Binding of Shp2 Tyrosine Phosphatase to FRS2 Is Essential for Fibroblast Growth Factor-Induced PC12 Cell Differentiation

FRS2 is a lipid-anchored docking protein that plays an important role in linking fibroblast growth factor (FGF) and nerve growth factor receptors with the Ras/mitogen-activated protein (MAP) kinase signaling pathway. In this report, we demonstrate that FRS2 forms a complex with the N-terminal SH2 domain of the protein tyrosine phosphatase Shp2 in response to FGF stimulation. FGF stimulation induces tyrosine phosphorylation of Shp2, leading to the formation of a complex containing Grb2 and Sos1 molecules. In addition, a mutant FRS2 deficient in both Grb2 and Shp2 binding induces a weak and transient MAP kinase response and fails to induce PC12 cell differentiation in response to FGF stimulation. Furthermore, FGF is unable to induce differentiation of PC12 cells expressing an FRS2 point mutant deficient in Shp2 binding. Finally, we demonstrate that the catalytic activity of Shp2 is essential for sustained activation of MAP kinase and for potentiation of FGF-induced PC12 cell differentiation. These experiments demonstrate that FRS2 recruits Grb2 molecules both directly and indirectly via complex formation with Shp2 and that Shp2 plays an important role in FGF-induced PC12 cell differentiation.     

Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2.

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.     

Integrin-mediated Activation of MAP Kinase Is Independent of FAK: Evidence for Dual Integrin Signaling Pathways in Fibroblasts

Integrin-mediated cell adhesion causes activation of MAP kinases and increased tyrosine phosphorylation of focal adhesion kinase (FAK). Autophosphorylation of FAK leads to the binding of SH2-domain proteins including Src-family kinases and the Grb2–Sos complex. Since Grb2–Sos is a key regulator of the Ras signal transduction pathway, one plausible hypothesis has been that integrin-mediated tyrosine phosphorylation of FAK leads to activation of the Ras cascade and ultimately to mitogen activated protein (MAP) kinase activation. Thus, in this scenario FAK would serve as an upstream regulator of MAP kinase activity. However, in this report we present several lines of evidence showing that integrin-mediated MAP kinase activity in fibroblasts is independent of FAK. First, a β1 integrin subunit deletion mutant affecting the putative FAK binding site supports activation of MAP kinase in adhering fibroblasts but not tyrosine phosphorylation of FAK. Second, fibroblast adhesion to bacterially expressed fragments of fibronectin demonstrates that robust activation of MAP kinase can precede tyrosine phosphorylation of FAK. Finally, we have used FRNK, the noncatalytic COOH-terminal domain of FAK, as a dominant negative inhibitor of FAK autophosphorylation and of tyrosine phosphorylation of focal contacts. Using retroviral infection, we demonstrate that levels of FRNK expression sufficient to completely block FAK tyrosine phosphorylation were without effect on integrin-mediated activation of MAP kinase. These results strongly suggest that integrin-mediated activation of MAP kinase is independent of FAK and indicate the probable existence of at least two distinct integrin signaling pathways in fibroblasts.     

A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells.

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline-rich domain-containing proteins such as Sos1. To define the difference in Grb2-associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST-Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST-Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST-Grb2 SH2 proteins exclusively bound to the p46(Shc), p52(Shc), and p66(Shc) are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N-SH3 and C-SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2-mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells.     

Sprouty2 binds Grb2 at two different proline-rich regions, and the mechanism of ERK inhibition is independent of this interaction

Sprouty2 has been widely implicated in the negative regulation of the fibroblast growth factor receptor-extracellular regulated kinase (ERK) pathway. Sprouty2 directly interacts with the adapter protein Grb2, member of the receptor tyrosine kinase-induced signaling pathways. In considering the functional role of Grb2, we investigated whether the interaction with this protein was responsible for ERK pathway inhibition. We found that the binding between Sprouty2 and Grb2 is constitutive, independent of Sprouty2 tyrosine phosphorylation, although it is increased when fibroblast growth factor receptor is activated. This connection is mediated by the N-terminal SH3 domain of Grb2 and two Sprouty2 proline-rich stretches (residues 59-64 and 303-307). Most importantly, a double Sprouty2 mutant (hSpry2 P59AP304A), which is unable to bind Grb2, developed at a similar inhibition level of fibroblast growth factor receptor-ERK pathway than that which originated from Sprouty2 wt. These results are evidence that the Sprouty2 mechanism of ERK inhibition is independent of Grb2 binding.     

TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos—but not RasGRP1—are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.     

The molecular functions of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway

Shp2 is a ubiquitous tyrosine phosphatase containing Src Homology 2 domains which plays major biological functions in response to various growth factors, hormones or cytokines. This is essentially due to its particularity of promoting the activation of the Ras/Mitogen-activated protein kinase pathway. Recent progresses have been made in the understanding of the molecular mechanisms involved in this regulation. We review here, and discuss the physiological relevance, of the following molecular functions of Shp2 that have been proposed to couple the phosphatase to Ras activation: promoter of Grb2/Sos recruitment through direct binding to Grb2, binding partner and regulator of SHPS-1, negative regulator of Sprouty, negative regulator of RasGAP recruitment, and activator of Src through dephosphorylation of Src-regulatory proteins.     

Gene dosage-dependent functions for phosphotyrosine-Grb2 signaling during mammalian tissue morphogenesis

BACKGROUND: The mammalian Grb2 adaptor protein binds pTyr-X-Asn motifs through its SH2 domain, and engages downstream targets such as Sos1 and Gab1 through its SH3 domains. Grb2 thereby couples receptor tyrosine kinases to the Ras-MAP kinase pathway, and potentially to phosphatidylinositol (PI) 3'-kinase. By creating a null (Delta) allele of mouse Grb2, we have shown that Grb2 is required for endoderm differentiation at embryonic day 4.0. RESULTS: Grb2 likely has multiple embryonic and postnatal functions. To address this issue, a hypomorphic mutation, first characterized in the Caenorhabditis elegans Grb2 ortholog Sem-5, was engineered into the mouse Grb2 gene. This mutation (E89K) reduces phosphotyrosine binding by the SH2 domain. Embryos that are compound heterozygous for the null and hypomorphic alleles exhibit defects in placental morphogenesis and in the survival of a subset of migrating neural crest cells required for branchial arch formation. Furthermore, animals homozygous for the hypomorphic mutation die perinatally because of clefting of the palate, a branchial arch-derived structure. Analysis of E89K/Delta Grb2 mutant fibroblasts revealed a marked defect in ERK/MAP kinase activation and Gab1 tyrosine phosphorylation following growth factor stimulation. CONCLUSIONS: We have created an allelic series within mouse Grb2, which has revealed distinct functions for phosphotyrosine-Grb2 signaling in tissue morphogenesis and cell viability necessary for mammalian development. The placental defects in E89K/Delta mutant embryos are reminiscent of those seen in receptor tyrosine kinase-, Sos1-, and Gab1-deficient embryos, consistent with the finding that endogenous Grb2 is required for efficient RTK signaling to the Ras-MAP kinase and Gab1 pathways.     

Significance of the Grb2 and son of sevenless (Sos) proteins in human bladder cancer cell lines

The epidermal growth factor (EGF) receptor has been suggested to have an important role in tumor initiation and progression of human bladder cancers. Grb2 protein, which is the downstream effector of the EGF receptor, acts as an adaptor protein between the EGF receptor and the Ras guanine-nucleotide exchange factor, son of sevenless (Sos) protein. Sos protein regulates the action of Ras protein by promoting the exchange of GDP for GTP. However, the significance of Grb2 and Sos proteins, which is related to EGF-triggered Ras activation, has not been elucidated in human bladder cancer. The aim of the present study is to clarify the significance of these proteins in human bladder cancer cell lines. In the present study, we used four human bladder cancer cell lines (T24, KU-7, UMUC-2, UMUC-6) and two kinds of cultured normal urothelial cells (HMKU-1, HMKU-2) isolated from patients with no malignancy. We examined the expression of EGF receptor, Grb2, and Sos proteins in these cells by Western blot analysis. Furthermore, the bladder cancer cell lines were subjected to sequence analysis to identify a point mutation in the c-H-ras gene at codon 12. There was no marked difference in the expression of the EGF receptor between human bladder cancer cell lines and cultured normal urothelial cells. On the other hand, expression of Grb2 and Sos proteins was substantially increased in all human bladder cancer cell lines examined in comparison with cultured normal urothelial cells, whether codon 12 of H-ras was mutated or not. These results suggest that the amplification of both Grb2 and SOS proteins plays an important role in the carcinogenesis of human bladder cancer.     

Multiple effector domains within SNT1 coordinate ERK activation and neuronal differentiation of PC12 cells

Differentiation of neuronal precursor cells in response to neurotrophic differentiation factors is accompanied by the activation of membrane-anchored SNT signaling adaptor proteins. Two classes of differentiation factors, the neurotrophins and fibroblast growth factors, induce rapid tyrosine phosphorylation of SNT1(FRS2alpha), which in turn enables SNT1 to recruit Shp2 tyrosine phosphatase and Grb2 adaptor protein in complex with the Ras GDP/GTP exchange factor Sos. To determine effector functions of SNT that promote neuronal differentiation of PC12 pheochromocytoma cells, we engineered a chimeric protein, SNT1(IRS)CX, bearing the effector region of SNT1 and the insulin receptor recognition domains of IRS2. Insulin promoted tyrosine phosphorylation of SNT1(IRS)CX in transfected PC12 cells accompanied by sustained activation of ERK1/2 mitogen-activated protein kinases and neuronal differentiation. The SNT1(IRS)CX-mediated response was dependent on endogenous Ras, MEK, and Shp2 activities. Mutagenesis of SNT1(IRS)CX identified three classes of effector motifs within SNT critical for both sustained ERK activation and neuronal differentiation: 1) four phosphotyrosine motifs that mediate recruitment of Grb2, 2) two phosphotyrosine motifs that mediate recruitment of Shp2, and 3) a C-terminal motif that functions by helping to recruit Sos. We discuss possible mechanisms by which three functionally distinct SNT effector motifs collaborate to promote a downstream biochemical and biological response.     

Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.     

Assembly of the Sos1-Grb2-Gab1 ternary signaling complex is under allosteric control

Allostery has evolved as a form of local communication between interacting protein partners allowing them to quickly sense changes in their immediate vicinity in response to external cues. Herein, using isothermal titration calorimetry (ITC) in conjunction with circular dichroism (CD) and macromolecular modeling (MM), we show that the binding of Grb2 adaptor—a key signaling molecule involved in the activation of Ras GTPase—to its downstream partners Sos1 guanine nucleotide exchange factor and Gab1 docker is under tight allosteric regulation. Specifically, our findings reveal that the binding of one molecule of Sos1 to the nSH3 domain allosterically induces a conformational change within Grb2 such that the loading of a second molecule of Sos1 onto the cSH3 domain is blocked and, in so doing, allows Gab1 access to the cSH3 domain in an exclusively non-competitive manner to generate the Sos1-Grb2-Gab1 ternary signaling complex.     

Molecular mechanism for the Shp-2 tyrosine phosphatase function in promoting growth factor stimulation of Erk activity

We have previously shown that activation of extracellular signal-regulated kinase (Erk) by epidermal growth factor (EGF) treatment was significantly decreased in mouse fibroblast cells expressing a mutant Shp-2 molecule lacking 65 amino acids in the SH2-N domain, Shp-2(Delta46-110). To address the molecular mechanism for the positive role of Shp-2 in mediating Erk induction, we evaluated the activation of signaling components upstream of Erk in Shp-2 mutant cells. EGF-stimulated Ras, Raf, and Mek activation was significantly attenuated in Shp-2 mutant cells, suggesting that Shp-2 acts to promote Ras activation or to suppress the down-regulation of activated Ras. Biochemical analyses indicate that upon EGF stimulation, Shp-2 is recruited into a multiprotein complex assembled on the Gab1 docking molecule and that Shp-2 seems to exert its biological function by specifically dephosphorylating an unidentified molecule of 90 kDa in the complex. The mutant Shp-2(Delta46-110) molecule failed to participate in the Gab1-organized complex for dephosphorylation of p90, correlating with a defective activation of the Ras-Raf-Mek-Erk cascade in EGF-treated Shp-2 mutant cells. Evidence is also presented that Shp-2 does not appear to modulate the signal relay from EGF receptor to Ras through the Shc, Grb2, and Sos proteins. These results begin to elucidate the mechanism of Shp-2 function downstream of a receptor tyrosine kinase to promote the activation of the Ras-Erk pathway, with potential therapeutic applications in cancer treatment.     

Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.

Growth factor receptor-binding protein-2 (Grb2) plays a key role in signal transduction initiated by Bcr/Abl oncoproteins and growth factors, functioning as an adaptor protein through its Src homology 2 and 3 (SH2 and SH3) domains. We found that Grb2 was tyrosine-phosphorylated in cells expressing BCR/ABL and in A431 cells stimulated with epidermal growth factor (EGF). Phosphorylation of Grb2 by Bcr/Abl or EGF receptor reduced its SH3-dependent binding to Sos in vivo, but not its SH2-dependent binding to Bcr/Abl. Tyr209 within the C-terminal SH3 domain of Grb2 was identified as one of the tyrosine phosphorylation sites, and phosphorylation of Tyr209 abolished the binding of the SH3 domain to a proline-rich Sos peptide in vitro. In vivo expression of a Grb2 mutant where Tyr209 was changed to phenylalanine enhanced BCR/ABL-induced ERK activation and fibroblast transformation, and potentiated and prolonged Grb2-mediated activation of Ras, mitogen-activated protein kinase and c-Jun N-terminal kinase in response to EGF stimulation. These results suggest that tyrosine phosphorylation of Grb2 is a novel mechanism of down-regulation of tyrosine kinase signaling.     

Regulation of sprouty expression by PLCgamma and calcium-dependent signals

Sprouty, an essential antagonist of fibroblast growth factor receptor signaling, is induced following fibroblast growth factor receptor activation. The signaling pathways that induce sprouty have been incompletely characterized. However, studies show that MAP kinase signaling stimulates sprouty induction in various cell lines. Here we report that activation of sprouty expression by basic fibroblast growth factor required phospholipase Cgamma (PLCgamma) and calcium-dependent signaling. We showed that the induction of sprouty was inhibited by chelation of intracellular or extracellular calcium and that a fibroblast growth factor receptor deficient for PLCgamma signaling only weakly induced sprouty expression. Additionally, inhibition of PLCgamma with a pharmacological antagonist repressed the induction of sprouty by basic fibroblast growth factor. These findings indicate that calcium-dependent signaling regulates sprouty expression and that PLCgamma is vital for this process. This pathway of sprouty induction may be critical at sites such as limb bud mesenchyme where MAP kinases are inactive.     

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.     

Evidence for direct interaction between Sprouty and Cbl

Sprouty (SPRY) was first identified in a genetic screen in Drosophila as an antagonist of fibroblast and epidermal growth factor receptors and Sevenless signaling, seemingly by inhibiting the receptor tyrosine kinase (RTK)/Ras/MAPK pathway. To date, four mammalian Sprouty genes have been identified; the primary sequences of the gene products share a well conserved cysteine-rich C-terminal domain with their Drosophila counterpart. The N-terminal regions do not, however, exhibit a large degree of homology. This study was aimed at identifying proteins with which human SPRY2 (hSPRY2) interacts in an attempt to understand the mechanism by which Sprouty proteins exert their down-regulatory effects. Here, we demonstrate that hSPRY2 associates directly with c-Cbl, a known down-regulator of RTK signaling. A short sequence in the N terminus of hSPRY2 was found to bind directly to the Ring finger domain of c-Cbl. Parallel binding was apparent between the Drosophila homologs of Sprouty and Cbl, with cross-species associations occurring at least in vitro. Coexpression of hSPRY2 abrogated an increase in the rate of epidermal growth factor receptor internalization induced by c-Cbl, whereas a mutant hSPRY2 protein unable to bind c-Cbl showed no such effect. Our results suggest that one function of hSPRY2 in signaling processes downstream of RTKs may be to modulate c-Cbl physiological function such as that seen with receptor-mediated endocytosis.     

Quantifying intramolecular binding in multivalent interactions: a structure-based synergistic study on Grb2-Sos1 complex

Numerous signaling proteins use multivalent binding to increase the specificity and affinity of their interactions within the cell. Enhancement arises because the effective binding constant for multivalent binding is larger than the binding constants for each individual interaction. We seek to gain both qualitative and quantitative understanding of the multivalent interactions of an adaptor protein, growth factor receptor bound protein-2 (Grb2), containing two SH3 domains interacting with the nucleotide exchange factor son-of-sevenless 1 (Sos1) containing multiple polyproline motifs separated by flexible unstructured regions. Grb2 mediates the recruitment of Sos1 from the cytosol to the plasma membrane where it activates Ras by inducing the exchange of GDP for GTP. First, using a combination of evolutionary information and binding energy calculations, we predict an additional polyproline motif in Sos1 that binds to the SH3 domains of Grb2. This gives rise to a total of five polyproline motifs in Sos1 that are capable of binding to the two SH3 domains of Grb2. Then, using a hybrid method combining molecular dynamics simulations and polymer models, we estimate the enhancement in local concentration of a polyproline motif on Sos1 near an unbound SH3 domain of Grb2 when its other SH3 domain is bound to a different polyproline motif on Sos1. We show that the local concentration of the Sos1 motifs that a Grb2 SH3 domain experiences is approximately 1000 times greater than the cellular concentration of Sos1. Finally, we calculate the intramolecular equilibrium constants for the crosslinking of Grb2 on Sos1 and use thermodynamic modeling to calculate the stoichiometry. With these equilibrium constants, we are able to predict the distribution of complexes that form at physiological concentrations. We believe this is the first systematic analysis that combines sequence, structure, and thermodynamic analyses to determine the stoichiometry of the complexes that are dominant in the cellular environment.