©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for a Differential Interaction of SHC and the Insulin Receptor Substrate-1 (IRS-1) with the Insulin-like Growth Factor-I (IGF-I) Receptor in the Yeast Two-hybrid System (*)

(Received for publication, April 28, 1995; and in revised form, July 31, 1995)

Sophie Tartare-Deckert (§) Dominique Sawka-Verhelle Joseph Murdaca Emmanuel Van Obberghen

From the From INSERM U145, Avenue de Valombrose, 06107 Nice Cedex 2, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using the yeast two-hybrid system, a genetic assay for studying protein-protein interactions, we have examined and compared the interaction of the insulin-like growth factor-I receptor (IGF-IR) and the insulin receptor (IR) with their two known substrates p52Shc and the insulin receptor substrate-1 (IRS-1). We also mapped the specific domains of the IGF-IR and p52Shc participating in these interactions. Our findings can be summarized as follows: (i) the tyrosine kinase activity of the IGF-IR is essential for the interaction with p52Shc and IRS-1, (ii) p52Shc and IRS-1 bind to the IGF-IR in the NPEY-juxtamembrane motif, (iii) contrary to p52Shc, IRS-1 binds also to the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR, and (iv) the amino-terminal domain of p52Shc is required for its association with the IR and the IGF-IR. We propose that (i) the IGF-IR and the IR share at least in part the same molecular mechanism underlying their interplay with their two substrates, p52Shc and IRS-1, and (ii) IRS-1 interacts with the IGF-IR in a fashion that is different from that used by p52Shc. Finally, our data highlight the crucial role of the juxtamembrane domain in signaling by both the IR and the IGF-IR.


INTRODUCTION

Insulin and insulin-like growth factor-I (IGF-I) (^1)elicit a wide variety of biological responses after binding to their respective cell surface receptors(1) . The insulin receptor (IR) and the IGF-I receptor (IGF-IR) are structurally related and are composed of two extracellular alpha-subunits and two transmembrane beta-subunits linked together by disulfide bonds(2, 3) . Both receptors are members of the tyrosine kinase receptor family. Binding of the ligand to the alpha-subunit stimulates the beta-subunit intrinsic tyrosine kinase activity, leading to multisite autophosphorylation of the beta-subunit and tyrosine phosphorylation of cellular substrates. The tyrosine autophosphorylation sites in the IR and in the IGF-IR are located at homologous positions in their corresponding domain. A cluster of three residues (Tyr-1146, -1150, and -1151 in the IR and Tyr-1131, -1135, and -1136 in the IGF-IR) is localized in the kinase domain. One residue, Tyr-960 in the IR, which corresponds to the Tyr-950 in the IGF-IR, is found in the juxtamembrane domain, and two sites (Tyr-1316 and -1322) are situated in the COOH terminus domain of the IR, whereas in the IGF-IR, only one (Tyr-1316) is present at this position. Receptor autophosphorylation is now generally believed to be essential for the biological activity and the action of the IR and the IGF-IR(4, 5, 6, 7) .

Known targets of IR and IGF-IR include a cytosolic 185-kDa protein, termed IRS-1 (insulin receptor substrate-1)(8) , and cytosolic proteins of 46, 52, and 66 kDa, termed Shc (Src-homology 2/alpha-collagen) (9, 10, 11) . p46Shc and p52Shc are both expressed from the same mRNA transcript by alternate translational initiation sites and thus differ in the extent of their amino-terminal domain sequences. p66Shc is likely to result from a distinct transcript. The three Shc proteins contain a carboxyl-terminal Src homology 2 (SH2) domain, a central glycine/proline-rich region homologous to the alpha1 chain of collagen, and an amino-terminal region containing a recently designated PID domain (for phosphotyrosine-interacting domain)(12) .

IRS-1 and Shc act as docking molecules linking the IR and the IGF-IR to downstream signaling pathways. Indeed, tyrosine phosphorylation sites in IRS-1 provide binding motifs for several distinct SH2 domain-containing proteins including the p85 regulatory subunit of phosphatidylinositol 3-kinase, the tyrosine-specific phosphatase Syp, and the small adaptor protein Grb-2(13) . By contrast, upon IR and IGF-IR activation tyrosine-phosphorylated Shc proteins appear to bind only to the SH2 domain of Grb-2(14, 15) . Association between Grb-2 and tyrosine-phosphorylated forms of IRS-1 and/or Shc have been directly implicated in the activation of the Ras signaling pathway(14, 16, 17) .

While it is well established that IRS-1 and Shc proteins are direct substrates for the IR and IGF-IR, little is known about the molecular nature of their interplay with these two receptors. However, these interactions appear to involve transient and/or weak binding since, using classical biochemical techniques, it has been difficult to detect in vivo physical association between the receptors and IRS-1 or Shc. Indeed, little or no IRS-1 can be co-immunoprecipitated with activated IR or IGF-IR by using anti-receptor antibodies, and only a small fraction of IR can be detected in anti-IRS-1 immunoprecipitates (18, 19) . Recently, using the yeast two-hybrid system as a genetic assay for detecting protein-protein interactions, O'Neill et al.(20) have revealed an interaction between the IR and IRS-1. In agreement with previous studies(21, 22) , they found that tyrosine 960 of the receptor juxtamembrane domain is involved and that the amino-terminal region of IRS-1 comprising amino acids 160-516 is sufficient for this interaction. Concerning the Shc proteins, it has been reported that these proteins do not co-immunoprecipitate with activated IR or IGF-IR(9, 10, 23) . However, Shc proteins co-immunoprecipitate with other activated tyrosine kinase receptors such as epidermal growth factor receptors, erbB-2/neu, and Trk(11, 15, 24, 25) . Since there is mounting evidence that Shc is a key factor in insulin and IGF-I receptor signaling(10, 26, 27) , unraveling the precise nature of the interplay between Shc and these receptors will certainly contribute to our understanding of insulin and IGF-I action.

The recently developed yeast two-hybrid system has rapidly turned out to be an exquisitely sensitive strategy for studying interacting proteins(28) . Therefore, we have used this technology to examine interactions between the two receptors and their substrates p52Shc and IRS-1. In addition, we have mapped the specific domains of the IGF-IR and p52Shc participating in these interactions.


EXPERIMENTAL PROCEDURES

Materials

The yeast strain L40 (MATa, trp1, leu2, his3, LYS2::lexA-HIS3, URA3::lexA-lacZ) and the yeast expression plasmid pBTM116 were provided by A. Vojtek (Seattle, WA), and the plasmid pACTII was provided by S. Elledge (Houston, TX). The L40 strain and the two-hybrid plasmids have been described earlier (29, 30) . The human Shc cDNA was kindly provided to us by P. G. Pelicci (Milan, Italy), and the rat IRS-1 cDNA was a gift from M. F. White and C. R. Kahn (Boston, MA). The human IR and IGF-IR cDNAs were obtained from A. Ullrich (Munich, Germany) and P. De Meyts (Copenhagen, Denmark), respectively. Synthetic defined dropout yeast media lacking the appropriate amino acids were obtained from BIO 101 (La Jolla, CA). Oligonucleotides were purchased from Eurogentec (Seraing, Belgium). All chemical reagents were from Sigma France, and enzymes were from New England Biolabs.

cDNA Constructs

Manipulations and sequencing of DNA were carried out according to standard protocols(31) . In most cases, we introduced convenient restriction endonuclease sites to each end of the desired cDNA fragment by polymerase chain reaction to allow the in-frame insertion into the two-hybrid expression plasmids. Reactions were performed using thermostable Deep Vent DNA polymerase (New England Biolabs). The coding sequences of the cytoplasmic domain of the IR (amino acids 944-1343) (2) and IGF-IR (amino acids 933-1337) (3) were polymerase chain reaction amplified and then inserted into the plasmid pBTM116 (30) in frame with the DNA binding domain of LexA yielding LexA-IR beta and LexA-IGF-IR beta hybrid constructs, respectively. The plasmid pBTM116 contains a Trp selection marker. All receptor mutants were generated by site-directed mutagenesis of double-stranded DNA using the Transformer kit (Clontech). Mutations were verified by DNA sequence analysis. The plasmid encoding the GAD-IRS-1 construct (IRS-1 amino acids 5-1235) (8) was obtained as follows. Rat IRS-1 cDNA cloned in pBluescript KS was cut by BspEI (position +600 of the IRS-1 sequence) and SalI (which cuts in the plasmid's polylinker). This fragment was then introduced in frame with the Gal4 activation domain between the XmaI and XhoI sites of the pACTII vector, which has a Leu selection marker. The GAD-Shc (Shc amino acids 1-473) (11) hybrid construct and derivatives were obtained by inserting polymerase chain reaction-generated fragments of the domains of interest into the pACTII vector in frame with the Gal4 activation domain.

Transformation of Yeast and Reporter Gene Expression

Growth conditions and maintenance of the yeast strain L40 were performed essentially as described(32) . L40 was transformed simultaneously with the two indicated hybrid plasmids by the improved lithium acetate method of Gietz et al.(33) . Cotransformants were selected on Trp, Leu plates to select for the pBTM116 and pACTII derivatives, respectively. Typically, after 3 days at 30 °C, three colonies of each transformation were patched to Trp, Leu master plates and incubated at 30 °C for 2 days. The transformants were then tested for beta-galactosidase activity by a color filter assay using the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as described (34) and for histidine prototrophy after replica plating on Trp, Leu, and His medium and incubating at 30 °C for 3 days. For quantitative studies of the beta-galactosidase activity, a solution assay using either o-nitrophenyl-beta-D-galactopyranoside or chlorophenol red-beta-D-galactopyranoside as substrate was carried out according to (34) , except that instead of using 100 µl of yeast cells from the liquid culture, 1 ml of yeast cell extract was used. When the assay was performed with chlorophenol red-beta-D-galactopyranoside, yeast extracts were incubated with 8 mM chlorophenol red-beta-D-galactopyranoside, and the increase in A was monitored after 30 min. Results were expressed as units, defined by Miller(35) .


RESULTS AND DISCUSSION

We have examined the interaction of the IGF-IR and IR with their substrates, i.e. p52Shc and IRS-1, by using the yeast two-hybrid system of Fields and Song(36) . In our study, the first hybrid is a fusion between the DNA binding domain of LexA and the cytoplasmic domain of the IR or the IGF-IR (LexA-IR beta or LexA-IGF-IR beta). The second hybrid is a fusion between the transcriptional activation domain of Gal4 and the full-length sequence of p52Shc (GAD-Shc) or IRS-1 (GAD-IRS-1). Both of these hybrid proteins are expressed in a yeast Saccharomyces cerevisiae strain. If the LexA receptor hybrid interacts with the GAD substrate hybrid, a functional transcription factor is created, leading to the transcription of reporter genes. As reporter strain, we used the L40 strain, which possesses two reporter genes, HIS3 and LacZ, containing upstream LexA binding sites(30) . Activation of LexA-LacZ and LexA-His3 genes can be easily monitored by the production of beta-galactosidase and growth in the absence of histidine, respectively.

First we verified that the two LexA receptor (IR and IGF-IR) fusions expressed in L40 were incapable by themselves, or in combination with an unrelated GAD fusion protein, of activating the expression of the two reporter genes (data not shown). We next investigated the interaction of LexA-IR beta and LexA-IGF-IR beta with GAD-Shc and GAD-IRS-1 in our two-hybrid assay. We found that coexpression of the LexA receptor construct together with GAD-Shc or GAD-IRS-1 in L40 resulted in an interaction detected by both the expression of beta-galactosidase and the growth in absence of histidine. We conclude that p52Shc interacts specifically with both the IR and the IGF-IR, and as recently shown(20) , IRS-1 associates with the IR but also with the IGF-IR (data not shown).

p52Shc and IRS-1 Bind to the NPEY Juxtamembrane Motif on the IGF-IR

Next, we characterized the interaction between the IGF-IR and its two substrates in more detail. In a first series of experiments, we examined whether the interaction of the IGF-IR with Shc and IRS-1 was dependent upon receptor tyrosine kinase activity. To address this issue, we mutated the conserved Lys (Lys-1003) in the ATP binding site of the LexA-IGF-IR beta to obtain a kinase-deficient construct (LexA-IGF-IR beta K1003T). This mutation has been previously shown to abolish the enzymatic activity of the IGF-IR(37) . We analyzed the tyrosine phosphorylation state of K1003T hybrid in the yeast, and we found that in contrast to the wild-type hybrid, which has a constitutively active tyrosine kinase, the mutated hybrid did not undergo autophosphorylation (data not shown). Then, we analyzed the ability of the K1003T construct to interact with GAD-Shc and GAD-IRS-1 by monitoring transcription of the LexA-LacZ gene using a quantitative solution assay (Fig. 1). Compared to the wild-type construct, the LexA-IGF-IR beta K1003T showed no efficient interaction with GAD-Shc (2.9 ± 0.75 units versus 177 ± 16), suggesting that the IGF-IR must be autophosphorylated to interact with GAD-Shc. Similar results were obtained by analyzing the interaction of LexA-IGF-IR beta K1003T and GAD-IRS-1 (2.1 ± 0.55 units versus 69 ± 0.6). Then, we mapped the p52Shc and IRS-1 binding sites on the cytoplasmic domain of the IGF-IR. To do so, several LexA-IGF-IR beta mutants were constructed by site-directed mutagenesis, and each mutated construct was tested in the two-hybrid assay for its ability to interact with GAD-Shc or with GAD-IRS-1 (Fig. 1). We found that the LexA-IGF-IR beta mutants Delta1290-1319 and Delta85 lacking the putative autophosphorylation site Tyr-1316 and the last COOH-terminal 85 amino acids, respectively, still interacted with GAD-Shc and GAD-IRS-1. Interestingly, these mutants exhibited a higher beta-galactosidase activity than that observed with the wild-type form, indicating that deletion of the COOH-terminal region increased the ability of the LexA-IGF-IR beta to interact with the two substrates. These results mean that the COOH-terminal domain of the IGF-IR is not involved in its association to p52Shc and IRS-1. In contrast, deletion of the NPEY motif within the juxtamembrane domain of the receptor (LexA-IGF-IR beta Delta947-950 construct) impaired the ability of both GAD-Shc and GAD-IRS-1 to interact with the receptor. To test the importance of the residues that form this motif, additional mutational analysis was performed. We tested two constructs, one with Tyr-950 mutated to Phe (LexA-IGF-IR beta Y950F) and one containing two point mutations at Asn-947 and Pro-948 (LexA-IGF-IR beta N947A/P948A). Neither bound detectably to GAD-Shc or to GAD-IRS-1. These results demonstrate that p52Shc and IRS-1 interact with the same site in the juxtamembrane domain of the IGF-IR and that the NPEY motif of the receptor plays a key role in p52Shc and IRS-1 recognition.


Figure 1: Quantitative analysis of the interaction of Shc and IRS-1 with different mutated IGF-IR forms. Left panel, schematic representation of the LexA-IGF-IR beta construct and its derivatives tested in the two-hybrid system. WT corresponds to the wild-type IGF-IR beta-subunit (amino acids 933-1337) fused to the DNA binding domain of LexA. K1003T contains a point mutation in the ATP binding site (Lys-1003 mutated to Thr). Delta 1290-1319 is a mutated form with amino acids 1290-1319 deleted. Delta 85 has a carboxyl-terminal deletion of the last 85 amino acids. Delta 947-950 is a mutated form with the NPEY motif deleted. Y950F has the Tyr-950 mutated to Phe. N947A/P948A contains two point mutations at Asn-947 and Pro-948. The juxtamembrane domain of the beta-subunit is shown as a shadedbox, the tyrosine kinase domain as an openbox, and the COOH-terminal domain as a hatchedbox. Right panel, determination of the beta-galactosidase activity in transformed yeast cells. The reporter strain L40 was cotransformed with the indicated panel of LexA-IGF-IR beta constructs and either the plasmid encoding GAD-Shc or the plasmid encoding GAD-IRS-1. Transformants were isolated on selective plates. The beta-galactosidase activities in cell lysates were measured using the substrate o-nitrophenyl-beta-D-galactopyranoside. The indicated activities were expressed as Miller's units (35) and were the average (±S.E.) calculated from samples prepared from five independent transformants. Similar results were obtained by analyzing growth on His plates.



The NPEY motif, located in the immediate juxtamembrane domain of the IGF-IR, is also found in the corresponding domain of the IR. Therefore, we mutated within this motif the Tyr-960 to Phe in the LexA-IR beta hybrid to test whether this mutation would also affect the interaction with p52Shc. We found that similar to the observation made with IGF-IR, mutation of Tyr-960 in LexA-IR beta prevented its interaction with GAD-Shc (data not shown). This mutant also failed to interact with GAD-IRS-1, which is consistent with the report of O'Neill et al.(20) . We interpret our data to mean that for both the IR and IGF-IR, mutation of the corresponding tyrosine within the NPEY motif receptor eliminates interaction with both IRS-1 and p52Shc. From this we conclude that both p52Shc and IRS-1 bind the same conserved region of the two receptors. Further, our data obtained with the LexA-IGF-IR beta K1003T mutant would suggest that receptor autophosphorylation of Tyr-950 is crucial for these interactions.

It has been proposed that the juxtamembrane domain of both the IGF-IR and the IR is the site of interaction with IRS-1. Indeed, mutation of Tyr-960 in the IR and of Tyr-950 in the IGF-IR has been shown to eliminate IRS-1 phosphorylation in intact cells, without significantly affecting the tyrosine kinase activity of the mutated receptors(21, 22, 38) . Further, the biological actions of insulin and of IGF-I were severely impaired in cells expressing these mutated receptors. Our present demonstration that Tyr-960 of the IR and Tyr-950 of the IGF-IR are not only the sites of interaction with IRS-1 but also the sites of interaction with p52Shc provides a key insight. Indeed, our data allow us to suggest that the profound alteration of the biological responses observed in these previous studies is likely to be due to the fact that the tyrosine phosphorylation of Shc proteins is also impaired.

Mutations of the Major Autophosphorylation Sites in the IGF-IR Affect the Interaction with IRS-1 and p52Shc Differently

Next, we asked if the interaction was also dependent on the major autophosphorylation sites (Tyr-1131, -1135, and -1136) of the IGF-IR. Indeed, it has been proposed for the IR that phosphorylation of these corresponding tyrosine residues contributes to Shc and IRS-1 phosphorylation in intact cells(5, 39) . We replaced in the LexA-IGF-IR beta the tyrosine residues (1131, 1135, and 1136) individually or in combination, and we analyzed the ability of the different constructs to interact with GAD-Shc and GAD-IRS-1 (Table 1). Interaction was monitored by a beta-galactosidase assay and the growth in the absence of histidine. Interestingly, the various mutated IGF-IR forms exhibited a different behavior concerning their interaction with p52Shc versus IRS-1. Indeed, we found that mutation of Tyr-1131 and Tyr-1135 (Y1131F construct and Y1135F construct, respectively) did not affect the interaction with GAD-Shc but severely impaired the interaction with GAD-IRS-1. Similar results were obtained with the LexA-IGF-IR beta Y1135/1136F construct, which contained two point mutations at Tyr-1135 and Tyr-1136. GAD-Shc interaction with the LexA-IGF-IR beta Y1131/1135F, although not detectable with the beta-galactosidase assay, was evident with the more sensitive measure of growth on medium lacking histidine. However, these data mean that mutation of both tyrosine residues 1131 and 1135 decreases the interaction with GAD-Shc and suggest that individually these tyrosine residues are not implicated in the interaction with GAD-Shc, but when combined they contribute to the optimal interaction. We conclude that the major tyrosine autophosphorylation sites of the IGF-IR are involved differently in the interaction with p52Shc and IRS-1, suggesting that individually these sites might contribute to the specificity of substrate recognition.



The Amino-terminal Domain of Shc Interacts with Both the IGF-IR and the IR

Finally, we investigated which domain of Shc is involved in its molecular interplay with the IR and the IGF-IR. We analyzed the interaction of different deletion hybrids of Shc with both LexA-IGF-IR beta and LexA-IR beta by the liquid beta-galactosidase assay. Analysis of the results shown in Fig. 2indicated that (i) deletion of the SH2 and collagen domains of Shc did not significantly affect the interaction with the IGF-IR and with the IR and (ii) the amino-terminal domain (amino acids 1-230) is necessary for the association between Shc and the IR and between Shc and the IGF-IR, and that constructs with more severe deletions in this domain did not bind to the two receptors. This suggests that interaction of p52Shc with the Tyr-960 of the IR and with the Tyr-950 of the IGF-IR is independent of the SH2 domain and that the amino-terminal region of Shc may be involved in interactions dependent on phosphotyrosine. While our work was in progress, different reports appeared that entirely support our view(40, 41, 42) . From these studies, the interaction motif in the amino-terminal domain of p52Shc has been defined as amino acids 46-209 and has been called PID (for phosphotyrosine interaction domain) or PTB (for phosphotyrosine binding domain)(12, 40) . Hence, it seems extremely likely that in our present work the motif in the amino-terminal domain of p52Shc that mediates interaction with both the IR and the IGF-IR is this very same PID/PTB domain.


Figure 2: Quantitative analysis of the interaction of IGF-IR and IR with Shc deletion mutants. Left panel, schematic representation of the GAD-Shc(1-473) and its derivatives tested in the two-hybrid system. Fragments of Shc corresponding to the indicated residues were obtained and expressed as a fusion protein with the Gal4 activation domain. The amino-terminal domain of Shc is shown as a shadedbox, the collagen domain as an openbox, and the SH2 domain as a hatchedbox. MET +1 and MET +2 correspond to the two translation initiation sites. Right panel, determination of the beta-galactosidase activity in transformed yeast cells. The reporter strain L40 was cotransformed with plasmid encoding the indicated panel of GAD-Shc constructs and either the plasmid encoding LexA-IGF-IR beta or the plasmid encoding LexA-IR beta. The beta-galactosidase activities in cell lysates were measured using the substrate o-nitrophenyl-beta-D-galactopyranoside and were calculated according to Miller(35) . Values represent the average (±S.E.) of five independent transformants. Similar results were obtained by analyzing growth on His plates.



After our manuscript was submitted, a report by Gustafson et al.(43) appeared in which the two-hybrid system was used to characterize the interaction between the IR and Shc. The findings obtained in this study are totally in agreement with our conclusion concerning the interaction between these two molecules.

In summary, using the two-hybrid system we have shown that p52Shc and IRS-1 bind to tyrosine-phosphorylated IGF-IR. Further, we found that the amino-terminal domain of p52Shc binds to the IGF-IR and to the IR in the juxtamembrane NPEY motif, and that IRS-1 and Shc bind this same motif in the two receptors. However, in contrast to Shc, IRS-1 shows no interaction with different IGF-IR forms mutated on the principal Tyr-autophosphorylation sites, suggesting that interaction between the IGF-IR and IRS-1 requires at least two major determinants on the receptor: a first one located in the juxtamembrane region and a second one in the kinase domain. Our conclusions have been inferred from observations made with the yeast two-hybrid system, and we can not exclude the possibility that the interactions detected are indirect and require intermediates within the yeast nucleus. One could also argue that the relevant environment of the cytoplasm of mammalian cells is different from the yeast nucleus. Despite this possibility, the yeast two-hybrid system is clearly the most efficient method allowing the detection of interaction between the IR or the IGF-IR and their two identified substrates, Shc and IRS-1.

Both Shc and IRS-1 have been implicated in mediation of insulin- or IGF-I-induced activation of Ras and of the mitogen-activated protein kinase cascade(14, 16, 17) . In response to insulin or IGF-I stimulation, tyrosine-phosphorylated Shc (or IRS-1) binds to the adaptor Grb-2, which forms a complex with Sos, a guanine nucleotide exchange factor of Ras. These events couple Shc to Sos or IRS-1 to Sos. Based on our results it is tempting to propose a model in which the interaction of Shc (or IRS-1) with the immediate juxtamembrane domain of the IGF-IR and the IR results in the targeting of the complex Grb-2/Sos near the membrane where Ras is localized. Hence, the interaction of Shc and IRS-1 with the juxtamembrane domains of the two receptors could represent an anchoring and pulling step in the membrane recruitment of Shc-Grb-2-Sos and IRS-1-Grb-2-Sos complexes to Ras.

Finally, our demonstration that for both the IGF-IR and the IR the two central proteins in signaling, IRS-1 and Shc, use at least in part the same receptor domain highlights the essential role of the juxtamembrane domain in receptor functioning.


FOOTNOTES

*
This research was supported in part by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur la Cancer Grant 4021, Université de Nice-Sophia Antipolis, La Ligue contre le Cancer, and Groupe LIPHA Contract 93123 (Lyon, France). 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. Tel.: 33-93-81-54-47; Fax: 33-93-81-54-32.

(^1)
The abbreviations used are: IGF-I, insulin-like growth factor-I; IR, insulin receptor; IGF-IR, insulin-like growth factor-I receptor; Shc, Src-homology 2/alpha-collagen; IRS-1, insulin receptor substrate-1; SH2 domain, Src homology 2 domain; GAD, Gal4 activation domain; Delta, deletion mutant.


ACKNOWLEDGEMENTS

We are grateful to Anne-Françoise Burnol and Jacques Camonis for helpful advice regarding the two-hybrid assay. We thank A. Vojtek and S. Elledge for the L40 strain and yeast plasmids. We thank P. G. Pelicci for the human Shc cDNA, M. F. White and C. R. Kahn for the rat IRS-1 cDNA, A. Ullrich for the human IR cDNA, and P. De Meyts for the human IGF-IR cDNA. We also sincerely thank E. Van Obberghen-Schilling and M. Deckert for critical reading of the manuscript.


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