts were incubated with 10 µg of purified JM and were processed as described in Fig. 3. Samples (30 µg) of total cell extracts and the material eluted from the immobilized JM were resolved by means of 7.5% SDS-PAGE and immunoblotted with anti-IRS-1 or anti-P-Tyr (alpha-pY) antibodies. B, quantitation of the intensity of the bands, corresponding to the content of the IRS-1 protein and the content of the Tyr-phosphorylated IRS-1 shown in A. Results of two independent experiments are presented as mean ± S.D.



Effects of Deletion of the PH Domain on Binding of IRS-1 to the JM Peptide

IRS-1 contains at its amino-terminal end a PH region, which has been implicated in mediating either protein-protein (35) or protein-lipid (36, 37) interactions. In previous studies we provided evidence suggesting that this domain presumably is not involved in direct binding to the IR(22) . To further support this notion, we compared the binding of the JM peptide either to the wild-type (WT) IRS-1 or to a mutated form of IRS-1 (IRS-1), in which four blocks of the PH domain (Pro^5-Pro) were deleted. As shown in Fig. 5, both WT and mutated IRS-1 interacted to the same extent with the JM peptide, indicating that partial deletion of the PH domain of IRS-1 does not affect its ability to bind to the isolated JM domain of the insulin receptor in vitro.


Figure 5: Binding of WT and mutated IRS-1 (IRS-1) to JM. A, cytosolic extracts were prepared from insulin-treated CHO cells overexpressing WT or a mutated form of IRS-1 (IRS-1). The extracts were incubated with 10 µg of purified JM and were processed as described in Fig. 3. Samples (20 µg) of total cell extracts and the material eluted from the immobilized JM were resolved by means of 7.5% SDS-PAGE and immunoblotted with IRS-1 antibodies. B, quantitation of the intensity of the bands corresponding to WT and mutated IRS-1 shown in A. Results of four independent experiments are presented as mean ± S.D.




DISCUSSION

In the present study, we demonstrate that the JM and CT regions, within the cytoplasmic portion of the insulin receptor, are functional independent entities that contain sufficient structural information to enable independent and direct binding of effector molecules.

The JM region most likely serves as a binding site for IRS-1, since IRS-1 preferentially interacts with the JM peptide and exhibits no significant binding to the CT peptide. Binding of IRS-1 presumably involves the NPEY motif present in this domain. Support for this notion is provided by the fact that (i) mutation of Tyr, within this motif, or deletion of 12 amino including this motif (38) impedes Tyr phosphorylation of IRS-1 by the insulin receptor kinase; (ii) IGF-1 and the IL-4 receptors, which contain LX(4)NPXYXS motifs, also phosphorylate IRS-1(11, 12) ; (iii) a fusion protein between GST and amino acids 424-561 of the IL-4 receptor, named P3 (which includes the NPXY motif), binds IRS-1(12) ; (iv) mutations of either Asn, Pro, or Tyr abolish IRS-1 interactions with IR in the yeast two-hybrid system(39) ; (v) a Y960F mutant of IR fails to bind the isolated PTB domain of IRS-1 (40) .

Our results indicate that the JM region, in its non-phosphorylated form, is sufficient to promote interactions with IRS-1. According to our model these interactions depend upon the presence of an intact NPEY motif per se, and might take place even when Tyr is not phosphorylated. Phosphorylation of the latter, most likely potentiates the interactions, but is not an absolute prerequisite. Three lines of evidence support this model. First, a number of studies from different laboratories indicate that Tyr is poorly phosphorylated in vivo(9, 41, 42, 43) , although the JM region is a major site for insulin-stimulated Ser phosphorylation(44) . Second, there is no direct evidence that Tyr is indeed phosphorylated when IR is expressed in the yeast two-hybrid system (39) under conditions that promote interaction with IRS-1. Third, the P3 GST-fusion peptide (vide supra), which apparently contains a non-phosphorylated NPXY motif, interacts with IRS-1(12) .

Recent studies have implicated a phosphopeptide (15 amino acids long) surrounding the NPEY motif of IR as a key contributor to the interactions of the JM region of IR with the PTB domains of IRS-1 and Shc(40) . The ID for the interactions of this phosphopeptide with IRS-1 is 170 µM, while the nonphosphorylated peptide exhibits only negligible binding (ID > 1 mM). Since we show a significant binding of nonphosphorylated JM to IRS-1 at a peptide concentration of 1 µM, it suggests that extending the size of the peptide from 15 to 41 amino acids, drastically increases, about 1000-fold, the affinity of the nonphosphorylated JM region to IRS-1. It therefore appears that sequences outside of the 15-amino acid peptide significantly contribute to the interactions of the JM region with the PTB domain of IRS-1 and these interactions take place even when a nonphosphorylated Tyr residue is present within the NPXY motif.

The role of the PH domain of IRS-1 in mediating interactions with the IR was also evaluated. We have previously shown that a mutated (DeltaPro^5-Pro) form of IRS-1 whose PH domain was partially deleted (IRS-1) undergoes significantly reduced insulin-dependent tyrosine phosphorylation in vivo, compared with wild-type (WT) IRS-1. In contrast, both WT IRS-1 and IRS-1 undergo comparable insulin-dependent Tyr phosphorylation in vitro when incubated with partially purified insulin receptor kinase(22) . These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that this domain is presumably not involved in direct interactions between IRS-1 and the IR. The present study supports this notion since we demonstrate that partial deletion of the PH region does not impair the capacity of IRS-1 to interact with the isolated JM region. These findings are consistent with a model in which the PH region mediates protein-lipid interactions and serves to dock IRS-1 in close proximity to the receptor, while the actual protein-protein interactions with the JM region, are mediated by the PTB domain of IRS-1(40, 45) .

The interactions of Shc with the IR are slightly more complex. Shc derived from untreated or from insulin-treated cells interacts with the CT peptide to similar extents. This suggests that Tyr-phosphorylated Shc isoforms interact with the CT peptide to the same extent as the nonphosphorylated forms; alternatively, the fraction of Tyr-phosphorylated Shc could be so low that it does not affect the overall pattern of Shc binding. We favor the latter possibility since preliminary studies indicated that Tyr-phosphorylated Shc fails to interact with the CT region.

Interaction of Shc with the CT region are enhanced about 2-fold when the CT region is subjected to in vitro Tyr phosphorylation by a partially purified insulin receptor kinase. Two autophosphorylation sites at Tyr and Tyr are located at this region, and one of them (Tyr) is placed in a context of YXXM motif, a potential binding site for SH2 domains(46) . The SH2 domain of Shc could therefore interact with the CT region of IR, presumably through binding to the region that includes Tyr. Involvement of the CT region of the IR in the interactions with Shc is also implicated by studies indicating that an IR mutant, in which 82 amino acids at the carboxyl-terminal end were deleted (IR), fails to promote Shc but not IRS-1 phosphorylation(30) . Similarly, deletion of the SH2 domain of Shc reduces its interaction with the IR more than 50% as assayed by the yeast two-hybrid system(45) . It should be noted, however, that the isolated Shc SH2-domain fails to interact with IR in the yeast two-hybrid system, suggesting that the native conformation of the whole Shc protein is required for optimal Shc binding.

Shc interacts, albeit to a very low extent, with the nonphosphorylated JM region of IR. Since the isolated JM region fails to undergo significant in vitro Tyr phosphorylation, we were unable to determine whether Tyr phosphorylation of this domain potentiates these interactions. Previous studies implicated the PTB domain of Shc in mediating interactions with NPXY motifs present in the JM region of IR(40) . Mutation of the Tyr residue within the NPXY motif of IR severely impairs Shc phosphorylation in cultured cells (30) and impedes interactions of Shc with either the IR (45) or the IGF-1 receptor (47) in the yeast two-hybrid system. Hence, the low extent of Shc binding to the nonphosphorylated JM region could be attributed to the fact that the Shc PTB domain interacts exclusively with Tyr-phosphorylated NPXY motifs(23, 24, 31, 40, 48) .

The low affinity of the Shc PTB domain for the nonphosphorylated JM region could be compensated by the interactions of Shc SH2 domains with the nonphosphorylated CT region of IR. A similar phenomenon was seen when the interactions of Shc with the EGFR were studied(24) . Mutations of the highly conserved FLVR sequence in the SH2 domain of Shc reduces the binding of Shc to the EGFR by approximately 90%. In contrast, deletion of the amino-terminal region (including a significant portion of the PTB domain) reduces the binding of Shc to the EGFR by only 50%. These observations suggest that the amino-terminal domain can cooperate with the SH2 domain to promote binding to growth factor receptors (24) .

In summary, although both IRS-1 and Shc interact with IR, significant differences in the nature of these interactions were observed. Shc, but not IRS-1, interact with the CT region, while IRS-1 interacts with a higher affinity with the nonphosphorylated JM region. These differences in binding to the JM region could be accounted for by the fact that PTB domain of Shc interacts with a 5-fold higher affinity with phosphopeptides containing NPEpY motifs(40) , while binding of the PTB domain of IRS-1 is also promoted by JM sequences distal to the NPEY motif. This unique feature of the PTB domain of IRS-1 enables it to selectively bind with a high affinity to the nonphosphorylated JM region of IR. The distinct binding characteristics of IRS-1 and Shc to the different regions of IR could account for the different efficiency of their phosphorylation in vivo, their different sensitivity to the action of kinase inhibitors, and the different biological responses mediated by these effector molecules(49) .

Finally, our results indicate that the interactions of IR with IRS-1, and presumably also with Shc, are regulated by Tyr phosphorylation of the effector proteins. Once IRS-1 is subjected to Tyr phosphorylation, its affinity for the IR is markedly reduced. This phenomenon is not surprising if we consider the fact that effectors like IRS-1 serve as substrates for the insulin receptor kinase. Our findings, therefore, suggest that once IRS-1 is phosphorylated, it translocates away from the receptor into a new subcellular location. This translocation could be part of insulin signal transduction mechanism that is mediated by effector proteins of the insulin receptor kinase.


FOOTNOTES

*
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.

§
Present address: Dept. of Pediatrics, Oregon Health Sciences University, Portland, OR 97201.

An incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. To whom correspondence should be addressed. Fax: 972-8-342-380; Lizick{at}weizmann.weizmann.ac.il.

(^1)
The abbreviations used are: IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRS-1, a mutated form of IRS-1 in which 60 amino acids (Pro^5-Pro) were deleted; CT, carboxyl-terminal; JM, juxtamembrane; PH, pleckstrin homology; PTB, phosphotyrosine binding; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; EGFR, epidermal growth factor receptor; WT, wild-type; WGA, wheat germ agglutinin.

(^2)
Restriction sites for BamHI and HindIII are underlined.


ACKNOWLEDGEMENTS

We thank Drs. Ronit Sagi-Eisenberg and Daniel Schindler for helpful discussions and a critical review of this manuscript. We also thank Dr. Dana Beitner-Johnson for invaluable comments and suggestions.


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