Active GTP-bound Form Research Articles

Electrostatic control of GTP and GDP binding in the oncoprotein p21 ras

Background: p21 ras is one of the GTP-binding proteins that act as intercellular molecular switches. The GTP-bound form of p21 ras sends a growth-promoting signal that is terminated once the protein is cycled back into its GDP-bound form. The interaction of guanine-nucleotide-exchange factors (GEFs) with p21 ras leads to activation of the protein by promoting GDP→GTP exchange. Oncogenic mutations of p21 ras trap the protein in its biological active GTP-bound form. Other mutations interfere with the activity of GEF. Thus, it is important to explore the structural basis for the action of different mutations. Results The crystal structures of p21 ras are correlated with the binding affinities of GTP and GDP by calculating the relevant electrostatic energies. It is demonstrated that such calculations can provide a road map to the location of ‘hot’ residues whose mutations are likely to change functional properties of the protein. Furthermore, calculations of the effect of specific mutations on GTP and GDP binding are consistent with those observed. This helps to analyze and locate functionally important parts of the protein. Conclusion Our calculations indicate that the protein main chain provides a major contribution to the binding energies of nucleotides and probably plays a key role in relaying the effect of GEF action. Analysis of p21 ras mutations in residues that are important for the proper function of GEFs suggests that the region comprising residues 62—67 in p21 ras is the major GEF-binding site. This analysis and our computer simulations indicate that the effect of GEF is probably propagated to the P-loop (residues 10–17) through interaction between Gly60 and Gly12. This then reduces the interaction between the main-chain dipoles of the P-loop and the nucleotide. Finally, the results also suggest a possible relationship between the GTP→GDP structural transition and the catalytic effect of the GTPase-activating protein.

Ras effectors

The search for proteins which interact with the active GTP-bound form of Ras in order to transmit signals for proliferation, differentiation and oncogenesis has been a long one. Now there are several strong candidates for Ras effectors that include protein kinases, lipid kinases and guanine nucleotide exchange factors. Structural information on how one Ras-binding domain in an effector interacts with Ras-GTP has recently been obtained. Recent data show that transformation by Ras oncoproteins requires the activation of several signal transduction pathways, including those which transmit signals via members of the Rho family of GTPases.

Regulation of Interaction of ras p21 with RalGDS and Raf-1 by Cyclic AMP-dependent Protein Kinase

RalGDS is a GDP/GTP exchange protein for ral p24, a member of small GTP-binding protein superfamily. We have recently shown that RalGDS interacts directly with the GTP-bound active form of ras p21 through the effector loop of ras p21 in vitro, in insect cells and in the yeast two-hybrid system. These results suggest that RalGDS functions as an effector protein of ras p21. Here, we report that RalGDS interacts with ras p21 in mammalian cells in response to an extracellular signal. Epidermal growth factor (EGF) induced the interaction of c-ras p21 and RalGDS in COS cells expressing both proteins, but not in the cells expressing RalGDS and c-ras p21T35A, which is an effector loop mutant of ras p21. We also found that cyclic AMP-dependent protein kinase (protein kinase A) regulated the selectivity of ras p21-binding to either RalGDS or Raf-1. Protein kinase A phosphorylated RalGDS as well as (1-149)Raf (amino acid residues 1-149). Although the phosphorylated (1-149)Raf had a lower affinity for ras p21 than the unphosphorylated (1-149)Raf, both the phosphorylated and unphosphorylated RalGDS had the similar affinities for ras p21. The phosphorylation of RalGDS did not affect its activity to stimulate the GDP/GTP exchange of ral p24. Pretreatment of COS cells with forskolin further stimulated the interaction of ras p21 and RalGDS induced by EGF under the conditions that EGF-dependent Raf-1 activity was inhibited. These results indicate that ras p21 distinguishes between RalGDS and Raf-1 by their phosphorylation by protein kinase A.

TRNA-ribosome interactions.

Direct measurements of the rates of dissociation of dipeptidyl-tRNA from the ribosome show that hyperaccurate SmP and SmD ribosomes have unstable A-site binding of peptidyl-tRNA, while P-site binding is extremely stable in relation to the wild type. Error-prone Ram ribosomes, on the other hand, have stable A-site and unstable P-site binding of peptidyl-tRNA. At least for these mutant ribosomes, we conclude that stabilization of peptidyl-tRNA in one site destabilizes binding in the other. Elongation factor Tu (EF-Tu) undergoes a dramatic structural transition from its GDP-bound form to its active GTP-bound form, in which it binds aa-tRNA (aminoacyl-tRNA) in ternary complex. The effects of substitution mutations at three sites in domain I of EF-Tu, Gln124, Leu120, and Tyr160, all of which point into the domain I-domain III interface in both the GTP and GDP conformations of EF-Tu, were examined. Mutations at each position cause large reductions in aa-tRNA binding. An attractive possibility is that the mutations alter the domain I-domain III interface such that the switching of EF-Tu between different conformations is altered, decreasing the probability of aa-tRNA binding. We have previously found that two GTPs are hydrolyzed per peptide bond on EF-Tu, the implication being that two molecules of EF-Tu may interact on the ribosome to catalyze the binding of a single aa-tRNA to the A-site. More recently we found that ribosomes programmed with mRNA constructs other than poly(U), including the sequence AUGUUUACG, invariably use two GTPs per peptide bond in EF-Tu function. Other experiments measuring the protection of aa-tRNA from deacylation or from RNAse A attack show that protection requires two molecules of EF-Tu, suggesting an extended ternary complex. To remove remaining ambiguities in the interpretion of these experiments, we are making direct molecular weight determinations with neutron scattering and sedimentation-diffusion techniques.

Identification of Rap1 as a Target for the Crk SH3 Domain-Binding Guanine Nucleotide-Releasing Factor C3G

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.

Interleukin-11 Induces Complex Formation of Grb2, Fyn, and JAK2 in 3T3L1 Cells

Previous studies suggested that interleukin-11 (IL-11) induces the activation of mitogen-activated protein kinase (MAPK) in mouse 3T3L1 cells. However, the mechanisms by which IL-11 activates MAPK remain elusive. Our present results show that IL-11 promotes the formation of the active GTP-bound form of Ras, suggesting that IL-11 actions may be transduced in part through the Ras/MAPK signaling pathway. By immunoblotting and immunoprecipitation, we further demonstrate the association of tyrosine phosphoproteins with Grb2, an adaptor protein serving as a key intermediate for Ras activation. These phosphotyrosine-containing proteins have been subsequently identified to be JAK2, Fyn, and Syp. JAK2 and Fyn are transiently associated with Grb2 upon stimulation with IL-11, suggesting that JAK2 and Fyn may be involved in transducing signals from the IL-11 receptor-glycoprotein 130 to the Ras system through Grb2. Taken together, these results suggest that IL-11-induced interactions of JAK2, Fyn, and Grb2 may not only provide a novel mechanism for the activation of the Ras/MAPK system but also indicate cross-talk among diverse signaling pathways.

Macrophage-Stimulating Protein Activates Ras by both Activation and Translocation of SOS Nucleotide Exchange Factor

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2 but not Shc, associated with the activated RON-β chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Pas might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.

Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

A GDP/GTP Exchange-stimulatory Activity for the Rab5-RabGDI Complex on Clathrin-coated Vesicles from Bovine Brain

Small GTPases of the Rab family are key regulators of intracellular transport. They are associated with the cytoplasmic surface of distinct exocytic and endocytic organelles and with transport vesicles connecting these compartments. Rab proteins are also present in the cytosol in the GDP-bound conformation complexed to Rab GDP dissociation inhibitor (RabGDI). Upon membrane association, RabGDI is released, and the Rab protein is converted into the GTP-bound form. In this paper we have investigated whether Rab5, which regulates the clathrin-coated vesicle-mediated pathway of endocytosis, can directly associate with the membrane of clathrin-coated vesicles (CCV) purified from bovine brain in vitro. We found that RabGDI can specifically deliver Rab5 but not Rab7, which is localized to late endosomes, to CCV. Furthermore, CCV contain a heat- and trypsin-sensitive activity that stimulates the dissociation of GDP from Rab5, but not from Rab7, and the subsequent binding of GTP. The activity was found to be associated with the CCV membrane but not with the coat components. CCV weakly stimulated GDP release from either post-translationally modified or unmodified Rab5 alone. However, maximal GDP dissociation stimulation required the presence of RabGDI, suggesting that the factor(s) responsible for the membrane association and GDP/GTP exchange of Rab5 recognize the protein complexed to RabGDI. These data demonstrate that CCV are competent for acquiring Rab5 and for converting the molecule into the GTP-bound active form.

8] Measurement of intrinsic nucleotide exchange and GTP hydrolysis rates

This chapter describes methods to measure nucleotide exchange rates and GTP hydrolysis rates of Rho, Rac, and G25K. Like all GTP-binding proteins, Rho-related GTPases exist in two conformational states: an inactive GDP-bound form and an active GTP-bound form; their interconversion occurs through a cycle of guanine nucleotide exchange and GTP hydrolysis. The intrinsic nucleotide exchange and GTPase activities of all members of the Ras superfamily are relatively slow, and in vivo, they are stimulated by guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs). GEFs and GAPs for the members of the Rho subfamily have been identified, although their cellular roles and exact specificities are still unclear. The first GAP described for this family (Rho–GAP) was purified biochemically from human spleen and was shown to be active on Rho, but it is also active on Rac and CDC42/G25K. Another important regulator of the Rho family of proteins is the Rho–GDP-dissociation inhibitor (GDI), Rho–GDI. Guanine nucleotide off rates are discussed in the chapter.

Negative regulation of p120GAP GTPase promoting activity by p210bcr/abl: implication for RAS-dependent Philadelphia chromosome positive cell growth.

The p210bcr/abl tyrosine kinase appears to be responsible for initiating and maintaining the leukemic phenotype in chronic myelogenous leukemia (CML) patients. p21ras-p120GAP interactions play a central role in transducing mitogenic signals. Therefore, we investigated whether p21ras and p120GAP are regulated by p210bcr/abl, and whether this activation is functionally significant for CML cell proliferation. We report that transient expression of p210bcr/abl in fibroblast-like cells induces simultaneous activation of p21ras and inhibition of GTPase-promoting activity of p120GAP, and confirm these data showing that downregulation of p210bcr/abl expression in CML cells with bcr/abl antisense oligodeoxynucleotides induces both inhibition of p21ras activation and stimulation of GTPase-promoting activity of p120GAP. Tyrosine phosphorylation of two p120GAP-associated proteins, p190 and p62, which may affect p120GAP activity, also depends on p210bcr/abl tyrosine kinase expression. Direct dependence of these effects on the kinase activity is proven in experiments in which expression of c-MYB protein in fibroblast-like cells or downregulation of c-MYB expression resulting in analogous inhibition of CML cell proliferation does not result in the same changes. Use of specific antisense oligodeoxynucleotides to downregulate p21ras expression revealed a requirement for functional p21ras in the proliferation of Philadelphia chromosome-positive CML primary cells. Thus, the p210bcr/abl-dependent regulation of p120GAP activity is responsible, in part, for the maintenance of p21ras in the active GTP-bound form, a crucial requirement for CML cell proliferation.

Role of Glutamine-61 in the Hydrolysis of GTP by p21H-ras: An Experimental and Theoretical Study

The active GTP-bound form of p21ras is converted to the biologically inactive GDP-bound form by enzymatic hydrolysis and this function serves to regulate the wild-type ras protein. The side chain of the amino acid at position 61 may play a key role in this hydrolysis of GTP by p21. Experimental studies that define properties of the Q61E mutant of p21H-ras are presented along with supporting molecular dynamics simulations. We find that under saturating concentrations of GTP the Q61E mutant of p21H-ras has a 20-fold greater rate of intrinsic hydrolysis (kcat = 0.57 min-1) than the wild type. The affinity of the Q61E variant for GTP (Kd = 115 microM) is much lower than that of the wild type. GTPase activating protein does not activate the variant. From molecular dynamics simulations, we find that both the wild type and Q61E mutant have the residue 61 side chain in transient contact with a water molecule that is well-positioned for hydrolytic attack on the gamma phosphate. Thr-35 also is found to form a transient hydrogen bond with this critical water. These elements may define the catalytic complex for hydrolysis of the GTP [Pai et al. (1990) EMBO J. 9, 2351]. Similarly, the G12P mutant, which also has an intrinsic hydrolysis rate similar to the wild type, is found to form the same complex in simulation. In contrast, molecular dynamics analysis of the mutants G12R, G12V, and Q61L, which have much lower intrinsic rates than the wild-type p21, do not show this complex.(ABSTRACT TRUNCATED AT 250 WORDS)

The adapter protein Shc interacts with the interleukin-2 (IL-2) receptor upon IL-2 stimulation.

Binding of interleukin-2 (IL-2) to the IL-2 receptor (IL-2R) stimulates Src family kinases, tyrosine phosphorylation of several proteins, conversion of Ras to its active GTP-bound form, and eventually c-fos, c-jun, and c-myc induction. The IL-2R beta chain plays a crucial role in IL-2R signaling. Within the cytoplasmic domain of the beta chain, a region essential for mitogenesis and another involved in binding the Src family kinase Lck have been defined. The beta chain itself is tyrosine-phosphorylated upon IL-2 stimulation. Since the adapter protein Shc acts upstream of Ras and is involved in T cell receptor-mediated Ras activation, we examined the role of Shc in IL-2 signaling. Shc was found to be tyrosine-phosphorylated upon IL-2 stimulation in CTLL-20 cells. After its phosphorylation, Shc interacted with another adapter protein, Grb2, and, via Grb2, with the Ras GTP/GDP exchange factor mSOS. After IL-2 stimulation, Shc also associated with the IL-2R beta chain. Thus, during IL-2 signaling, the interaction of Shc with the IL-2R beta chain and its simultaneous association with Grb2 and mSOS may couple IL-2R stimulation to Ras signaling.

23] Identification of mutant forms of G-protein α subunits in human neoplasia by polymerase chain reaction-based techniques

Publisher Summary G-protein α subunits bind guanosine-5'-triphosphate (GTP) and guanosine-diphosphate (GDP) and cycle between an inactive GDP-bound form and an active GTP-bound form. The role of α subunits in signal transduction may be defined genetically and biochemically, when a G-protein α subunit is expressed in a given cell type, it may interact with an appropriate set of activated receptors (G-protein receptors), bind GTP for GDP, and transduce its signal to second messengers via a relatively slow intrinsic hydrolysis of GTP to GDP, thereby instigating a complex sorting and amplification cascade resulting, finally, in either mitogenic effects, hormone secretion, or cell differentiation. Members of the α-subunit families may be divided into four different groups based on amino acid sequences. In the first group the αs and the olfactory subunits activate adenylyl cyclase and elevate levels of cAMP within the cell. The α subunits of the inhibitory class are largely ADP-ribosylated by pertussis toxin and include Gi1, Gi2, Gi3, Go, and transducin. ADP-ribosylation at the carboxyl terminus by pertussis toxin results in the modification of amino acid sequences in the α chain and uncouples the α subunit from its receptor and, consequently, from its signal transduction pathway. A further group of α subunits has been cloned, the Gq class. This group of α subunits activates phospholipase C and is pertussis toxin insensitive. A fourth group, consisting of Gα12 and Gα13, has been cloned, and their sequences have been shown to be unique from the other three groups.

From EF-Tu to P21ras and back again

Guanine nucleotide binding proteins are a class of proteins sharing three general features (for reviews see 11,21): first, they bind strongly to guanine nucleotides; second, they have a slow intrinsic GTPase activity that converts the active GTP-bound form into the inactive GDP-bound form and that in many cases is stimulated by GTPase-activating proteins (GAPS); and third, the transition between the GTP-bound and GDP-bound forms is accompanied by a conformation change that dramatically alters their affinity for other macromolecules.

Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small G protein superfamily that is implicated in regulated secretion, particularly in neurotransmitter release. Rabphilin-3A contains at least two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal C2 domain, which interacts with both Ca2+ and phospholipid. Because Rabphilin-3A interacts preferentially with GTP-Rab3A rather than with GDP-Rab3A, we have examined here whether Rabphilin-3A affects the GTPase activity of Rab3A. Rabphilin-3A and its N-terminal fragment, but not its C-terminal fragment, very weakly stimulated the basal GTPase activity of Rab3A. However, Rabphilin-3A and its N-terminal fragment strongly inhibited the Rab3A GAP-stimulated GTPase activity of Rab3A. Ca2+ and phospholipid showed no effect on these activities of Rabphilin-3A. The physiological significance of the GAP activity of Rabphilin-3A is obscure, but it is likely that Rabphilin-3A inhibits Rab3A GAP activity and keeps Rab3A in the GTP-bound active form during its action as a target molecule for Rab3A.

Studies of the Ras-GDP and Ras-GTP noncovalent complexes by electrospray mass spectrometry

A novel MS based methodology utilizing electrospray ionization is described for the detection of the noncovalent interaction between a host protein ( ras) and its guest ligands (GDP and GTP). The ras proteins are regulatory guanine nucleotide binding proteins which serve as signal transducers controlling cell proliferation or differentiation. They can exist in two interconvertible states of GDP-bound (inactive form) and GTP-bound (active form). The presence of the noncovalent complexes of ras-GDP and ras-GTP, as well as the unbound apo- ras protein, in various sample solutions containing biological buffers were confirmed by the observed average molecular weights of 19295, 19374, and 18852 Da, respectively. The stability of the observed GDP-bound and GTP-bound complexes is a combined function of solution pH and organic modifier content.

Combination of Arachidonic Acid and Guanosine 5′-O-(3-Thiotriphosphate) Induces Translocation of rac p21s to Membrane and Activation of NADPH Oxidase in a Cell-Free System

The superoxide-generating NADPH oxidase system in phagocytes consists of membrane-associated cytochrome b558 and three cytosolic components named p67-phox, p47-phox, and rac p21s. In a cell-free system consisting of membrane and cytosol, the oxidase can be activated with guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and an unsaturated fatty acid such as arachidonic acid (AA). Incubation of cytosol and membrane with AA alone caused clear translocation of p47-phox and p67-phox to the membrane, but only slight translocation of rac p21s. GTPγS alone did not significantly induce the iranslocation of rac p21s. However, GTPγS in combination with AA markedly augmented mac p21s translocation to the membrane. The translocation of rac p21s is not induced by GDP or GDPβS. These results indicate that the GTP-bound active form of mac p21s is the entity that is translocated to the membrane by the action of AA.

A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.

The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs that recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.

Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2

Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (alpha ASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (alpha IRS-1) from the insulin-stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1.ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated 170-kDa protein that was recognized by alpha IRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1.ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in alpha ASH immunoprecipitates, suggesting the formation of an ASH.IRS-1.PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.