©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tyrosine Phosphorylation of Insulin Receptor Substrate-1 in Vivo Depends upon the Presence of Its Pleckstrin Homology Region (*)

(Received for publication, May 8, 1995 )

Hedva Voliovitch (1) Daniel G. Schindler (1) Yaron R. Hadari (1) Simeon I. Taylor (2) Domenico Accili (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

To characterize the structural basis for the interactions between the insulin receptor (IR) and its major substrate, insulin receptor substrate-1 (IRS-1), a segment of the NH-terminal region of IRS-1 (Pro-Pro) was deleted. This region contains the first four conserved boxes of a pleckstrin homology (PH) domain, located at the NH-terminal part of IRS-1. COS-7 cells were then cotransfected with the genes coding for IR and a wild-type (WT) or a mutated form of IRS-1. IRS-1 underwent significantly reduced insulin-dependent tyrosine phosphorylation compared with WT IRS-1. The reduced in vivo tyrosine phosphorylation of IRS-1 was accompanied by reduced association between IRS-1 and its downstream effector p85 regulatory subunit of phosphatidylinositol-3 kinase. In contrast, both WT IRS-1 and IRS-1 underwent comparable insulin-dependent tyrosine phosphorylation in vitro when incubated with partially purified insulin receptor kinase. These findings suggest that the overall structure of IRS-1 is not altered by deletion of its PH domain and that the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro. In conclusion, the PH region might facilitate in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. This could account for the fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions remain unaffected.


INTRODUCTION

The insulin receptor (IR)()is a transmembrane glycoprotein composed of two extracellular subunits and two transmembrane subunits linked by a disulfide bond to form an heterotetrameric structure. The subunits contain the insulin-binding domain, whereas the transmembrane subunits function as tyrosine-specific protein kinase (insulin receptor kinase), which undergoes autophosphorylation following insulin binding (compare (1, 2, 3, 4) for reviews). Autophosphorylation activates the insulin receptor kinase (5) and enables it to phosphorylate protein substrates, including insulin receptor substrate-1 (IRS-1) (6) and Shc (7) . IRS-1 is a key element in insulin action, and mutations of IR (e.g. mutation of Tyr) that abolish IRS-1 phosphorylation inhibit insulin-induced metabolic and growth-promoting effects(4, 8, 9) . IRS-1 and its homologue called 4PS are also essential for insulin-, IGF-1-, and IL-4-stimulated mitogenesis (10, 11, 12, 13) . In contrast, the phenotype of mice lacking IRS-1 is surprisingly mild(14, 15) . The mice suffer some retardation of intrauterine growth and show impaired glucose tolerance. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin.

In non-treated cells, IRS-1 is serine-phosphorylated, and, after insulin stimulation, tyrosine and serine phosphorylation of IRS-1 is increased(13) . IRS-1 has 21 potential tyrosine phosphorylation sites, including six in YMXM motifs and three in YXXM motifs. These motifs belong to a common consensus sequence (D/E)(D/E)Y(M/V)(P/D/E)M also present in the platelet-derived growth factor, colony-stimulating factor-1, and Kit receptors(16) . Phosphorylation of these Tyr residues of IRS-1 by insulin receptor kinase is believed to create a binding site for the SH2 domains of the p85 regulatory subunit of phosphatidylinositol 3-kinase(17, 18) , Grb2, Nck, and the protein tyrosine phosphatase SH-PTP2 (Syp)(19) . The association between p85 and IRS-1 results in activation of phosphatidylinositol 3-kinase (17, 18) in a mechanism independent of the direct activation of phosphatidylinositol 3-kinase by Ras(20) . Similarly, binding of the ras activator, son of sevenless (mSos), to the Grb2IRS-1 complexes links the insulin receptor to the ras signaling pathway(21) . Both Grb-2 and phosphatidylinositol 3-kinase can simultaneously bind distinct tyrosine-phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could serve as a docking site for multiple SH2 proteins to further propagate the insulin signal(22) .

IRS-1 also contains a pleckstrin homology (PH) domain (23, 24) at its extreme NH-terminal region(25) . This domain of about 100 amino acids is found in a number of signaling proteins including mSos and Gap(26) . It consists of six weakly conserved blocks separated by insertions of different lengths. This domain has been termed ``pleckstrin homologue'' because it was originally detected as an internal repeat in pleckstrin, a 47-kDa protein that is the major substrate of protein kinase C in platelets(27) . It is unlikely that the PH domain could function catalytically because of its small size and lack of conserved residues. Instead, it may be involved in molecular recognition similarly to SH2 and SH3 domains. As with these domains, the PH domain does not occupy a specific position in the host molecule, suggesting that it is functionally independent(28) .

To study the role of the PH domain of IRS-1, a truncated form of IRS-1 was generated where the first four ``blocks'' of the PH region were deleted. Here we demonstrate that such a deletion selectively impairs the ability of IRS-1 to undergo in vivo insulin-dependent phosphorylation and markedly inhibits the association between the truncated form of IRS-1 and phosphatidylinositol 3-kinase, but does not affect the capacity of the mutated IRS-1 to serve as an in vitro substrate for the insulin receptor kinase.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes were purchased from Boeheringer Mannheim and Promega. Radiolabeled nucleotides and [S]methionine were from Amersham Corp. All other reagents were from Sigma unless stated otherwise. Wheat germ agglutinin coupled to agarose was from BioMakor (Kiryat Weizmann, Rehovot, Israel).

Antibodies

Antibodies to IRS-1 (anti-IRS-1) were raised in rabbits according to standard procedures (29) by injection of a keyhole limpet hemocyanin-coupled peptide CYASINFQKQPEDRQ that corresponds to the carboxyl-terminal 14 amino acids of rat liver IRS-1 (6) (and an additional Cys residue at the NH-terminal site). Antibodies were affinity-purified from the serum by adsorption onto a column of peptide coupled to Affi-Gel 10, elution with 100 mM HCl glycine, pH 2.7, and immediate neutralization. Monoclonal mouse anti-Tyr(P) antibodies (PY-20) were purchased from Affinity Research Products (Nottingham, UK) and were used at 1:1000 dilution.

Cloning of the Mouse IRS-1 Gene

The IRS-1 gene was cloned from a 129SV mouse genomic library made in the Fix vector (Stratagene). The library was screened with a 0.7-kilobase pair cDNA probe corresponding to base pairs 670-1314 of the rat IRS-1 sequence (6) . Positive clones were identified, isolated by three rounds of screening, digested with different restriction enzymes, and subcloned into a pGEM-11Z vector (Promega). The insert (denoted pGEM-11Z-gIRS1) represented the sequence from -2.7 to 4.7 kilobase pairs of the IRS-1 gene. Because the coding segment of IRS-1 lacks introns, the plasmid contained the entire coding region of the 129SV mouse IRS-1 gene (base pairs 478-4700, numbered according to the rat IRS-1 sequence(6) ). It should be noted that, similar to the human IRS-1 gene(30) , the mouse genomic sequence also contains an intron located 20 nucleotides 3` to the TAG termination codon.

Generation of IRS-1 Expression Vectors

To generate expression vectors for IRS-1, sequences upstream of the ATG initiation codon were deleted because they contain several in-frame initiation and termination codons that might interfere with efficient translation (10) . In order to delete these regions, the sequence from 512 to 812, including the ATG start codon at 589 and the XhoI site at 785, was amplified by polymerase chain reaction (PCR). The 5` primer (5`-GGAAGTCGACAAGCTTAGCATGGCGAG-3`) was located at nucleotide 586 and included HindIII and SalI sites. The 3` primer (5`-CCGCTTGTTGAGTTGAAACAGCT-3`) was located at nucleotide 812 of the IRS-1 gene. Both pGEM-11Z-gIRS1 and the PCR product were digested with HindIII and XhoI, and the 5` end of the genomic IRS-1 was replaced with the PCR fragment (nucleotides 586-785). Proper, in-frame ligation of the PCR product into this new plasmid (pGEM-11Z-cIRS1) was confirmed by restriction map analysis and sequencing. To allow expression of the IRS-1 molecule in COS-7 cells, the IRS-1 gene (0.58-4.7 kilobase pairs) was excised from pGEM-11Z-cIRS1 with HindIII and SalI, gel-purified, and ligated into pcDNAI/Amp or pcDNAIII (Invitrogen) to generate pcDNAI-IRS-1 or pcDNAIII-IRS-1, respectively.

To generate a construct expressing IRS-1, where 60 amino acids from the NH-terminal region of IRS-1 (Pro-Pro) are deleted, pcDNAI-IRS-1 was digested with HindIII and XhoI, and a 200-base-pair piece was deleted and replaced by a double-stranded synthetic oligonucleotide containing matching overhangs:

The initiation codon is indicated in bold. The correctness of the construct (pcDNAI-IRS1) was verified by restriction mapping.

To generate pcDNAIII-IRS-1 , the IRS1 gene was excised from pcDNAI-IRS-1 with HindIII and SalI, gel-purified, and ligated into pcDNAIII (Invitrogen). To generate a plasmid for transient expression of IR in COS-7 cells, the IR coding sequence was excised from pGEM4Z-IR with XhoI. pcDNAI plasmid was linearized with XhoI, gel purified, dephosphorylated with calf intestinal alkaline phosphatase, and ligated to the IR coding sequence. The correctness of the construct (pcDNA-IR) was verified by restriction mapping.

Transient Expression of the IRS-1 Gene in COS-7 Cells

COS-7 cells were cotransfected using DEAE-dextran (31) with pcDNA-IRS1, pcDNA-IRS1, or pcDNA-IR (alone or in the combinations indicated in the figure legends). After 36 h the cells were starved for 8 h in serum-free Dulbecco's modified Eagle's medium and stimulated for 1 min with 10M insulin. Cells were then washed three times with phosphate-buffered saline and were frozen in liquid nitrogen. Cell extracts were made in 250 µl of lysis buffer I containing 0.15 M sucrose, 50 mM Hepes, pH 7.4, 80 mM -glycerophosphate, 10 mM NaF, 1% Triton X-100, 0.1% SDS, 2 mM each of EDTA, EGTA, and sodium orthovanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged for 15 min at 4 °C in an Eppendorf centrifuge at 12,000 g, and protein concentrations were determined with the Bradford dye binding assay(32) . 50 µg of protein from the supernatants were loaded on a 7.5% SDS-PAGE and immunoblotted with anti-phosphotyrosine antibodies or with anti-IRS-1 antibodies.

Stable Expression of the IRS-1 Gene in CHO Cells

CHO cells were transfected by electroporation (33) with pcDNAIII-IRS1, pcDNAIII-IRS1, or pcDNA-IR. 4 10 cells were electroporated at 960 microfarads and 250V using 10 µg of DNA. Following transfection, cells were incubated in nonselective medium. 48 h later cells were divided into 8 plates, and G418 (Life Technologies, Inc., 500 µg/ml active units) was added for selection of stable colonies. G418-resistant clones that overexpress IRS-1 or IRS1 were isolated and further propagated. For in vitro phosphorylation by insulin receptor kinase, cell extracts were prepared from cells grown to confluence in 15-cm plates. The cells were freeze-thawed three times in 800 µl of lysis buffer containing 0.15 M sucrose, 50 mM Hepes, pH 7.4, 80 mM -glycerophosphate, 10 mM NaF, 2 mM each of EDTA, EGTA, and sodium orthovanadate, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged for 15 min at 4 °C in an Eppendorf centrifuge at 12,000 g, and protein concentrations were determined with the Bradford dye binding assay (32) .

Immunoprecipitation

Anti-IRS-1 antibodies (at a 1:10 dilution) were added to 60 µl of 50% protein A-Sepharose in 0.1 M Tris buffer, pH 8.5, and were incubated for 1 h at 4 °C. Cell extracts, prepared in buffer I, were incubated for 2 h with the antibody-protein A-Sepharose complex. Immunocomplexes were washed, suspended in sample buffer(34) , resolved on 7.5% SDS-PAGE (35) , and transferred to nitrocellulose for Western blotting.

Protein Electrophoresis and Blotting

Immunoblotting was carried out essentially as described(18) . The blotted proteins were incubated with monoclonal anti-Tyr(P) or polyclonal anti IRS-1 antibodies at 4 °C for 16 h, followed by intensive washing. To detect antibody binding, a horseradish peroxidase-conjugated goat anti-mouse antibodies ECL kit (Amersham) was used according to the manufacturer's instructions.

Partial Purification of Insulin Receptor

Insulin receptors were partially purified from rat liver plasma membranes. The preparation of membranes, solubilization in Triton X-100, and affinity chromatography of insulin receptors on wheat germ agglutinin coupled to agarose were carried out as described previously(36) .

In Vitro Phosphorylation of IRS-1

The reaction was carried out essentially as described previously(37) . Briefly, 60-µl aliquots of partially purified insulin receptors (50-200 µg/ml) were incubated in the presence or absence of 10M insulin (in 50 mM Hepes, 0.1% bovine serum albumin, 0.1% Triton X-100, pH 7.6) for 30 min, 22 °C). Cytosolic extracts derived from CHO cells that overexpress either WT-IRS-1 or IRS-1 were added, and phosphorylation in a final volume of 450 µl was initiated with 80 µl of a ``reaction mix'' to yield the following final concentrations: 50 mM Hepes, 1 mM ATP, 10 mM magnesium acetate, 4 mM manganese acetate, and 0.05% Triton X-100. Reactions were allowed to proceed for 10 min at 22 °C and were terminated by adding EDTA to a final concentration of 20 mM. IRS-1 was precipitated with anti-IRS-1 antibodies, and the samples were further processed as described above.

Phosphatidylinositol 3-Kinase Assay

Assay of phosphatidylinositol 3-kinase activity was performed as described previously.(18) . Briefly, whole cell lysates were prepared as above, and the amount of IRS-1 in each extract was estimated based upon immunoblots with IRS-1 antibodies. Equal amounts of IRS-1 were immune precipitated with anti-IRS-1 antibodies, and phosphatidylinositol 3-kinase activity was assayed in the immunoprecipitates using phosphatidyl inositol and [-P]ATP as substrates. The reaction was carried out for 10 min at 24 °C. Reaction products were extracted and separated by thin layer chromatography. Autoradiograms were scanned by densitometry.


RESULTS

Insulin-dependent Tyrosine Phosphorylation of IRS-1 in COS-7 Cells

Stimulation of untransfected COS-7 cells with insulin resulted in a very low insulin-dependent tyrosine phosphorylation of both IR and IRS-1 (Fig. 1, lane 3). Transfection of these cells with an IR expression plasmid yielded an insulin-dependent prominent band whose molecular mass (95 kDa) is consistent with that of native subunits of the insulin receptor and a fainter band whose molecular mass (180 kDa) is consistent with that of IRS-1 (Fig. 1, lane 2). When the cells were cotransfected with expression plasmids for both IR and WT-IRS-1, the level of insulin-induced IR phosphorylation remained largely unaltered, whereas IRS-1 phosphorylation was markedly (16-fold) enhanced (Fig. 1, lane 1). These findings show that transient expression of either IR or IRS-1 generates functional full-length proteins. Moreover, the expressed IRS-1 serves as a substrate for the insulin receptor kinase. Comigration of the native (Fig. 1, lane 2) and transfected forms of IRS-1 (Fig. 1, lane 1) suggests that transfected IRS-1 undergoes the same post-translational modification as the native endogenous one.


Figure 1: Insulin-dependent phosphorylation of IRS-1 in COS-7 cells. COS-7 cells remained untransfected (lane 3), or were transfected with pcDNA-IR (IR) alone (lane 2) or in combination with pcDNA-IRS1 (IRS-1) (lane 1). After 36 h cells were incubated for 8 h in serum-free Dulbecco's modified Eagle's medium and then stimulated for 1 min with 10M insulin. Cell extracts were prepared; 50 µg of protein were resolved on 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose. Immunoblotting with anti-Tyr(P) was performed as described under ``Experimental Procedures.''



Partial Deletion of the PH Region Impairs Insulin-induced Tyrosine Phosphorylation of IRS-1

To study the functional role of the PH region of IRS-1, a truncated form of the IRS-1 gene was generated where 61 amino acids encoding for the NH-terminal region of the PH domain (Pro-Pro) were deleted (IRS-1). WT or mutant mouse IRS-1 genes were then transiently expressed in COS-7 cells together with the IR gene. Although both WT and mutated IRS-1 molecules were expressed at comparable levels (detected by immunoblotting with anti IRS-1 antibodies) (Fig. 2, bottom), IRS-1 underwent significantly reduced insulin-dependent tyrosine phosphorylation (detected by immunoblotting with anti-Tyr(P) antibodies) (Fig. 2, top). Analysis by densitometry revealed that the insulin-induced increase in tyrosine phosphorylation of WT-IRS-1 is 13-fold greater than the increase in IRS-1. These results therefore suggest that the PH domain of IRS-1 is required for normal in vivo tyrosine phosphorylation of IRS-1 by IR.


Figure 2: Reduced insulin-dependent phosphorylation of IRS-1. COS-7 cells were cotransfected with pcDNA-IR in combination with pcDNA-IRS1 (WT) or pcDNA-IRS1 (PH). After 36 h cells were incubated for 8 h in serum-free Dulbecco's modified Eagle's medium and then stimulated for 1 min with 10M insulin. Cell extracts were prepared; 50 µg of protein were resolved on 10% SDS-PAGE under reducing conditions and transferred to nitrocellulose. Immunoblotting with anti-Tyr(P) (Blot -P-Ty, top) and anti-IRS-1 antibodies (Blot -IRS-1, bottom) was performed as described under ``Experimental Procedures.'' The intensity of the bands corresponding to tyrosine-phosphorylated IRS-1 and IRS1 was quantitated by densitometry. These values were: 9 for IRS-1 without insulin, 109 for IRS-1 with insulin, 11 for IRS-1 without insulin, and 1433 for IRS-1 with insulin.



IRS-1Exhibits Reduced Association with Phosphatidylinositol 3-Kinase

In view of the fact that phosphatidylinositol 3-kinase associates with and is activated upon interaction with tyrosine-phosphorylated IRS-1(18) , the functional consequences of the reduced phosphorylation of IRS-1 were assessed by assaying for the phosphatidylinositol 3-kinase activity associated with the truncated form of IRS-1. As shown in Fig. 3, the reduced tyrosine phosphorylation of IRS-1 was accompanied by reduced phosphatidylinositol 3-kinase activity detected in IRS-1 immunoprecipitates (Fig. 3). Although comparable amounts of both WT IRS-1 and IRS-1 were precipitated by anti IRS-1 antibodies (not shown), the increase in phosphatidylinositol 3-kinase activity, associated with insulin-stimulated IRS-1, was 2-fold higher than the activity associated with the insulin-stimulated IRS-1. These results indicate that deletion of the PH region of IRS-1 impairs its interactions with downstream effectors like phosphatidylinositol 3-kinase.


Figure 3: Phosphatidylinositol 3-kinase activity in IRS-1 immunoprecipitates from insulin-treated COS-7 cells. COS-7 cells were cotransfected with pcDNA-IR in combination with pcDNA-IRS1 or pcDNA-IRS1. Following stimulation for 1 min with 10M insulin, cell extracts were prepared and immunoprecipitated with anti-IRS-1 antibodies. Phosphatidylinositol 3-kinase activity was assayed in the immunoprecipitates by in vitro phosphorylation of phosphatidylinositol as described under ``Experimental Procedures.'' The intensity of the spots corresponding to phosphatidylinositol 3-phosphate was quantitated by densitometry. These values were: 10 for IRS-1 without insulin, 116 for IRS-1 with insulin, 14 for IRS-1 without insulin, and 234 for IRS-1 with insulin.



IRS-1Is Phosphorylated in Vitro to Levels Comparable to Those of WT IRS-1

To rule out the possibility that IRS-1 fails to undergo in vivo phosphorylation due to gross alterations in its overall conformation, we compared the abilities of wild-type and mutated IRS-1 to serve as in vitro substrates for insulin receptor kinase. Increasing amounts of cytosolic extracts derived from CHO cells that overexpress either IRS-1 or WT IRS-1 were subjected to phosphorylation by partially purified insulin receptor kinase. As shown in Fig. 4(top) both forms of IRS-1 underwent insulin-dependent tyrosine phosphorylation in a dose-dependent manner. However, there was no significant difference between the extent of their phosphorylation (when normalized according to the amount of IRS-1) (Fig. 4, bottom). Hence, IRS-1 appears to maintain the overall structural organization of WT IRS-1 to the extent that it serves as a comparable in vitro substrate for insulin receptor kinase.


Figure 4: In vitro phosphorylation of WT IRS-1 and IRS-1 by insulin receptor kinase. Top, aliquots (100-300 µl) of cytosolic extracts derived from CHO cells overexpressing WT IRS-1 (lanes A-D) or IRS-1 (lanes E-H) were subjected to in vitro phosphorylation by partially purified insulin receptor kinase in the presence (+) or the absence (-) of insulin as described under ``Experimental Procedures.'' IRS-1 immunoprecipitates were resolved by means of SDS-PAGE and blotted with either Tyr(P) or IRS-1 antibodies. Bottom, the intensity of the bands was quantified by densitometry, and the results are presented in arbitrary units. P-Tyr, Tyr(P); , IRS-1; , IRS-1.




DISCUSSION

The present study provides evidence that deletion of four out of six PH subdomains of IRS-1 largely impairs its capacity to serve as an in vivo substrate for the insulin receptor kinase. The reduced phosphorylation could not be accounted for by deletion of tyrosine phosphorylation sites, because only 2 (Tyr and Tyr) out of 21 such potential sites were deleted in IRS-1.

The immediate outcome of the reduced phosphorylation of IRS-1 is reduced phosphatidylinositol 3-kinase activity that remains associated with the mutated form of IRS-1. This result is somewhat expected in view of the fact that the SH2 domains of p85, the regulatory subunit of phosphatidylinositol 3-kinase, presumably interact with Tyr residues 460, 608, 939, and 987 of IRS-1, placed in the context of YMXM or YXXM motifs (38) . This association results in activation of pp110, the catalytic unit of phosphatidylinositol 3-kinase, by an as yet unknown mechanism. Our results therefore suggest that the reduced phosphorylation of IRS-1 also involves impaired phosphorylation of at least some of these residues. Still, we cannot rule out the possibility that deformation of the overall native structure of IRS-1 induced by the truncation impairs the capability of IRS-1 to interact with phosphatidylinositol 3-kinase. The reduction in phosphatidylinositol 3-kinase activity is less dramatic than the reduction in Tyr(P) content of IRS-1. This apparent discrepancy could be attributed to the fact that the residual amount of tyrosine-phosphorylated IRS-1 is still high enough to complex with the limited amount of available endogenous phosphatidylinositol 3-kinase.

Partial deletion of the PH region could alter the overall structure of IRS-1 such that it fails to interact with and to serve as a substrate for IR. Alternatively, the PH domain itself could serve as the binding domain to IR. In such a case, its deletion could prevent interactions between IRS-1 and IR, even though the rest of the IRS-1 molecule still maintains its overall native structure. To address these possibilities, we compared the abilities of wild-type and mutated IRS-1 to serve as in vitro substrates for insulin receptor kinase. We could readily demonstrate that both IRS-1 forms serve as comparable in vitro substrates for insulin receptor kinase, indicating that the partial deletion of the PH region does not affect, in any major way, the in vitro interactions between IRS-1 and insulin receptor kinase. These findings serve to make two points: (i) the overall structure of IRS-1 is not grossly modified by the mutation and (ii) the PH domain is not the main site for protein-protein interactions between the insulin receptor and IRS-1, at least in vitro.

Although the solution structure of two PH domains has been resolved (39, 40) , the function of the PH domain is largely unknown. On the basis of structural similarities between PH domains and lipid-binding proteins, it has been proposed that the PH domain binds to lipophilic molecules(39) . Consistent with this hypothesis, PH domains were shown to bind to phosphatidylinositol 4,5-bisphosphate(41) . Alternatively, it has been suggested that PH domains function in the recognition of phosphorylated Ser/Thr residues in specific peptide sequences(42) . Indeed, a PH domain localized at the COOH-terminal end of -adrenergic receptor kinase is essential for targeting -adrenergic receptor kinase to the plasma membrane(25) , presumably through interaction with the -subunits of heterotrimeric G-proteins. These findings have set the stage for the hypothesis that, similar to SH2 and SH3 domains, PH regions might be involved in protein-protein interactions. Support for this notion is provided by the fact that, when tested in vitro, the PH domain of IRS-1, expressed as a GST-fusion protein, interacts, though to a rather low extent, with G(43) . In contrast, a 356-amino-acid region encompassed by amino acids 160-516 of IRS-1 outside the PH region is sufficient for interactions with the receptor in the yeast two-hybrid assay(44) .

At present it is difficult to reconcile these alternatives. However, our studies support the hypothesis that the PH region facilitates in vivo binding of IRS-1 to membrane phospholipids or other cellular constituents in close proximity to the IR, whereas the actual interactions with the IR are presumably mediated through other domains of the IRS-1 molecule. The fact that partial deletion of the PH domain selectively impairs the in vivo interactions between the insulin receptor and IRS-1, whereas their in vitro interactions are unaffected, strongly supports this hypothesis.


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.

§
Incumbent of the Philip Harris and Gerald Ronson Career Development Chair in Diabetes Research. To whom correspondence should be addressed: Dept. of Chemical Immunology, Weizmann Institute of Science, P. O. Box 26, Rehovot 76100, Israel. Fax: 972-8-342-380; E-Mail: Lizick@weizmann.weizmann.ac.il.

The abbreviations used are: IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRS-1, IRS-1 with a segment of the NH-terminal region (Pro-Pro) deleted; PH, pleckstrin homologue; WT, wild-type; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Dr. Ronit Sagi-Eisenberg for helpful discussions and a critical review of this manuscript. We thank Yael Biener and Keren Paz for helpful advice and invaluable assistance.


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