Site-Directed Mutagenesis and Yeast Two-Hybrid Studies of the Insulin and Insulin-Like Growth Factor-1 Receptors: The Src Homology-2 Domain-Containing Protein hGrb10 Binds to the Autophosphorylated Tyrosine Residues in the Kinase Domain of the Insulin Receptor

Lily Q. Dong, Sarah Farris, Jeff Christal and Feng Liu1

Department of Pharmacology The University of Texas Health Science Center at San Antonio San Antonio, Texas 78284-7764


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To characterize the structural basis for the interaction between hGrb10 and the insulin receptor and the insulin-like growth factor-1 receptor, different mutant receptors containing a segment of deletion in either the juxtamembrane domain or in the C terminus of the receptors, or containing tyrosine-to-phenylalanine point mutations in these regions of the insulin receptor, were generated. Yeast two-hybrid and in vitro binding studies of the interaction between the mutant receptors and hGrb10 revealed that tyrosine residues in these regions are not essential for the binding of hGrb10. To further identify the binding site for hGrb10, all conserved tyrosine residues in the kinase domain of the insulin receptor were replaced with either phenylalanine or alanine by site-directed mutagenesis. Mutations of all tyrosine residues in this region, except at positions 1162/1163, did not inhibit the binding of the receptor to hGrb10. The binding of the Src homology 2 domain of hGrb10 to the receptors was significantly enhanced in the presence of an intact pleckstrin homology domain. Our findings suggest that, unlike other Src homology 2 domain-containing proteins, hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the insulin receptor, and the pleckstrin homology domain plays an important role in hGrb10/receptor interaction. Because the autophosphorylated tyrosine residues are critical for the autophosphorylation and kinase activity of the receptor, the binding of hGrb10 at these sites may suggest a role for the protein in the transduction or regulation of insulin receptor signaling.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The insulin receptor (IR) and insulin-like growth factor I receptor (IGF-1R) are heterotetrameric transmembrane proteins that show a high degree of structural and functional similarities. They both consist of two extracellular {alpha}-subunits and two transmembrane ß-subunits. The juxtamembrane and the kinase domains of these two receptors are highly homologous. Both receptors contain a NPXY motif in their juxtamembrane regions that has been shown to bind IR substrate-1 (IRS-1) (1), Shc (2), and GTPase-activating protein (GAP) (3). Mutation of the conserved tyrosine residue in the NPXY motif of the IR abolishes the interaction between the receptor and IRS-1 in vitro (4) and in the yeast two-hybrid system (5) and impairs insulin-stimulated metabolic and mitogenic effects in cells (6, 7). Both receptors also contain three tyrosine residues in close proximity in the kinase domains (Tyr1158, Tyr1162, and Tyr1163 in the IR and Tyr1131, Tyr1135, and Tyr1136 in the IGF-I. [(The numbering systems used for IR and IGF-1R are Ebina et al.(8) and Ullrich et al.(9), respectively.] x-Ray crystallography of the human IR indicates that Tyr1162 is bound to the active site of the receptor kinase (10). Autophosphorylation of these residues has been shown to play a critical role in the activation of the receptor kinases toward their cellular substrates (7, 11, 12).

Despite the high sequence homology in the juxtamembrane and kinase domains, the C-terminal region of the two receptors has only limited (44%) homology (9). For example, there are two autophosphorylation sites (Tyr1328 and Tyr1334) in the C-terminal domain of the IR, but only one of them (Tyr1334) is conserved between the IR and IGF-1R. The two tyrosine residues in the C-terminal of the IR have been shown to be the binding sites for Src homology 2 (SH2) domain-containing proteins such as the p85 subunit of phosphatidylinositol (PI) 3-kinase, syp (3), and Shc (13), and have been suggested to play a role in modulating mitogenic function (14, 15, 16).

To identify potential molecules involved in the IR-signaling pathway, we have recently used the yeast two-hybrid technique with the IR cytoplasmic domain as bait to find its interacting proteins. We identified a SH2 domain-containing protein hGrb10 that binds specifically to tyrosine-phosphorylated IR (17). Unlike other SH2 domain-containing proteins such as p85 and syp, hGrb10 does not bind to IRS-1 in vivo. Several isoforms of hGrb10, which differ in their pleckstrin homology (PH) domain and in the N-terminal region, have been found in skeletal muscle, fat, and HeLa cells (17, 18, 19). Expression in cells of the isoform containing a deletion in the PH domain (Grb-IR/hGrb10{alpha}) inhibits insulin-stimulated substrate tyrosine phosphorylation and PI 3-kinase activity, suggesting that this protein may play a role in the regulation of insulin action (17).

To better understand the mechanisms of hGrb10 involvement in the IR or the IGF-1R signal transduction pathways, we decided to further characterize the interaction between hGrb10 and these receptors. The data presented in this paper show that, unlike other SH2 domain-containing proteins, which bind to either the juxtamembrane domain or the C-terminal region of the IR or IGF-1R, hGrb10 binds specifically to the autophosphorylated tyrosine residues in the kinase domain of the receptors. Because the autophosphorylated tyrosine residues in the kinase domain of the receptors are critical for receptor autophosphorylation and kinase activity, the direct binding of hGrb10 to these residues may provide a mechanism for the regulation of receptor signaling.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Binding of hGrb10 to the IR and IGF-1R Is through a Direct Interaction between the SH2 Domain of the Protein and the Phosphotyrosine Residues on the Receptors
We have previously shown that the binding of the SH2 domain-containing protein hGrb10 to the IR requires the tyrosine phosphorylation of the receptor (17). Although this finding suggests that the binding is probably through a direct interaction between the phosphorylated tyrosine residue(s) of the receptor and the SH2-domain of hGrb10, it is also possible that the phosphorylation-induced conformational change of the receptor, rather than the autophosphorylated tyrosine residue itself, is required for hGrb10 to bind. To test whether the SH2 domain of hGrb10 is directly involved for binding to the IR and IGF-1R, we replaced the conserved arginine residue (Arg474) within this domain to glutamine and studied the interaction of the mutant SH2 domain to the IR using the yeast two-hybrid system. We found that the point mutation within the SH2 domain completely abrogated the binding of hGrb10(SH2) to the IRcyto and IGF-1Rcyto (data not shown). To further investigate this interaction, we generated a mutant IR in which the conserved lysine residue within the ATP-binding site of the IR was mutated to alanine (IRK1030A). Yeast two-hybrid studies revealed that this IR mutant, which was unable to autophosphorylate itself, failed to bind to hGrb10(SH2) (data not shown). These findings provide further evidence that the binding of hGrb10 to the receptor is through a direct interaction between the SH2 domain of hGrb10 and the autophosphorylated tyrosine residues on the IR.

Tyrosine Residues in the Juxtamembrane or the C Terminus of the IR and IGF-1R Are Not Essential for the Binding to hGrb10
There are a total of 13 tyrosine residues in the cytoplasmic domain of the human IR (15 in the cytoplasmic domain of the IGF-1R). At least six of these tyrosine residues, Tyr972 in the juxtamembrane domain, Tyr1158, Tyr1162 and Tyr1163 in the kinase domain, and Tyr1328 and Tyr1334 in the C-terminal region, have been shown to undergo insulin-stimulated autophosphorylation. To determine which residue(s) is involved in binding hGrb10, we first mapped the regions on the receptor involved in the binding. We constructed several yeast two-hybrid plasmids encoding the cytoplasmic domains of IR or IGF-1R with deletions at either the juxtamembrane domain or the C-terminal region (Fig. 1AGo). Significant ß-Gal activity was detected when all of these GAL4BD/IR fusion proteins, except for GAL4BD/IR{Delta}CT, which has a 47-amino acid deletion in the C-terminal region of the IR, were coexpressed in yeast cells with GAL4AD/hGrb10(SH2) fusion protein (Fig. 1AGo). Our data suggest that tyrosine residues in either the juxtamembrane domains of the IR and IGF-1R or in the C-terminal region of the IGF-1R are not essential for the binding of hGrb10.



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Figure 1. Interaction of hGrb10 with the IR and IGF-IR in the Yeast Two-Hybrid System

A, Mapping the interacting domains of the IR and IGF-1R with hGrb10. Yeast two-hybrid plasmids containing deletions in either the juxtamembrane domain (JM) or the carboxyl-terminal (CT) of the receptors were constructed by PCR and subcloned as described in Materials and Methods. Six individual colonies of yeast transformants were streaked onto a Trp-Leu- plate and incubated at 30 C overnight. The interaction was determined by ß-galactosidase filter assays. (++) indicates high binding affinity (blue), and (-) indicates no apparent binding (white) in the assays. B, Construction of GAL4 activation (AD)/hGrb10 yeast two-hybrid plasmids. The PH domain and the SH2 domain are indicated.

 
The observation in the two-hybrid system that a deletion in the C-terminal region of the IR abolished the binding of the receptor to hGrb10 suggests that either tyrosine residues in this region or a certain conformation of the receptor, which was abolished by the truncation, are required for the binding. To test whether the two tyrosine residues in the C-terminal region of the IR are involved in binding hGrb10, two experiments were carried out. First, we studied the in vitro interaction between the GST/hGrb10(SH2) fusion protein and a mutant IR in which the C-terminal 69-amino acid residues were deleted (IR{Delta}69). Lysates from insulin-treated Chinese hamster ovary (CHO) cells overexpressing IR{Delta}69 (CHO.IR{Delta}69) were incubated with immobilized glutathione-S-transferase (GST) or GST/hGrb10 fusion proteins or with wheat germ agglutinin (WGA) agarose to precipitate the total IR{Delta}69 in the lysate. The hGrb10-associated proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by immunoblotting using the antibody against phosphotyrosine (Fig. 2AGo) or the {alpha}-subunit of the IR (Fig. 2BGo). As shown in Fig. 2Go, GST/hGrb10(SH2) precipitated a significant amount of the mutant IR{Delta}69 in insulin-stimulated cells (lane 4). No IR{Delta}69 was precipitated by the GST control (Fig. 2Go, lanes 1 and 2) or by the GST/hGrb10(SH2) fusion protein from non-insulin-treated cells (Fig. 2Go, lane 3). The conclusion that Tyr1328 and Tyr1334 are not the binding sites for hGrb10 was further confirmed by site-directed mutagenesis and yeast two-hybrid studies. As shown in Fig. 3AGo, substitution of the two tyrosine residues with phenylalanine did not inhibit the binding of the mutant receptors to hGrb10 in the yeast two-hybrid system, suggesting that these residues were not essential for the binding to hGrb10. On the other hand, mutation of Tyr1334 to phenylalanine abolished the interaction between the receptor and the p85 subunit of PI 3-kinase (Fig. 3BGo). Our results are consistent with the data of Staubs et al. (3), who found that the interaction between p85 and the IR was inhibited by the peptide containing a phosphotyrosine at position 1334. These findings also provide evidence that the two SH2 domain-containing proteins, hGrb10 and p85, bind to the IR at different sites.



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Figure 2. Interaction of the IR Mutant with a 69-Amino Acid Deletion at the C-Terminal Region (IR{Delta}69) with GST-hGrb10 Fusion Protein in Vitro

Lysates from insulin-treated (+) or nontreated (-) CHO cells expressing IR{Delta}69 were incubated with GST or GST/hGrb10(SH2) bound to glutathione-agarose beads or with WGA-agarose. The agarose-bound proteins were examined by immunoblotting with antibody to phosphotyrosine (A) or to the {alpha}-subunit of the IR (B). The {alpha}-subunit (IR{alpha}) and ß-subunit (IRß) of the mutant IR are indicated.

 


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Figure 3. Test of hGrb10 (A) or p85 (B) Interaction with the IR-Containing Tyrosine to Phenylalanine Mutations at the C-Terminal of the Receptor

The interaction was determined by ß-galactosidase liquid assays as described in Materials and Methods. The ß-galactosidase (ß-Gal) values are means ± SD of two to six independent assays, and each assay is an average of triplicate determinations. The data were analyzed by one-way ANOVA and the post hoc analysis was conducted using Fisher’s protected least-significant difference (PLSD) to determine the significance of difference between the wild type and the individual mutants. **, P < 0.01.

 
Identification of Tyr1162/1163 on the IR as the Binding Site for hGrb10
Having found that tyrosine residues in either the juxtamembrane domain or the C-terminal region of the IR or IGF-1R are not essential for the binding of hGrb10, we focused our attention on the tyrosine residues in the kinase domain of the receptors. Sequence comparison between IR and IGF-1R indicated that seven of the eight tyrosine residues in the kinase domain of the IR, including Tyr1011, Tyr1087, Tyr1122, Tyr1158, Tyr1162, Tyr1163, and Tyr1210, are conserved between the two receptors (Fig. 4Go). To determine which tyrosine residues are involved in the binding of hGrb10, we replaced all the conserved tyrosine residues, individually or in combination, with either phenylalanine or alanine by site-directed mutagenesis. Yeast two-hybrid studies of these mutant IRs with hGrb10 showed that mutation of tyrosine residues at position 1011, 1087, 1122, 1158, and 1210 of the IR had almost no effect on the binding of the receptor to hGrb10 (Fig. 5Go). In contrast, substitution of tyrosine residues at position 1162 and 1163 with alanine or phenylalanine significantly decreased the binding (Fig. 5Go).



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Figure 4. The Alignment of the Kinase Domain (KD) of the IR and IGF-1R

The conserved tyrosine residues are shown in bold characters.

 


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Figure 5. Yeast Two-Hybrid Studies of the Interaction between hGrb10 and the IR Kinase Domain Mutants

The interaction was determined by ß-galactosidase liquid assays as described in Materials and Methods. The ß-galactosidase (ß-Gal) values are means ± SD of three to six independent assays, and each assay is an average of triplicate determinations. The data were analyzed by one-way ANOVA, and the post hoc analysis was conducted using Fisher’s PLSD to determine the significance of difference between the wild type and the individual mutants. **, P < 0.01.

 
The Involvement of the PH Domain in hGrb10/Receptor Interaction
We have previously shown that there are at least two isoforms of hGrb10: one has a 46-amino acid deletion in the PH domain (hGrb10) and another contains an intact PH domain (17). Using a 0.9-kb hGrb10a cDNA (17) as probe, we screened a human skeletal muscle cDNA library and cloned the isoform containing an intact PH domain (hGrb10{gamma}, GenBank accession number AF001543 and Fig. 1BGo). To test whether the difference in the PH domain plays a role for hGrb10 binding to the IR or IGF-1R, we studied the interaction between the IR or IGF-1R and the full-length hGrb10{gamma} isoforms with or without an intact PH domain by the yeast two-hybrid system. As shown in Fig. 5Go, the presence of an intact PH domain significantly enhanced the binding of hGrb10 to both the IR and IGF-1R. The interaction, however, was abolished when the conserved arginine residue in the SH2 domain of hGrb10{gamma} was mutated to a glutamine or Tyr1162/1163 of the IR were replaced with phenylalanine (Fig. 6Go). On the other hand, truncation mutation at the juxtamembrane domain or point mutations at tyrosine residues 1011, 1087, 1122, 1158, 1210, and 1328/1334 of the IR had similar or increased activities compared with that of the wild type IR in the ß-gal liquid assays (data not shown), suggesting that these tyrosine residues are not directly involved in the binding. These data provide further evidence that the interaction between hGrb10 and the IR is through a direct interaction between the SH2 domain of the protein and Tyr1162/1163 of the IR.



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Figure 6. Interaction of hGrb10 Isoforms with the IR and IGF-1R in the Yeast Two-Hybrid System

The interaction was determined by ß-galactosidase liquid assays as described in Materials and Methods. The ß-galactosidase (ß-Gal) values are means ± SD of four to six independent assays, and each assay is an average of triplicate determinations. The data were analyzed by one-way ANOVA using the SPSS computer program (Prentice Hall, Upper Saddle River, NJ).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
hGrb10 is a newly identified SH2 domain-containing protein that binds with high affinity to the IR and IGF-1R. Our previous studies showed that expression of hGrb10 in cells expressing the IR inhibited insulin-stimulated GAP-associated p60 protein tyrosine phosphorylation and PI 3-kinase activity (17). However, the underlying mechanism of the inhibition has not been elucidated.

One possible explanation for the inhibition of insulin action by hGrb10 is that hGrb10 binds to a site on the IR that blocks the binding of other downstream signaling molecules. To test this hypothesis, we attempted to identify the binding site of hGrb10 to the receptor. Our results show that, unlike other SH2 domain-containing proteins such as p85, Grb2, and Shc, which bind either to the juxtamembrane domain or the C-terminal of the IR, hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the IR. This finding is consistent with the result from the recent study of O’Neill et al. (18), who showed that the binding of the hGrb10{alpha} splice variant Grb10/IR-SV1 (hGrb10ß) to the IR and IGF-1R was independent of the juxtamembrane domain and the C-terminal region of the receptors and was significantly reduced when the tyrosine residues at positions 1162 and 1163 of the IR were changed to phenylalanines, although they could not exclude the possibility that other tyrosine residues in the region may be involved in the binding. Data presented in this study showed that substitution of all the conserved tyrosine residues at positions 1011, 1087, 1122, and 1210 in the kinase domain of the IR did not inhibit the binding of the receptor to hGrb10, suggesting that these tyrosine residues are not the binding site for hGrb10. On the other hand, mutation of Tyr1162 and Tyr1163 significantly inhibited the binding. The binding of the SH2 domain of hGrb10 to the autophosphorylated tyrosine residues in the kinase domain was supported by the observation that a phosphopeptide corresponding to the sequence of the IR containing Tyr1158/1162/1163 bound to the GST-hGrb10(SH2) but not to the GST fusion protein in vitro (our unpublished observations). This conclusion is also consistent with the recent finding that the binding of the SH2 domain of hGrb10ß was inhibited by the same IR activation loop phosphopeptide (19). This finding, however, is contradictory to that of Hansen et al. (20), who reported that the hGrb10 mouse homolog mGrb10, whose SH2 domain sequence is 99% identical to that of hGrb10, binds to the phosphotyrosine residue at 1334 in the C terminus of the IR. The reason for the discrepancy between our results and those of Hansen et al. (20) is unclear. However, a recent study has shown that Tyr1316 of the human IGF-1R (equivalent to Tyr1334 of the human IR) is not the site for mGrb10 to bind (21).

Our data have shown that replacement of the two tyrosine residues at the C-terminal regions resulted in a 2-fold gain of function for binding of the receptor to hGrb10 (Fig. 3AGo). It is interesting to note that this same mutant was 2-fold more active than the wild type for poly(Glu/Tyr) phosphorylation in vitro (22). These data suggest that the two C-terminal tyrosine residues may play a role in modulating affinities of the IR, probably by blocking the critical residues in the activation loop of the receptor from their downstream substrates or binding proteins. These findings are consistent with the results from many studies that show that the C-terminal region of the IR plays an important role in insulin action (23, 24).

The observation that the interaction between hGrb10 and the IR or IGF-1R was significantly increased in the presence of an intact PH domain suggests that the PH domain may play a role in the interaction, either due to a direct interaction of this motif to the IR or to the generation of a PH-domain-induced conformation of hGrb10 that assisted the interaction. The finding that a single amino acid mutation in the SH2 domain of hGrb10 completely abolished the binding of the protein to the receptors suggests that the latter hypothesis is more likely. This conclusion is consistent with our findings that either an intact PH domain, or the full-length protein of hGrb10{gamma} with a 180-amino acid deletion at the C terminus [which includes the SH2 domain and the insert between the PH and the SH2 domain (IPS)] did not interact with the IR or IGF-1R in the yeast two-hybrid system (S. Farris and L. Q. Dong, unpublished data). These results suggest that neither the PH domain nor the N-terminal region of hGrb10 isoforms was sufficient to bind to the IR and that the SH2 domain of hGrb10 is directly involved and is sufficient to bind to the phosphotyrosine residues on the receptors. It is possible that a new binding site, which recognizes the autophosphorylated IR, may be generated in hGrb10 after the protein binds to the IR. This hypothesis is consistent with the recent finding that the IPS region of hGrb10 can bind to the IR in the yeast two-hybrid system (24a, 24b).

The direct binding of hGrb10 to the activation loop of the IR may show its physiological relevance. x-Ray crystallography of the IR showed that the autophosphorylated tyrosine residues are part of the active site of the receptor tyrosine kinase (10). Numerous studies have also shown that the autophosphorylated tyrosine residues in the kinase domain of the IR or IGF-1R play a critical role in receptor autophosphorylation and receptor kinase activity (1). The binding of hGrb10 to this region may suggest a role for the protein in the IR and other growth factor receptor signaling. For example, the binding of hGrb10 at the active site of the receptor tyrosine kinase may prevent some downstream substrates from binding to the receptor or to be phosphorylated by the receptor tyrosine kinase and thus plays a regulatory role in signaling. This hypothesis is consistent with the findings that overexpression of Grb-IR/hGrb10{alpha} or microinjection of the GST fusion protein containing the SH2 domain of hGrb10 in cells inhibits insulin-stimulated PI 3-kinase activity (17) or mitogenesis (18), respectively. On the other hand, the binding of these hGrb10 isoforms to the autophosphorylated tyrosine residues in the kinase domain of the receptors may bring certain other substrates closer to the active site so that specific signaling cascades will continue. hGrb10 isoforms may thus function as a molecular switch to control specific signaling pathways. As hGrb10 contains multiple functional domains, including the SH2 domain, the PH domain, and a proline-rich sequence at its N terminus, it is capable of binding different signaling molecules in cells. Identification and characterization of hGrb10 downstream interacting proteins should provide a better understanding of the physiological role of of the protein in signaling processes initialized by insulin or other growth factors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Cell Lines
Yeast two-hybrid plasmids pGBT9, pGAD424, and pGADGH and the host strain SFY526 were from CLONTECH (Palo Alto, CA). Restriction enzymes, DNA ligase, and T4 DNA polymerase are from Life Technologies (Gaithersburg, MD) and New England Biolabs (Beverly, MA). All site-directed mutagenesis primers and sequencing primers were synthesized by Life Technologies. Human IR and IGF-1R cDNAs and CHO.IR{Delta}69 cells, a Chinese hamster ovary cell line overexpressing the mutant IR with a 69-amino acid deletion in the C-terminal region (25), were gifts of R. A. Roth (Stanford University, Stanford, CA). The pGAD/p85 two-hybrid plasmid was provided by J. Koland (University of Iowa, Iowa City, IA) and was described previously (26). The construction of plasmids pGBD9/IR, pGAD/hGrb10(SH2), and pGEX/hGrb10(SH2) and the expression of the GST/hGrb10(SH2) fusion protein were described previously (17).

Site-Directed Mutagenesis
A 2.4-kb BamHI-XbaI cDNA fragment encoding the cytoplasmic domain of the human IR was subcloned into plasmid pBluescript (Stratagene, La Jolla, CA) and used as a template for site-directed mutagenesis. Mutagenesis was carried out according to the protocol as described by Kunkel et al. (27) using customized primers. Complementary DNA fragments encoding different mutant IRs were generated by PCR and fused to the sequence encoding the Gal4 DNA-binding domain in the plasmid pGBT9. Complementary DNAs encoding IRY1162/1163A and IRY1158/62/63A were provided by Dr. B. Zhang (Merck Research Laboratories, Rahway, NJ). All PCR and site-directed mutagenesis products were confirmed by restriction mapping and DNA sequencing (detailed mutagenesis and cloning strategies are available upon request).

Construction of the IR, IGF-1R, and hGrb10 Truncation Mutants
cDNAs encoding the cytoplasmic domain of the IR or IGF-1R with truncation mutations in either the juxtamembrane domain or in the C-terminal region were generated by PCR using human IR or IGF-1R cDNAs as templates, respectively (Fig. 1Go). The PCR primers used were: 1) 5'-GCGAATTCGATGGGCCGCTGGGA-3'; 2) 5'-GCGAATTCGTGCCGGACGAGTGGG-3'; 3) 5'-CAGCGTCGACAGTGCGAGGAACG-3'; 4) 5'-CAGCGTCGACATGGTAGAGTCGT-3'; 5) 5'-GCGAATTCAGCAGGCTGG-GGAATG-3'; 6) GCGAATTCGTTCCTGATGAGTGG-3'; 7) GAGCGTCGACAGGCTGTCTCTCGTCG-3'; 8) 5'-GAGCGTCGACAGATTCAGGATCCA-3', with the added restriction sites underlined. After restriction digestion with EcoRI and SalI, the cDNA fragments were subcloned into the yeast two-hybrid plasmid pGBT9 to generate different GAL4 DNA binding domain/IR mutant fusion protein constructs (Fig. 1AGo). To generate different hGrb10 yeast two-hybrid constructs, the following PCR primers were used: 9) 5'-GCGAATTCCTTTTTGCACCATCC-3'; 10) GCGAATTCTCGACGCCAGTG-3'; 11) 5'-GCGGATCCATTGCCACGAGG-3'; 12) 5'-GACCTCGAGAGGACATCTGCG-3', with the added restriction sites underlined (Fig. 1BGo).The full-length hGrb10{gamma} cDNA (GenBank accession number AF001543) was obtained by screening a human muscle cDNA library (Stratagene) using a 0.9-kb Grb-IR/hGrb10{alpha} cDNA as probe (17). To generate the SH2-domain mutant hGrb10{gamma}, we replaced the conserved arginine residue within the SH2-domain (FLLR529DS) with a glutamine residue. The cDNA encoding the wild type or mutant hGrb10{gamma} was amplified by PCR and subcloned into the plasmid pGADGH (CLONTECH). Full-length or different truncated versions of hGrb10 were generated by PCR using cDNAs encoding for Grb-IR/hGrb10{alpha} or its PH domain-containing isoform hGrb10{gamma} as templates. After digestion with the corresponding restriction enzymes, the cDNA fragments were subcloned into plasmids pGAD GH or pGAD424 to generate GAL4 activation domain (AD)/hGrb10 constructs (Fig. 1BGo). Transformation of SFY526 yeast cells was carried out by electroporation.

ß-Galactosidase Filter and Liquid Assays
The recombinant pGBT9/hGrb10 plasmids were used to transform the yeast host strain SFY526 with plasmids pGAD/hGrb10 by electroporation. Single colonies of transformants were picked, selected in minimal medium lacking tryptophan and leucine, and grown in yeast pepton dextrose (YPD) medium to OD600 of 0.5–1.0. ß-Galactosidase activity (Miller unit) was assayed using o-nitrophenyl ß-D-galactopyranoside as substrates (28). The values are the means ± SD of three to six independent assays.

In Vitro Binding of IR{Delta}69 with GST/hGrb10 Fusion Protein
CHO.IR{Delta}69 cells were grown in Ham’s F12 medium containing 10% newborn calf serum to 90% confluence in 100-mm plates. After being serum starved for 1 h at 37 C, the cells were treated with 10-8 M insulin for 8 min and lysed in lysis buffer containing 50 mM HEPES, (pH 7.6), 1 mM EDTA, 150 mM NaCl, 1% Triton-X-100, 10 mM sodium fluoride, 20 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Cell lysates were incubated with 15 µg GST or GST-hGrb10(SH2) coupled to glutathione agarose or with 25 µl of WGA agarose to precipitate the total IR in the cells. After incubation at 4 C for 4 h, the agarose beads were washed three times with WGA buffer (50 mM HEPES, pH 7.6, 150 mM NaCl and 1% Triton X-100) and then boiled in SDS sample buffer. The precipitated proteins were separated by SDS-PAGE and blotted to a nitrocellulose membrane. The hGrb10-associated IR{Delta}69 were detected by antibodies to either the phosphotyrosine (RC20, Transduction Laboratories, Lexington, KY) or to the {alpha}-subunit of the IR (3B11, gift of Dr. K. Shii).


    ACKNOWLEDGMENTS
 
We thank Dr. Bei Zhang for the IRY1158F and IRY1162/1163A cDNAs, Dr. Jeffrey E. Pessin for permission to use, and Dr. John G. Koland for circulating, the p85 yeast two-hybrid plasmid, and Dr. Kozui Shii for the monoclonal antibody (3B11) to the IR. We would also like to thank Dr. Richard A. Roth for the IR and IGF-1R cDNAs and his continuous support and helpful suggestions.


    FOOTNOTES
 
Address requests for reprints to: Feng Liu, Department of Pharmacology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7764.

This research was supported in part by a Grant-in-Aid from the American Heart Association, Texas Affiliate, Inc., and by a Research Grant from the Juvenile Diabetes Foundation International.

1 Recipient of the Lyndon Baines Johnson Research Award from the American Heart Association, Texas Affiliate. Back

Received for publication May 13, 1997. Revision received July 1, 1997. Accepted for publication July 30, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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