Inhibition of hGrb10 Binding to the Insulin Receptor by Functional Domain-mediated Oligomerization*

Lily Q. Dong, Sarah Porter, Derong Hu, and Feng LiuDagger

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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

hGrb10 is a newly identified Src homology 2 (SH2) and pleckstrin homology (PH) domain-containing protein that binds to autophosphorylated receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors. To identify potential downstream proteins that interact with hGrb10, we screened a yeast two-hybrid cDNA library using the full-length hGrb10gamma as bait. A fragment of hGrb10, which included the IPS (insert between the PH and SH2 domain) and the SH2 domains, was found to bind with high affinity to the full-length protein. The interaction between the IPS/SH2 domain and the full-length hGrb10 was further confirmed by in vitro glutathione S-transferase fusion protein binding studies. Gel filtration assays showed that hGrb10 underwent tetramerization in mammalian cells. The interaction involved at least two functional domains, the IPS/SH2 region and the PH domain, both of which interacted with the NH2-terminal amino acid sequence of hGrb10gamma (hGrb10gamma Delta C, residues 4-414). Competition studies showed that hGrb10gamma Delta C inhibited the binding of hGrb10 to the tyrosine-phosphorylated insulin receptor, suggesting that this region may play a regulatory role in hGrb10/insulin receptor interaction. We present a model for hGrb10 tetramerization and its potential role in receptor tyrosine kinase signal transduction.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Growth factors and hormones initiate and regulate cell growth, differentiation, and metabolism by binding to receptors on the cell membrane. The binding of ligands to their receptors results in receptor tyrosine phosphorylation and kinase activation. Through an intracellular molecule relay, signals are amplified and transmitted from receptors to downstream targets. Under physiological conditions, these signaling processes are accurately regulated through mechanisms such as phosphorylation/dephosphorylation, coordinate localization of enzymes/substrates, and assembly of signaling molecule complexes by scaffolding, anchoring, and adaptor proteins (1).

Many of the adaptor proteins involved in receptor tyrosine kinase signaling contain specific function modules such as the Src homology 2 (SH2)1 domain and the PH domain. One such example is Grb10 (2). Grb10 was first identified by screening a bacterial expression library with the autophosphorylated carboxyl terminus of the epidermal growth factor receptor as a probe (3). This newly identified adaptor protein has been shown to bind directly to several autophosphorylated receptor tyrosine kinases, including the IR (4-8), IGF-1R (5, 6, 9), ELK (10), and Ret (11, 12). Grb10 contains several functional domains including an SH2 domain at the extreme COOH terminus, a PH domain in the central region, and a proline-rich sequence near the NH2 terminus, suggesting that it is capable of interacting with multiple signaling proteins. The SH2 domain of the protein has been shown to be essential for the protein to interact with the autophosphorylated IR and IGF-1R (4-6, 13), whereas the proline-rich sequence of hGrb10 has been shown to bind SH3 containing sequence (6). The function of the PH domain of hGrb10 is currently unknown but may also play an important role in signaling.

Several isoforms of Grb10 have recently been identified from human species, including Grb-IR/hGrb10alpha (4), hGrb10beta (5, 6), and hGrb10gamma (14). These isoforms differ in their PH domains and extreme NH2-terminal sequences, probably because of alternative splicing events. By using the yeast two-hybrid system and site-directed mutagenesis, we have recently shown that, unlike other SH2 domain-containing proteins such as Shc and the 85-kDa subunit of phosphatidylinositol 3-kinase, hGrb10 binds to the autophosphorylated tyrosine residues in the kinase domain of the IR (13). We also found that hGrb10 isoforms are expressed differentially in cells, suggesting that these isoforms may play different roles in receptor tyrosine kinase signal transduction (14). hGrb10 undergoes insulin-stimulated phosphorylation (14) and membrane translocation (6, 14), suggesting that the protein is a potential signaling molecule downstream of the IR. However, little is known about the functional role of Grb10 in signal transduction pathways. We previously found that overexpression of Grb-IR/hGrb10alpha , which has a 46-amino acid deletion in the PH domain, in Chinese hamster ovary cells overexpressing the insulin receptor (CHO/IR) inhibited insulin-stimulated phosphatidylinositol 3-kinase activity (4). On the other hand, microinjection of the SH2 domain of the protein in fibroblasts was found to inhibit insulin and IGF-1-mediated mitogenesis, suggesting that the endogenous protein may have a positive role in signaling (5). More recently, Baserga and colleagues (15) have shown that overexpression of mGrb10 in mouse embryo fibroblast cells inhibits IGF-1-mediated cell proliferation by causing a delay in the S and G2 phases of the cell cycle. The inhibitory effect of Grb10 has also been suggested from recent genomic imprinting studies. The gene coding for mouse Grb10 has been identified as a maternally expressed gene located on proximal chromosome 11 (16). Genetic studies have shown that maternal duplication of chromosome 11 proximal to the translocation breakpoint cause prenatal growth retardation (17). In humans, Grb10 is located on chromosome 7p11.2-12 (14, 18). Maternal disomy of human chromosome 7 has been shown to cause Silver-Russell syndrome whose symptoms include pre- and postnatal growth retardation and other dysmorphologies (19).

To further understand the functional role of Grb10, we attempted to identify molecules that interacted with the protein using the yeast two-hybrid system. Here we present evidence that hGrb10 undergoes tetramerization in cells. In addition, we have found that the interaction involves at least two functional domains: the IPS/SH2 domain and the PH domain. Furthermore, we have shown that the NH2-terminal sequence of hGrb10 inhibited the binding of the protein to the autophosphorylated IR, suggesting that the oligomerization may play a role in the regulation of insulin signaling. We present a model for the mechanism of the tetramerization and its potential role in receptor signal transduction.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The yeast two-hybrid system were from CLONTECH. Bacterial protein expression vectors pGEX-4T-1 and pET21a were from Amersham Pharmacia Biotech and Novagen (Madison, WI), respectively. A polyclonal antibody to the COOH terminus of hGrb10 and Chinese hamster ovary cells expressing the IR and hGrb10 isoforms (CHO/IR/hGrb10alpha and CHO/IR/hGrb10gamma ) were described previously (4, 14).

Construction of Plasmids-- The cDNAs encoding the full-length (residues 4-594) and various truncated mutants of hGrb10gamma were generated by polymerase chain reaction using hGrb10gamma cDNA (14) as a template (Fig. 3A). Convenient restriction endonuclease sites were introduced to allow the in-frame insertion of the cDNAs into the yeast two-hybrid plasmids pGBT9 and pGAD424, the GST fusion protein expression plasmid pGEX-4T-1, and the bacterial expression vector pET21a. All recombinant plasmid constructs were verified by restriction mapping and/or DNA sequencing (detailed cloning strategies available upon request).

Yeast Two-hybrid Studies-- A yeast two-hybrid cDNA library derived from HeLa cells was screened with plasmid pGBT9-hGrb10gamma as bait. Positive clones were identified by selection of transformants on minimum medium without Leu, Trp, and His and by beta -galactosidase filter assays. For interaction studies, SFY526 cells were cotransfected with various truncated forms of hGrb10gamma fused in frame with either the DNA binding domain or the activation domain of GAL4 protein. Interactions were monitored by beta -galactosidase filter or liquid assays described previously (13).

Expression of GST Fusion Proteins-- DH5alpha cells containing plasmids encoding for various recombinant GST/hGrb10 fusion proteins were grown in LB medium at 37 OC overnight. This culture was diluted 1:10 and grown at 30 °C for 80 min. Expression of the fusion protein was induced by the addition of isopropyl-beta -D-thiogalactoside to a final concentration of 1 mM. After 3.5-h induction, cells were harvested by centrifugation at 5,000 × g for 10 min, washed with 10 mM Tris-HCl, pH 8.0, and suspended in bacterial lysis buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM dithiothreitol, 5 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 0.1% (v/v) Triton X-100, and 1 mg/ml lysozyme and lysed by sonication. Cell lysates were clarified by centrifugation at 12,000 × g for 15 min. The GST/hGrb10 fusion proteins were purified by affinity chromatography with glutathione-agarose beads (Sigma).

Expression and Purification of the Full-length and the IPS/SH2 Truncated Mutant of hGrb10gamma in Bacterial Cells-- BL21(DE3) bacterial cells expressing the full-length hGrb10gamma with a His tag at its COOH terminus and a truncated form of hGrb10gamma with a deletion in the IPS/SH2 region (hGrb10gamma Delta (IPS/SH2), Fig. 3A) were grown in LB medium. The expression and cell lysis procedures were similar to those used to purify the GST fusion protein described above. The His-tagged hGrb10gamma was affinity-purified with Ni-NTA-agarose beads according to the protocol of the manufacturer (Qiagen, Chatsworth, CA). To purify hGrb10gamma Delta (IPS/SH2), solid (NH4)2SO4 was added to cell lysates to a final concentration of 10% (v/v). The suspension was centrifuged at 15,000 × g for 20 min. Additional solid (NH4)2SO4 was added to the supernatant to a final concentration of 15% (v/v). The suspension was centrifuged for 20 min at 15,000 × g, and the pellet containing the recombinant protein was retained and resuspended in a minimal volume of buffer containing 20 mM Hepes, pH 7.0, 50 mM KCl, 1 mM beta -mercaptoethanol, and 0.1% (v/v) Triton X-100. The suspension was loaded onto a gel filtration Sephacryl S-300 column, and elution of the recombinant protein was performed at a flow rate of 0.2 ml/min in WGA buffer (50 mM Hepes, pH 7.6, 0.15 NaCl, and 0.1% (v/v) Triton X-100). The fractions containing the protein were pooled and the purity of the protein was determined by Coomassie Blue staining.

In Vitro Binding Studies-- Lysates (0.3 ml) from CHO/IR/hGrb10alpha or CHO/IR/hGrb10gamma cells (in 100-mm plates) were mixed with 15 µg of freshly made GST or GST/hGrb10 fusion proteins coupled to glutathione-agarose. After incubation at 4 °C overnight, the beads were washed extensively with cold WGA buffer. hGrb10 isoforms associated with the GST/hGrb10 fusion proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by immunoblot with anti-hGrb10 antibody.

Competition Studies-- To purify the IR, cell lysate from CHO/IR cells were incubated with WGA beads for 4 h at 4 OC. After extensive washing with WGA buffer, the IR attached to the beads was in vitro phosphorylated by incubation at room temperature for 1 h with kinase buffer containing 50 mM Hepes, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 5 mM MgCl2, 5 mM MnCl2, 1 mM ATP, and 1 µM insulin and then eluted with 0.3 M N-acetylglucoseamine. The partially purified, tyrosine-phosphorylated IR (10 µg) was added to tubes containing the bacteria expressed His-tagged hGrb10gamma (15 µg) coupled to Ni-NTA agarose beads and different concentrations of purified hGrb10gamma Delta C. After incubation at 4 OC overnight, the beads were washed extensively with WGA buffer, and the IR associated with hGrb10gamma was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by a polyclonal antibody to the beta -subunit of the receptor (Santa Cruz Biotechnology, Santa Cruz, CA).

Size Exclusion Chromatography-- A Sephacryl S-300 column (16 × 60 cm) was equilibrated with buffer containing 50 mM Hepes, pH 7.0, and 0.15 M NaCl and calibrated with standards: thyroglobulin, 669 kDa; ferritin, 440, kDa; catalase, 232 kDa; aldolase, 158 kDa; and ovalbumin, 44 kDa. Lysates from CHO/IR/hGrb10alpha or CHO/IR/hGrb10gamma cells were clarified by centrifugation at 12,000 × g for 15 min at 4 °C. The clarified supernatant (0.5 ml) was applied to the column and eluted with the same buffer at a flow rate of 0.2 ml/min. Fractions (1 ml) were collected and proteins were precipitated by the addition of 100 µl of 0.15% sodium deoxycholate and 100 µl of 72% trichloric acid, separated by SDS-PAGE, and the position of hGrb10alpha or hGrb10gamma in the elution profile was determined by Western blot using an anti-hGrb10 antibody.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of hGrb10 Dimerization by the Yeast Two-hybrid System-- To identify hGrb10-associated proteins, we screened a yeast two-hybrid library derived from HeLa cell cDNAs using the full-length hGrb10gamma cDNA as bait. From 4 million colonies screened, we identified over 50 positives that grew on synthetic medium without Leu, Trp, and His and remained positive during the beta -galactosidase filter assays.

DNA sequence analysis of one of the positive clones (clone B4) revealed an unexpected finding: the cDNA insert encoded the COOH terminus of hGrb10 including the IPS and the SH2 domain (residues 415-594 of hGrb10gamma , Fig. 3A). Re-cotransformation of the cloned plasmid into yeast strain SFY526 with plasmid pGBT9, pLAM5 (an unrelated plasmid), or pGBT9-hGrb10gamma showed that the interaction (determined by beta -galactosidase filter assays) was completely dependent on the expression of both the positive clone and the GAL4-hGrb10gamma fusion protein. These results suggested that hGrb10 may be dimerized in cells and that the dimerization may be mediated by the IPS/SH2 domain of the protein.

hGrb10 Dimerized in Vitro-- To test whether the dimerization was through a direct interaction between two hGrb10 molecules or mediated by an auxiliary protein(s) in yeast cells, we first investigated whether the IPS/SH2 domain interacted with hGrb10 in vitro by GST fusion protein pull-down studies. As shown in Fig. 1, both Grb-IR/hGrb10alpha (lanes 1 and 2) or hGrb10gamma (lanes 3 and 4) were precipitated by the GST-IPS/SH2 fusion proteins. Under similar condition, no hGrb10 isoforms were pulled down by the control GST (data not shown). The GST-IPS/SH2 fusion protein precipitated a lesser amount of Grb-IR/hGrb10alpha than hGrb10gamma (Fig. 1, lanes 1-4), probably because of a lesser expression of this isoform in cells (Fig. 1, lanes 9-12). However, no significant difference was observed between hGrb10 isoforms precipitated from cells treated with or without insulin (Fig. 1, lanes 1-4).


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Fig. 1.   Binding of hGrb10 isoforms to the GST-IPS/SH2 and the GST-PH domains of the protein. Fifteen micrograms of GST-hGrb10(IPS/SH2) (lanes 1-4) or GST-hGrb10(PH) (lane 5-8) bound to glutathione-conjugated agarose beads were incubated with lysates (400 µl) from insulin-treated (+) or nontreated (-) CHO/IR/hGrb10alpha (lanes 1, 2, 5, 6) or CHO/IR/hGrb10gamma (lanes 3, 4, 7, 8) cells. hGrb10 isoforms bound to the beads were eluted, separated by SDS-PAGE, and analyzed by Western blotting using a polyclonal antibody against hGrb10. Cell lysates (20 µl) were loaded as a control (lanes 9-12).

hGrb10 Underwent Tetramerization in Mammalian Cells-- To see whether the dimerization also occurred in mammalian cells, Grb-IR/hGrb10alpha and hGrb10gamma expressed in either CHO/IR/hGrb10alpha or CHO/IR/hGrb10gamma cells were subjected to size exclusion chromatography analysis. As shown in Fig. 2A, hGrb10gamma was eluted as two peaks of approximately 320 and 80 kDa, respectively, suggesting that hGrb10gamma exists as both a tetramer and a monomer in mammalian cells. Similar elution profile was observed for hGrb10alpha (Fig. 2B), suggesting that this isoform can also form a tetramer in mammalian cells. Although no apparent peaks were observed in the 120-240 kDa range, we could not exclude the possibility that some dimeric and trimeric Grb10 might also be present in cells. It is possible that the signal of these dimers and trimers may be masked by the smearing peaks of the more abundant tetramers and monomers.


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Fig. 2.   Tetramerization of hGrb10 isoforms in mammalian cells. CHO/IR/hGrb10gamma (A) or CHO/IR/hGrb10alpha (B) cells from five 100-mm plates were grown to subconfluent. Cell lysates (0.5 ml) were subjected to fast protein liquid chromatography size exclusion chromatography analysis. Proteins from each fractions (1 ml) were collected and precipitated with trichloric acid in the presence of sodium deoxycholate, separated by SDS-PAGE, and detected by Western blotting analysis using a polyclonal antibody to hGrb10 (insets). The relative amount of hGrb10 isoforms in each fraction was estimated by NIH Image analysis. The data shown are representative of at least three separate experiments.

The IPS/SH2 Domain of hGrb10 Interacts with the NH2 Terminus of the Protein-- Having found that hGrb10 tetramerized in cells and that the IPS/SH2 domain was involved in the interaction, we attempted to identify the sequence in hGrb10 that interacts with this domain. We constructed various yeast two-hybrid plasmids in which different regions of hGrb10 were fused in frame with either the GAL4 DNA binding domain (in plasmid pGBT9) or with the GAL4 transcription activation domain (in plasmid pGAD424, Fig. 3A). We tested the interaction between these fusion proteins by the yeast two-hybrid system. As shown in Fig. 3B, The IPS/SH2 region interacted with both the wild-type and a mutant form of hGrb10gamma in which the critical arginine residue in the SH2 domain was changed to glycine (R520G). This mutant of hGrb10 did not bind the IR in the yeast two-hybrid system (13). These data suggest that the structural requirement for hGrb10 dimerization and for its interaction with the IR are different. The IPS/SH2 protein also bound to a truncated form of hGrb10 in which the IPS/SH2 region was deleted (hGrb10gamma Delta (IPS/SH2)). In addition, no beta -galactosidase activity was detected between two IPS/SH2 domains using the yeast two-hybrid system (Fig. 3B). These results suggest that the dimerization is not through a direct interaction between two IPS/SH2 regions. To delimit the boundary of the IPS/SH2 interaction sequence, additional truncated versions were generated. However, no interaction was detected between these mutants and the IPS/SH2 domain in the yeast two-hybrid system (Fig. 3B), suggesting that a specifically folded structure of the N terminus, which could be disrupted by the truncation, may be required for the IPS/SH2 region to bind. The interaction seems also to require an intact IPS/SH2 domain as neither the SH2 nor the IPS region alone bound with a significant affinity to the full-length form of hGrb10gamma (data not shown).


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Fig. 3.   Mapping of hGrb10 subdomains involved in the dimerization by the yeast two-hybrid system. A, diagram of the domain structures of the wild-type and mutant hGrb10. The PH, IPS, and the SH2 domains are shown. The NH2-terminal proline-rich sequence (residues 136-143) is indicated as a hatched box. A and C, interaction of the IPS/SH2 domain (B) and the PH domain (C) with different regions of Grb10 in the yeast two-hybrid system. Yeast two-hybrid plasmids containing the full-length or truncated forms of hGrb10 fused to the DNA binding domain (in pGBT9) or the activation domain (in pGAD424) of GAL4 were constructed as described under "Experimental Procedures." The interaction was determined by beta -galactosidase liquid assays. Values are means ± S.D. of two to six independent assays, and each assay was an average of triplicate determinations.

The Involvement of the PH Domain in hGrb10 Dimerization-- The domain structure of hGrb10gamma consists of several functional regions: a COOH-terminal SH2 domain, a central PH domain, and an NH2-terminal proline-rich sequence (Fig. 3A). Although our data showed that the IPS/SH2 domain was sufficient to bind the full-length protein, we also found that the full-length hGrb10gamma underwent dimerization with a higher beta -galactosidase activity in the yeast two-hybrid system (data not shown). This observation suggested that additional region(s) may also be involved in the interaction. To test this hypothesis, we subcloned cDNAs encoding various truncated form of hGrb10 (Fig. 3A) into both the "bait" (pGBT9) and the "prey" (pGAD424) plasmids and tested the interaction between these yeast two-hybrid fusion proteins. Our studies showed that, in addition to the IPS/SH2 region, the PH domain (residues 290-414) was sufficient to interact the full-length hGrb10 (Fig. 3C). This observation was confirmed by in vitro binding studies, which showed that the GST-hGrb10(PH) fusion protein interacted with both hGrb10 isoforms expressed in CHO/IR/hGrb10 cells (Fig. 1, lanes 5-8). No beta -galactosidase activities were observed when other fragments were tested against the full-length Grb10 in the yeast two-hybrid system (data not shown). To map the binding region for the PH domain, we tested the interaction between the PH domain and various truncated forms of hGrb10 in the yeast two-hybrid system. We found that the minimum sequence that retained binding to the PH domain was between residues 4-290 (Delta (PH/IPS/SH2); Fig. 3, A and C). These data suggest that although both the IPS/SH2 and the PH domain interacts with the NH2-terminal region of the protein, the detailed structural requirement for the binding of the IPS/SH2 and the PH domains may be different.

The NH2-terminal Region Negatively Regulates the Binding Affinity of hGrb10 to the IR-- We previously showed that the SH2 domain of hGrb10 was essential for hGrb10 binding to the autophosphorylated IR in vitro and in cells (13). The finding that the IPS/SH2 region of hGrb10 interacts with the NH2-terminal region of the protein raised an interesting question of whether this interaction affects the binding of the SH2 domain to the IR. To address this question, we expressed a truncated form of hGrb10gamma with a deletion in the IPS/SH2 region (hGrb10gamma Delta (IPS/SH2), Fig. 3A) in bacteria and purified it to near homogeneity (Fig. 4A). We tested the effect of the recombinant protein on the interaction between hGrb10gamma and the IR in vitro. As expected, significant autophosphorylated IR was pulled down by the His-tagged hGrb10gamma protein bound to Ni-NTA-agarose beads (Fig. 4B, lane 2), but not by the Ni-NTA beads control (Fig. 4B, lane 1). The interaction between the IR and hGrb10gamma , however, was competitively inhibited by increased concentrations of hGrb10gamma Delta (IPS/SH2) (Fig. 4B, lanes 3-7). The inhibition was specific as unrelated proteins such as bovine serum albumin at similar concentrations had no effect on the binding (data not shown). In addition, the hGrb10gamma Delta (IPS/SH2) recombinant protein did not bind to the IR in vitro (data not shown), suggesting that the inhibition was because of a competitively binding of the polypeptide to a site on hGrb10gamma which affected the binding of the protein to the tyrosine-phosphorylated IR.


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Fig. 4.   Inhibition of hGrb10 binding to the IR by the NH2-terminal region of the protein. A, Coomassie Blue staining of the purified hGrb10Delta (IPS/SH2). The IPS/SH2 domain truncated hGrb10 was overexpressed in BL21(DE3) cells under the control of T7 promoter. The protein was purified by ammonium sulfate fractionation and size exclusion fast protein liquid chromatography as described under "Experimental Procedures." The molecular mass and the purified protein (1.5 µg) are shown in lanes 1 and 2, respectively. B, His-tagged hGrb10gamma (15 µg) coupled to Ni-NTA-agarose beads was incubated with 10 µg of WGA purified, in vitro tyrosine-phosphorylated IR in the presence of 0 (lane 2), 32 nM (lane 3), 160 nM (lane 4), 320 nM (lane 5), 480 nM (lane 6), and 640 nM (lane 6) of purified hGrb10Delta (IPS/SH2) protein, respectively. After incubation at 4 OC overnight, the beads were washed three times with WGA buffer, and the IR associated with hGrb10gamma was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by a polyclonal antibody to the beta -subunit of the receptor.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

We report here the oligomerization of an adaptor protein, hGrb10. To our knowledge, this is the first indication that an SH2 and PH domain-containing adaptor protein undergoes tetramerization in cells, and the oligomerization is mediated by at least two functional domains, the IPS/SH2 and the PH domain. These findings are quite interesting as oligomerization of adaptor proteins in cells may have important physiological relevance. One possible function for hGrb10 tetramerization may be to provide a reservoir of latent hGrb10 molecules and prevent the protein from nonspecific binding to cellular phosphotyrosine-containing proteins. We have shown previously that the SH2 domain of hGrb10 binds the autophosphorylated tyrosine residues in the kinase domain of the IR (13). Recently, Dr. Gustafson and colleagues (20) have found that the IPS region alone is also capable of interacting with the IR and IGF-1R in a kinase dependent manner. The finding that the IPS/SH2 domain is one of the determinants involved in the hGrb10 tetramerization suggests that the tetramerization may regulate the binding of the protein to the autophosphorylated IR. Consistent with this hypothesis, we have found that the NH2 terminus of hGrb10 (hGrb10Delta (IPS/SH2)), which interacted with the IPS/SH2 domain of the protein (Fig. 3B), competitively inhibited the binding of hGrb10 to the IR (Fig. 4B). These data suggest that the tetramerized hGrb10 may be unable to interact with phosphotyrosine-containing peptides because of the blockage of its IPS/SH2 domain by the NH2-terminal sequence. The stimulation of cells by insulin or other growth factors may induce a conformational change of hGrb10 so that the SH2 domain of the protein is accessible for binding to phosphotyrosine-containing receptors. Tetramerization may thus provide a mechanism to regulate the interaction between the adaptor protein and the receptor in cells.

Another possible role of hGrb10 oligomerization in cells may be to serve as a docking complex to recruit multiple signaling molecules. As Grb10 monomer contains several functional domains such as an SH2 domain, a PH domain, and a proline-rich sequence, all of which are able to interact with other cellular proteins, oligomerization of Grb10 should enhance the binding capacity of the protein to these signaling molecules. Although we were unable to determine whether hGrb10 binds to the IR or IGF-1R as a monomer or oligomer because of technical difficulties, this hypothesis is quite interesting, especially with the findings that hGrb10 is capable of binding simultaneously to the IR and other signaling proteins in cells (5, 6, 21). Therefore, oligomerization of the adaptor protein may provide a novel mechanism to colocalize multiple signaling molecules to regulate cell signaling processes.

In this study, we have also found that the PH domain is sufficient to interact with the full-length hGrb10 (Fig. 1, lanes 5-8, and Fig. 3C). PH domains have been shown to be involved in both protein-lipids or protein-protein interactions (22). Examples of the latter include beta -adrenergic receptor kinase, IRS-1, and the Btk tyrosine kinase, whose PH domains bind to the beta gamma -subunits of trimeric G proteins (23), the IR (24), and protein kinase C (25), respectively. The finding that the PH domain is involved in hGrb10 dimerization in vitro and in the yeast two-hybrid system provides new evidence that PH domains are functional motifs and can function independently to mediate regulated protein-protein interactions in signaling. However, it should be pointed out that the IPS/SH2 domain of hGrb10 is sufficient to interact with the full-length protein and that Grb10alpha , the isoform which has a 46-amino acid deletion, including part of the PH domain, was still be able to undergo tetramerization in cells (Fig. 2B). These data suggest that the PH domain may not be essential for hGrb10 tetramerization. One possible role for the involvement of the PH domain may be to provide additional specificity for the interaction. Further studies will be needed to test this hypothesis.

A simple model, which accounts for the role of hGrb10 tetramerization, is depicted in Fig. 5. In this model, hGrb10 tetramerization in cells involves two functional motifs: the IPS/SH2 domain and the PH domain, both interacting with the NH2-terminal region of its partner. The tetramerization may bury the functional domains of hGrb10 and prevent them from nonspecifically interacting with the tyrosine-phosphorylated receptors or other cellular signaling molecules. The balance between monomeric and oligomeric hGrb10 may thus provide a specificity for the adaptor to transduce or regulate receptor tyrosine kinase signaling processes. This balance may be regulated by mechanisms such as phosphorylation, which could result from stimulation by insulin or other growth factors (14). The modification may cause a conformational change of the protein and lead to the access of the SH2 domain by the autophosphorylated receptor. The binding affinity of the SH2/phosphotyrosine containing sequence interaction may be much greater than that of the interaction between the SH2 domain and the non-tyrosine-phosphorylated hGrb10 NH2-terminal sequence so that the hGrb10-receptor complex is stabilized. The binding of hGrb10 to the IR may result in a further conformational change for hGrb10, which may allow other functional domains such as the PH in the protein to interact with downstream signaling molecules. It should be pointed out that the tetramerization may not be unique for Grb10 but may occur in other adaptor molecules as well. For example, the COOH-terminal region of Grb10 has recently been shown to bind to the full-length Grb7 in the yeast two-hybrid system.2 It would be interesting to see whether oligomerization is a general mechanism for other adaptor proteins in the regulation of cellular signaling processes.


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Fig. 5.   Model for hGrb10 tetramerization as an adaptor protein. hGrb10 forms a tetramer in cells through the interaction between the IPS/SH2 domain, the PH domain, and the NH2-terminal region of the protein. Binding of insulin to the receptor may induce a conformational change in hGrb10 and shift the balance from tetramer to monomer. The dissociation of hGrb10 tetramer may lead to the access of the IPS/SH2 domain to the tyrosine-phosphorylated insulin receptor. Further conformational changes may be induced in hGrb10 after its binding to the receptor, which could result in the exposure of other functional regions for interaction with downstream signaling molecules.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK52933 (to F. L. and L. Q. D.) and by a Research Grant from the South Texas Health Research Center for Diabetes Research (to F. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, UTHSCSA, 7703 Floyd Curl Dr., San Antonio, TX 78284-7764. Tel.: 210-567-3097; E-mail: liuf{at}uthscsa.edu.

1 The abbreviations used are: SH2, Src homology 2; GST, glutathione S-transferase; IR, insulin receptor; IGF-1, insulin-like growth factor-1; IPS, insert between the PH domain and the SH2 domain; PAGE, polyacrylamide gel electrophoresis; PH, pleckstrin homology; WGA, wheat germ agglutinin; CHO, Chinese hamster ovary; Ni-NTA, nickel-nitrilotriacetic acid.

2 J. Cooper, personal communication.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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