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The adaptor protein Grb10 plays important roles in mitogenic signaling. However, its roles in acute myeloid leukemia (AML) are predominantly unknown. Here we describe the role of Grb10 in FLT3-ITD-mediated AML. We observed that Grb10 physically associates with FLT3 in response to FLT3-ligand (FL) stimulation through FLT3 phospho-tyrosine 572 and 793 residues and constitutively associates with oncogenic FLT3-ITD. Furthermore endogenous Grb10-FLT3 association was observed in OCI-AML-5 cells. Grb10 expression did not alter FLT3 receptor activation or stability in Ba/F3-FLT3 cells. However, expression of Grb10 enhanced FL-induced Akt phosphorylation without affecting Erk or p38 phosphorylation in Ba/F3-FLT3-WT and Ba/F3-FLT3-ITD. Selective Grb10 depletion reduced Akt phosphorylation in Ba/F3-FLT3-WT and OCI-AML-5 cells. Grb10 transduces signal from FLT3 by direct interaction with p85 and Ba/F3-FLT3-ITD cells expressing Grb10 exhibits higher STAT5 activation. Grb10 regulates the cell cycle by increasing cell population in S-phase. Expression of Grb10 furthermore resulted in an increased proliferation and survival of Ba/F3-FLT3-ITD cells as well as increased colony formation in semisolid culture. Grb10 expression was significantly increased in AML patients compared to healthy controls and was also elevated in patients carrying FLT3-ITD mutants. The elevated Grb10 expression partially correlated to relapse as well as to poor prognosis. These results suggest that Grb10 binds to both normal and oncogenic FLT3 and induces PI3K-Akt and STAT5 signaling pathways resulting in an enhanced proliferation, survival and colony formation of hematopoietic cells., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The effects of insulin and type 1 insulin-like growth factor (IGF-1) on metabolism, growth and survival are mediated by their association with specific receptor tyrosine kinases, which results in both receptor and substrate phosphorylation. Phosphotyrosine residues on receptors and substrates provide docking sites for signaling proteins containing SH2 (Src homology 2) domains, including molecular adaptors. This review focuses on the regulation of insulin/IGF-1 signaling and action by two adaptor families with a similar domain organization: the growth factor receptor-bound proteins Grb7/10/14 and the SH2B proteins. Both Grb10/14 and SH2B1/B2 associate with the activation loop of insulin/IGF-1 receptors through their SH2 domains, but association of Grb10/14 also involves their unique BPS domain. Consistent with Grb14 binding as a pseudosubstrate to the kinase active site, insulin/IGF-induced activation of receptors and downstream signaling pathways in cultured cells is inhibited by Grb10/14 adaptors, but is potentiated by SH2B1/B2 adaptors. Accordingly, Grb10 and Grb14 knockout mice show improved insulin/IGF sensitivity in vivo, and, for Grb10, overgrowth and increased skeketal muscle and pancreatic β-cell mass. Conversely, SH2B1-depleted mice display insulin and IGF-1 resistance, with peripheral depletion leading to reduced adiposity and neuronal depletion leading to obesity through associated leptin resistance. Grb10/14 and SH2B1 adaptors also modulate insulin/IGF-1 action by interacting with signaling components downstream of receptors and exert several tissue-specific effects. The identification of Grb10/14 and SH2B1 as physiological regulators of insulin signaling and action, together with observations that variants at their gene loci are associated with obesity and/or insulin resistance, highlight them as potential therapeutic targets for these conditions., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Human antigen R (HuR) is a post-transcriptional regulator of gene expression that plays a key role in stabilizing mRNAs during cellular stress, leading to enhanced survival. HuR expression is tightly regulated through multiple transcription and post-transcriptional controls. Although HuR is known to stabilize a subset of mRNAs involved in cell survival, its role in the survival pathway of PI3-kinase/Akt signaling is unclear. Here, we show that in renal proximal tubule cells, HuR performs a central role in cell survival by amplifying Akt signaling in a positive feedback loop. Key to this feedback loop is HuR-mediated stabilization of mRNA encoding Grb10, an adaptor protein whose expression is critical for Akt activation. Stimulation of Akt by interaction with Grb10 then activates NF-κB, which further enhances HuR mRNA and protein expression. This feedback loop is active in unstressed cells, but its effects are increased during stress. Therefore, this study demonstrates a central role for HuR in Akt signaling and reveals a mechanism by which modest changes in HuR levels below or above normal may be amplified, potentially resulting in cell death or cellular transformation., (Copyright © 2012 Wiley Periodicals, Inc.)

The LIM homeodomain transcription factor Lmx1a is a very potent inducer of stem cells towards dopaminergic neurons. Despite several studies on the function of this gene, the exact in vivo role of Lmx1a in mesodiencephalic dopamine (mdDA) neuronal specification is still not understood. To analyse the genes functioning downstream of Lmx1a, we performed expression microarray analysis of LMX1A-overexpressing MN9D dopaminergic cells. Several interesting regulated genes were identified, based on their regulation in other previously generated expression arrays and on their expression pattern in the developing mdDA neuronal field. Post analysis through in vivo expression analysis in Lmx1a mouse mutant (dr/dr) embryos demonstrated a clear decrease in expression of the genes Grb10 and Rgs4, in and adjacent to the rostral and dorsal mdDA neuronal field and within the Lmx1a expression domain. Interestingly, the DA marker Vmat2 was significantly up-regulated as a consequence of increased LMX1A dose, and subsequent analysis on Lmx1a-mutant E14.5 and adult tissue revealed a significant decrease in Vmat2 expression in mdDA neurons. Taken together, microarray analysis of an LMX1A-overexpression cell system resulted in the identification of novel direct or indirect downstream targets of Lmx1a in mdDA neurons: Grb10, Rgs4 and Vmat2., (© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The imprinted gene Grb10 influences fetal growth, adult behavior and tumor formation. Investigating this gene often involves consecutive passaging of cells in vitro. We analyzed changes in Grb10 DNA methylation and histone modifications in serially passaged adult mouse tail-tip fibroblast cells with bisulfite genomic sequencing method and chromatin immunoprecipitation (ChIP) respectively. There was a significant reduction in global DNA methylation, but no changes in methylation at CpG island 1 (CGI1) of Grb10. There were no changes in methylation at histone H3K4me2, but less methylated histone H3K27me3 was detected, along with more of the acetylated histones H3Ac and H4Ac. The major-type transcript level of Grb10 in fibroblasts increased mildly after extended cultivation. These results suggest consecutive passaging may affect epigenetic modifications of Grb10 in adult fibroblast cells., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Defects in insulin secretion and reduction in β-cell mass are associated with type 2 diabetes in humans, and understanding the basis for these dysfunctions may reveal strategies for diabetes therapy. In this study, we show that pancreas-specific knockout of growth factor receptor-binding protein 10 (Grb10), which is highly expressed in pancreas and islets, leads to elevated insulin/IGF-1 signaling in islets, enhanced β-cell mass and insulin content, and increased insulin secretion in mice. Pancreas-specific disruption of Grb10 expression also improved glucose tolerance in mice fed with a high-fat diet and protected mice from streptozotocin-induced β-cell apoptosis and body weight loss. Our study has identified Grb10 as an important regulator of β-cell proliferation and demonstrated that reducing the expression level of Grb10 could provide a novel means to increase β-cell mass and reduce β-cell apoptosis. This is critical for effective therapeutic treatment of both type 1 and 2 diabetes.

GRB10 is an imprinted gene differently expressed from two promoters in mouse and human. Mouse Grb10 is maternally expressed from the major promoter in most tissues and paternally expressed from the brain-specific promoter within specific regions of the fetal and adult central nervous system. Human GRB10 is biallelically expressed from the major promoter in most tissues except in the placental villus trophoblast where it is maternally expressed, whereas the brain-specific promoter is paternally expressed in the fetal brain. This study characterized the ortholog of GRB10 in a marsupial, the tammar wallaby (Macropus eugenii) to investigate the origin and evolution of imprinting at this locus. The protein coding exons and predicted amino acid sequence of tammar GRB10 were highly conserved with eutherian GRB10. The putative first exon, which is located in the orthologous region to the eutherian major promoter, was found in the tammar, but no exon was found in the downstream region corresponding to the eutherian brain-specific promoter, suggesting that marsupials only have a single promoter. Tammar GRB10 was widely expressed in various tissues including the brain but was not imprinted in any of the tissues examined. Thus, it is likely that GRB10 imprinting evolved in eutherians after the eutherian-marsupial divergence approximately 160 million years ago, subsequent to the acquisition of a brain-specific promoter, which resides within the imprinting control region in eutherians.

Background: Infant birth weight is a complex quantitative trait associated with both neonatal and long-term health outcomes. Numerous studies have been published in which candidate genes (IGF1, IGF2, IGF2R, IGF binding proteins, PHLDA2 and PLAGL1) have been associated with birth weight, but these studies are difficult to reproduce in man and large cohort studies are needed due to the large inter individual variance in transcription levels. Also, very little of the trait variance is explained. We decided to identify additional candidates without regard for what is known about the genes. We hypothesize that DNA methylation differences between individuals can serve as markers of gene "expression potential" at growth related genes throughout development and that these differences may correlate with birth weight better than single time point measures of gene expression., Methods: We performed DNA methylation and transcript profiling on cord blood and placenta from newborns. We then used novel computational approaches to identify genes correlated with birth weight., Results: We identified 23 genes whose methylation levels explain 70-87% of the variance in birth weight. Six of these (ANGPT4, APOE, CDK2, GRB10, OSBPL5 and REG1B) are associated with growth phenotypes in human or mouse models. Gene expression profiling explained a much smaller fraction of variance in birth weight than did DNA methylation. We further show that two genes, the transcriptional repressor MSX1 and the growth factor receptor adaptor protein GRB10, are correlated with transcriptional control of at least seven genes reported to be involved in fetal or placental growth, suggesting that we have identified important networks in growth control. GRB10 methylation is also correlated with genes involved in reactive oxygen species signaling, stress signaling and oxygen sensing and more recent data implicate GRB10 in insulin signaling., Conclusions: Single time point measurements of gene expression may reflect many factors unrelated to birth weight, while inter-individual differences in DNA methylation may represent a "molecular fossil record" of differences in birth weight-related gene expression. Finding these "unexpected" pathways may tell us something about the long-term association between low birth weight and adult disease, as well as which genes may be susceptible to environmental effects. These findings increase our understanding of the molecular mechanisms involved in human development and disease progression.

Aims/hypothesis: The physiological significance of growth factor receptor-bound protein-10 (GRB10) in the pancreas is unclear. We hypothesised that GRB10 is involved in pancreatic apoptosis, as GRB10 binds with a family of cell-survival-related proteins implicated in apoptosis., Methods: Lentiviral vector small hairpin RNA (shRNA) targeting Grb10 was injected in vivo via an intraductal pancreatic route to target pancreatic tissues in adult mice, which were studied 2 weeks post-injection., Results: Using the TUNEL assay, we demonstrated for the first time that in vivo injection of lentivirus shRNA Grb10 directly into the adult mouse pancreas induced apoptosis in both exocrine and endocrine (alpha and beta) cells. This effect was more pronounced in alpha cells. Levels of the pro-apoptotic protein BCL2-interacting mediator of cell death (BIM) in islets was higher in lentivirus shRNA Grb10 than in lentivirus shRNA scramble mice. In the apoptotic pathway, BIM initiates apoptosis signalling, leading to activation of the caspase cascade. We propose that, when complexed with GRB10, BIM is inactive. On activation by stress signalling or, in the present study, following injection of lentivirus shRNA Grb10 into pancreas, BIM becomes unbound from GRB10 and activates the caspase cascade. Indeed, caspase-3 activity in islets was higher in the experimental than in the control group. Apoptosis induced by shRNA Grb10 resulted in a 34% decrease in fasting plasma glucagon. Mice injected with shRNA Grb10 had improved glucose tolerance despite reduced insulin secretion compared with shRNA scramble control mice., Conclusions/interpretation: GRB10 is critically involved in alpha cell survival and, as a result, plays an important role in regulating basal glucagon secretion and glucose tolerance in adult mice.

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.

The evolutionarily conserved serine-threonine kinase mammalian target of rapamycin (mTOR) plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phosphoproteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently down-regulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

Imprinted genes, defined by their preferential expression of a single parental allele, represent a subset of the mammalian genome and often have key roles in embryonic development, but also postnatal functions including energy homeostasis and behaviour. When the two parental alleles are unequally represented within a social group (when there is sex bias in dispersal and/or variance in reproductive success), imprinted genes may evolve to modulate social behaviour, although so far no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Here we demonstrate that within the brain Grb10 is expressed from the paternal allele from fetal life into adulthood and that ablation of this expression engenders increased social dominance specifically among other aspects of social behaviour, a finding supported by the observed increase in allogrooming by paternal Grb10-deficient animals. Grb10 is, therefore, the first example of an imprinted gene that regulates social behaviour. It is also currently alone in exhibiting imprinted expression from each of the parental alleles in a tissue-specific manner, as loss of the peripherally expressed maternal allele leads to significant fetal and placental overgrowth. Thus Grb10 is, so far, a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, owing to actions of the two parental alleles in different tissues.

In addition to inhibiting insulin receptor and IGF1R kinase activity by directly binding to the receptors, GRB10 can also negatively regulate insulin and IGF1 signaling by mediating insulin receptor and IGF1R degradation through ubiquitination. It has been shown that GRB10 can interact with the C2 domain of the E3 ubiquitin ligase NEDD4 through its Src homology 2 (SH2) domain. Therefore, GRB10 might act as a connector, bringing NEDD4 close to IGF1R to facilitate the ubiquitination of IGF1R by NEDD4. This is the first case in which it has been found that an SH2 domain could colocalize a ubiquitin ligase and its substrate. Here we report the crystal structure of the NEDD4 C2-GRB10 SH2 complex at 2.0 Å. The structure shows that there are three interaction interfaces between NEDD4 C2 and GRB10 SH2. The main interface centers on an antiparallel β-sheet composed of the F β-strand of GRB10 SH2 and the C β-strand of NEDD4 C2. NEDD4 C2 binds at nonclassical sites on the SH2 domain surface, far from the classical phosphotyrosine-binding pocket. Hence, this interaction is phosphotyrosine-independent, and GRB10 SH2 can bind the C2 domain of NEDD4 and the kinase domain of IGF1R simultaneously. Based on these results, a model of how NEDD4 interacts with IGF1R through GRB10 has been proposed. This report provides further evidence that SH2 domains can participate in important signaling interactions beyond the classical recognition of phosphotyrosine.

We previously reported an important role of RQCD1 in mammary carcinogenesis through the interaction with Grb10 interacting GYF protein 1 (GIGYF1), Grb10 interacting GYF protein 2 (GIGYF2) and growth factor receptor binding protein 10 (Grb10). In this study, we investigated the biological mechanism of RQCD1 in regulation of the Akt activity as the downstream signal of epidermal growth factor receptor (EGFR). Knockdown of RQCD1 reduced the Akt phosphorylation level that was induced by epidermal growth factor (EGF) stimulation. We found a possible formation of the big complex involved in the Akt activity including Akt, EGFR, GIGYF1 and GIGYF2, Grb10 and RQCD1. We subsequently defined that a region corresponding to 620-665th amino acids of GIGYF1 and 667-712th amino acids of GIGYF2 interacted with RQCD1. Furthermore, we found that RQCD1 was required for enhancement of the interaction of Grb10 with GIGYF1 and GIGYF2. Our findings in this study imply the functional mechanism of RQCD1 in the Akt activity regulation as a mediator in the EGFR-signaling pathway.

Bim is a proapoptotic member of the Bcl-2 family and is primarily involved in the regulation of the intrinsic apoptotic pathway. However, the detail of regulation of Bim's proapoptotic activity has not been clarified yet. Using Bim L as bait, we screened a human fetal cDNA library for interacting proteins and identified Grb10 as an interactor. This interaction was verified by co-immunoprecipitation and intracellular co-localization studies. The potential segment of Bim L that binds Grb10 was identified via a yeast mating test. Grb10 interacted with the DBD (dynein binding domain) of Bim and inhibited apoptosis triggered by overexpression of DBD containing Bim isoforms. The putative phosphorylation sites on DBD of Bim play a role for the anti-proapoptotic activity of Grb10. Our results suggest that Grb10 interacts with Bim L and inhibits its proapoptotic activity in a phosphorylation-dependant manner.

GIGYF2 has been reported as a candidate gene for PARK11-linked Parkinson's disease (PD). Heterozygous knockout of GIGYF2 results in neurodegeneration, suggesting important roles for GIGYF2 (Grb10 interacting GYF protein 2) in the CNS. In this study, we used novel GIGYF2 antibodies to clarify the distribution and function of GIGYF2. GIGYF2 was widely expressed, most highly in the pancreas and testis, and moderately in brain, lung, liver, kidney and spleen. In the brain, GIGYF2 was tightly associated with membrane in the S3 fraction, and localised in neuronal perikarya and proximal dendrites. Immunohistochemical analysis indicated sites of GIGYF2 localisation throughout the mouse brain, with high levels in the cerebral cortex, hippocampus, cerebellum, olfactory bulb and brainstem nuclei, but low levels in the substantia nigra and striatum. GIGYF2 was present in endosomes immunopositive for Rab4 and Grb10. Expression of GIGYF2 altered insulin-like growth factor-1 (IGF-1) receptor trafficking and enhanced IGF-1-induced extracellular signal-regulated kinase 1/2 phosphorylation, but not IGF-1 receptor or serine/threonine protein kinase Akt phosphorylation. There were no significant differences in signalling activation between cells expressing wild-type and putative PD-associated mutant GIGYF2. In PD brains, GIGYF2 did not localise to Lewy bodies. Our findings indicate a role for GIGYF2 in the regulation of signalling at endosomes, but no contribution of GIGYF2 to the pathogenesis of PD., (© 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.)

In our backstage experiment with differential display method among the differentially expressed genes we found the gene of GRB10 (Growth factor Receptor-Bound protein 10). The GRB10 protein binds to insulin and insulin-like growth factor receptors and acts as a negative regulatory protein. Besides, GRB10 gene polymorphisms are connected to the development of type 2 diabetes mellitus. In this experiment we investigated the allele frequency of RS 2237457, +11275G > A polymorphism in Hungarian healthy and type 2 diabetic populations (healthy: n = 77, diabetics: n = 85). We also searched for the connections between the genotype and glucose homeostasis measured by hyperinsulinemic - normoglycemic clamps in healthy volunteers (n = 88), glucose intolerant (IFG n = 15; IGT n = 29) and non-treated type 2 diabetic patients (n = 9). We did not find significant differences in allele frequencies between the Hungarian healthy and diabetic populations (healthy: g vs. a: 62% vs. 38%; 2DM g vs. a: 70% vs. 30%). In case of females, glucose utilization did not depend on GRB10 gene polymorphisms. Insulin production after oral glucose load was increased among males with gg alleles, and not after iv. glucose administration. The glucose disposal in muscle tissue was lower and the metabolic clearance rate was also lower calculated either for total body or muscle tissue in this group. In both genders gg alleles were associated with a disadvantageous lipid profile of decreased levels of large, buoyant LDL molecules and HDL levels in females. Metabolic changes related to the polymorphism of GRB10 gene support a gender specific role of this gene in insulin sensitivity and insulin signal transduction. It may be hypothesized on the basis of the differences in insulin release after oral and iv. glucose loads that GRB10 is involved in incretin signaling pathway.

Mono-allelic expression of imprinted genes from either the paternal or the maternal allele is mediated by imprinting control regions (ICRs), which are epigenetically marked in an allele-specific fashion. Although, in somatic cells, these epigenetic marks comprise both DNA methylation and histone methylation, the relationship between these two modifications in imprint acquisition and maintenance remains unclear. To address this important question, we analyzed histone modifications at ICRs in mid-gestation embryos that were obtained from Dnmt3L(-/-) females, in which DNA methylation imprints at ICRs are not established during oogenesis. The absence of maternal DNA methylation imprints in these conceptuses led to a marked decrease and loss of allele-specificity of the repressive H3K9me3, H4K20me3 and H2A/H4R3me2 histone modifications, providing the first evidence of a mechanistic link between DNA and histone methylation at ICRs. The existence of this relationship was strengthened by the observation that when DNA methylation was still present at the Snrpn and Peg3 ICRs in some of the progeny of Dnmt3L(-/-) females, these ICRs were associated with the usual patterns of histone methylation. Combined, our data establish that DNA methylation is involved in the acquisition and/or maintenance of histone methylation at ICRs.

Growth factor receptor bound (Grb)10 and Grb14 are closely related adaptor proteins that bind directly to the insulin receptor (IR) and regulate insulin-induced IR tyrosine phosphorylation and signaling to IRS-1 and Akt. Grb10- and Grb14-deficient mice both exhibit improved whole-body glucose homeostasis as a consequence of enhanced insulin signaling and, in the case of the former, altered body composition. However, the combined physiological role of these adaptors has remained undefined. In this study we utilize compound gene knockout mice to demonstrate that although deficiency in one adaptor can enhance insulin-induced IRS-1 phosphorylation and Akt activation, insulin signaling is not increased further upon dual ablation of Grb10 and Grb14. Context-dependent limiting mechanisms appear to include IR hypophosphorylation and decreased IRS-1 expression. In addition, the compound knockouts exhibit an increase in lean mass comparable to Grb10-deficient mice, indicating that this reflects a regulatory function specific to Grb10. However, despite the absence of additive effects on insulin signaling and body composition, the double-knockout mice are protected from the impaired glucose tolerance that results from high-fat feeding, whereas protection is not observed with animals deficient for individual adaptors. These results indicate that, in addition to their described effects on IRS-1/Akt, Grb10 and Grb14 may regulate whole-body glucose homeostasis by additional mechanisms and highlight these adaptors as potential therapeutic targets for amelioration of the insulin resistance associated with type 2 diabetes.

Genomic imprinting may have evolved not only to regulate fetal growth and development, but also behaviour. The mouse Grb10 gene provides a remarkable model to explore this idea because it shows paternal expression in brain, whereas in the placenta and most other embryonic tissues, expression is from the maternal allele. To assess the biological relevance of this reciprocal pattern of imprinting, we explored its conservation in humans. As in mice, we find the human GRB10 gene to be paternally expressed in brain. Maternal allele-specific expression is conserved only in the placental villous trophoblasts, an essential part of the placenta involved in nutrient transfer. All other fetal tissues tested showed equal expression from both alleles. These data suggest that the maternal GRB10 expression in placenta is evolutionarily important, presumably in the control of fetal growth. As in the mouse, the maternal transcripts originate from several kilobases upstream of the imprinting control region (ICR) of the domain, from a promoter region at which we find no allelic chromatin differences. The brain-specific paternal expression from the ICR shows mechanistic similarities with the mouse as well. This conserved CpG island is DNA-methylated on the maternal allele and is marked on the paternal allele by developmentally regulated bivalent chromatin, with the presence of both H3 lysine-4 and H3 lysine-27 methylation. The strong conservation of the opposite allelic expression in placenta versus brain supports the hypothesis that GRB10 imprinting evolved to mediate diverse roles in mammalian growth and behaviour.

Growth factor receptor-binding proteins Grb7, Grb10 and Grb14 are adaptor proteins containing a Ras-associating (RA) domain, a pleckstrin-homology (PH) domain, a family-specific BPS (between PH and SH2) region and a C-terminal Src-homology-2 domain. Previous structural studies showed that the Grb14 BPS region binds as a pseudosubstrate inhibitor in the tyrosine kinase domain of the insulin receptor to suppress insulin signaling. Here we report the crystal structure of the RA and PH domains of Grb10 at 2.6-A resolution. The structure reveals that these two domains, along with the intervening linker, form an integrated, dimeric structural unit. Biochemical studies demonstrated that Grb14 binds to activated Ras, which may serve as a timing mechanism for downregulation of insulin signaling. Our results illuminate the membrane-recruitment mechanisms not only of Grb7, Grb10 and Grb14 but also of MIG-10, Rap1-interacting adaptor molecule, lamellipodin and Pico, proteins involved in actin-cytoskeleton rearrangement that share a structurally related RA-PH tandem unit.

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K(+) channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K(+) channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K(+) channels.

Background: Neurotrophins are important regulators of growth and regeneration, and acutely, they can modulate the activity of voltage-gated ion channels. Previously we have shown that acute brain-derived neurotrophic factor (BDNF) activation of neurotrophin receptor tyrosine kinase B (TrkB) suppresses the Shaker voltage-gated potassium channel (Kv1.3) via phosphorylation of multiple tyrosine residues in the N and C terminal aspects of the channel protein. It is not known how adaptor proteins, which lack catalytic activity, but interact with members of the neurotrophic signaling pathway, might scaffold with ion channels or modulate channel activity., Results: We report the co-localization of two adaptor proteins, neuronal Src homology and collagen (nShc) and growth factor receptor-binding protein 10 (Grb10), with Kv1.3 channel as demonstrated through immunocytochemical approaches in the olfactory bulb (OB) neural lamina. To further explore the specificity and functional ramification of adaptor/channel co-localization, we performed immunoprecipitation and Western analysis of channel, kinase, and adaptor transfected human embryonic kidney 293 cells (HEK 293). nShc formed a direct protein-protein interaction with Kv1.3 that was independent of BDNF-induced phosphorylation of Kv1.3, whereas Grb10 did not complex with Kv1.3 in HEK 293 cells. Both adaptors, however, co-immunoprecipitated with Kv1.3 in native OB. Grb10 was interestingly able to decrease the total expression of Kv1.3, particularly at the membrane surface, and subsequently eliminated the BDNF-induced phosphorylation of Kv1.3. To examine the possibility that the Src homology 2 (SH2) domains of Grb10 were directly binding to basally phosphorylated tyrosines in Kv1.3, we utilized point mutations to substitute multiple tyrosine residues with phenylalanine. Removal of the tyrosines 111-113 and 449 prevented Grb10 from decreasing Kv1.3 expression. In the absence of either adaptor protein, channel co-expression reciprocally down-regulated expression and tyrosine phosphorylation of TrkB kinase and related insulin receptor kinase. Finally, through patch-clamp electrophysiology, we found that the BDNF-induced current suppression of the channel was prevented by both nShc and Grb10., Conclusion: We report that adaptor protein alteration of kinase-induced Kv1.3 channel modulation is related to the degree of direct protein-protein association and that the channel itself can reciprocally modulate receptor-linked tyrosine kinase expression and activity.

Functions of signaling mediators Grb10 or Gab1 have been described in mitogenesis but remained disconnected. Here, we report the peptide hormone-dependent direct association between Grb10 and Gab1 and their functional connection in mitogenic signaling via MAP kinase using cultured fibroblasts as a model. In response to PDGF-, IGF-I, or insulin increased levels of Grb10 potentiated cell proliferation or survival whereas dominant-negative, domain-specific Grb10 peptide mimetics attenuated cell proliferation. This response was sensitive to p44/42 MAPK inhibitor but not to p38 MAPK inhibitor. In response to IGF-I or insulin Raf-1, MEK 1/2, and p44/42 MAPK were regulated by Grb10 but not Ras or p38 MAPK. In response to PDGF MEK 1/2, p44/42 MAPK and p38 MAPK were regulated by Grb10 but not Ras or Raf-1. Peptide hormone-dependent co-immunoprecipitation of Grb10 and Gab1 was demonstrated and specifically blocked by a Grb10 SH2 domain peptide mimetic. This domain was sufficient for direct, peptide hormone-dependent association with Gab1 via the Crk binding region. In response to PDGF, IGF-I, or insulin, in a direct comparison, elevated levels of mouse Grb10 delta, or human Grb10 beta or zeta equally potentiated fibroblast proliferation. Proliferation was severely reduced by Gab1 gene disruption whereas an elevated Gab1 gene dose proportionally stimulated Grb10-potentiated cell proliferation. In conclusion, Gab1 and Grb10 function as direct binding partners in the regulation of the mitogenic MAP kinase signal. In cultured fibroblasts, elevated levels of human Grb10 beta, zeta or mouse Grb10 delta comparably potentiate mitogenesis in response to PDGF, IGF-I, or insulin.

Genomic imprinting is a developmental mechanism that mediates parent-of-origin-specific expression in a subset of genes. How the tissue specificity of imprinted gene expression is controlled remains poorly understood. As a model to address this question, we studied Grb10, a gene that displays brain-specific expression from the paternal chromosome. Here, we show in the mouse that the paternal promoter region is marked by allelic bivalent chromatin enriched in both H3K4me2 and H3K27me3, from early embryonic stages onwards. This is maintained in all somatic tissues, but brain. The bivalent domain is resolved upon neural commitment, during the developmental window in which paternal expression is activated. Our data indicate that bivalent chromatin, in combination with neuronal factors, controls the paternal expression of Grb10 in brain. This finding highlights a novel mechanism to control tissue-specific imprinting.

The ubiquitin ligase Nedd4 has been proposed to regulate a number of signaling pathways, but its physiological role in mammals has not been characterized. Here we present an analysis of Nedd4-null mice to show that loss of Nedd4 results in reduced insulin-like growth factor 1 (IGF-1) and insulin signaling, delayed embryonic development, reduced growth and body weight, and neonatal lethality. In mouse embryonic fibroblasts, mitogenic activity was reduced, the abundance of the adaptor protein Grb10 was increased, and the IGF-1 receptor, which is normally present on the plasma membrane, was mislocalized. However, surface expression of IGF-1 receptor was restored in homozygous mutant mouse embryonic fibroblasts after knockdown of Grb10, and Nedd4(-/-) lethality was rescued by maternal inheritance of a disrupted Grb10 allele. Thus, in vivo, Nedd4 appears to positively control IGF-1 and insulin signaling partly through the regulation of Grb10 function.

Experimental reduction in placental growth often leads to increased placental efficiency measured as grams of fetus produced per gram of placenta, although little is known about the mechanisms involved. This study tested the hypothesis that the smallest placenta within a litter is the most efficient at supporting fetal growth by examining the natural intra-litter variation in placental nutrient transfer capacity in normal pregnant mice. The morphology, nutrient transfer and expression of key growth and nutrient supply genes (Igf2P0, Grb10, Slc2a1, Slc2a3, Slc38a1, Slc38a2 and Slc38a4) were compared in the lightest and heaviest placentas of a litter at days 16 and 19 of pregnancy, when mouse fetuses are growing most rapidly in absolute terms. The data show that there are morphological and functional adaptations in the lightest placenta within a litter, which increase active transport of amino acids per gram of placenta and maintain normal fetal growth close to term, despite the reduced placental mass. The specific placental adaptations differ with age. At E16, they are primarily morphological with an increase in the volume fraction of the labyrinthine zone responsible for nutrient exchange, whereas at E19 they are more functional with up-regulated placental expression of the glucose transporter gene, Slc2a1/GLUT1 and one isoform the System A family of amino acid transporters, Slc38a2/SNAT2. Thus, this adaptability in placental phenotype provides a functional reserve capacity for maximizing fetal growth during late gestation when placental growth is compromised.

The adaptor protein Grb10 is an interacting partner of the IGF-I receptor (IGF-IR) and the insulin receptor (IR). Previous work from our laboratory has established the role of Grb10 as a negative regulator of IGF-IR-dependent cell proliferation. We have shown that Grb10 binds the E3 ubiquitin ligase Nedd4 and promotes IGF-I-stimulated ubiquitination, internalization, and degradation of the IGF-IR, thereby giving rise to long-term attenuation of signaling. Recent biochemical evidence suggests that tyrosine-kinase receptors (RTK) may not be polyubiquitinated but monoubiquitinated at multiple sites (multiubiquitinated). However, the type of ubiquitination of the IGF-IR is still not defined. Here we show that the Grb10/Nedd4 complex upon ligand stimulation mediates multiubiquitination of the IGF-IR, which is required for receptor internalization. Moreover, Nedd4 by promoting IGF-IR ubiquitination and internalization contributes with Grb10 to negatively regulate IGF-IR-dependent cell proliferation. We also demonstrate that the IGF-IR is internalized through clathrin-dependent and-independent pathways. Grb10 and Nedd4 remain associated with the IGF-IR in early endosomes and caveosomes, where they may participate in sorting internalized receptors. Grb10 and Nedd4, unlike the IGF-IR, which is targeted for lysosomal degradation are not degraded and likely directed into recycling endosomes. These results indicate that Grb10 and Nedd4 play a critical role in mediating IGF-IR down-regulation by promoting ligand-dependent multiubiquitination of the IGF-IR, which is required for receptor internalization and regulates mitogenesis., ((c) 2008 Wiley-Liss, Inc.)

By combining a tissue-specific microarray screen with mouse uniparental duplications, we have identified a novel imprinted gene, Dopa decarboxylase (Ddc), on chromosome 11. Ddc_exon1a is a 2-kb transcript variant that initiates from an alternative first exon in intron 1 of the canonical Ddc transcript and is paternally expressed in trabecular cardiomyocytes of the embryonic and neonatal heart. Ddc displays tight conserved linkage with the maternally expressed and methylated Grb10 gene, suggesting that these reciprocally imprinted genes may be coordinately regulated. In Dnmt3L mutant embryos that lack maternal germ line methylation imprints, we show that Ddc is overexpressed and Grb10 is silenced. Their imprinting is therefore dependent on maternal germ line methylation, but the mechanism at Ddc does not appear to involve differential methylation of the Ddc_exon1a promoter region and may instead be provided by the oocyte mark at Grb10. Our analysis of Ddc redefines the imprinted Grb10 domain on mouse proximal chromosome 11 and identifies Ddc_exon1a as the first example of a heart-specific imprinted gene.

Grb10 is a pleckstrin homology and Src homology 2 domain-containing protein that interacts with a number of phosphorylated receptor tyrosine kinases, including the insulin receptor. In mice, Grb10 gene expression is imprinted with maternal expression in all tissues except the brain. While the interaction between Grb10 and the insulin receptor has been extensively investigated in cultured cells, whether this adaptor protein plays a positive or negative role in insulin signaling and action remains controversial. In order to investigate the in vivo role of Grb10 in insulin signaling and action in the periphery, we generated Grb10 knockout mice by the gene trap technique and analyzed mice with maternal inheritance of the knockout allele. Disruption of Grb10 gene expression in peripheral tissues had no significant effect on fasting glucose and insulin levels. On the other hand, peripheral-tissue-specific knockout of Grb10 led to significant overgrowth of the mice, consistent with a role for endogenous Grb10 as a growth suppressor. Loss of Grb10 expression in insulin target tissues, such as skeletal muscle and fat, resulted in enhanced insulin-stimulated Akt and mitogen-activated protein kinase phosphorylation. Hyperinsulinemic-euglycemic clamp studies revealed that disruption of Grb10 gene expression in peripheral tissues led to increased insulin sensitivity. Taken together, our results provide strong evidence that Grb10 is a negative regulator of insulin signaling and action in vivo.

Both VEGF and insulin are implicated in the pathogenesis of diabetic retinopathy. While it has been established for many years that the number of cell surface receptors impacts upon VEGF and insulin action, little is known about the precise machinery and proteins driving VEGF-R2 and IR degradation. Here, we investigate the role of Hepatocyte growth factor-Regulated tyrosine kinase Substrate (Hrs), a regulator of RTK trafficking, in VEGF and insulin signaling. We report that ectopic expression of Hrs increases VEGF-R2 and IR number and tyrosine phosphorylation, leading to amplification of their downstream signaling. The UIM (Ubiquitin Interacting Motif) domain of Hrs is required for Hrs-induced increases in VEGF-R2, but not in IR. Furthermore, Hrs is tyrosine-phosphorylated in response to VEGF and insulin. We show that the UIM domain is required for Hrs phosphorylation in response to VEGF, but not to insulin. Importantly, Hrs co-localizes with both VEGF-R2 and IR and co-immunoprecipitates with both in a manner independent of the Hrs-UIM domain. Finally, we demonstrate that Hrs inhibits Nedd4-mediated VEGF-R2 degradation and acts additively with Grb10. We conclude that Hrs is a positive regulator of VEGF-R2 and IR signaling and that ectopic expression of Hrs protects both VEGF-R2 and IR from degradation.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a component of cell-surface receptors for Wnt proteins and Wnt is known to promote recruitment of Axin by LRP6 thereby inhibiting beta-catenin's degradation. We show here that growth factor receptor-bound protein10 (GRB10), a multi-modular adaptor protein that is known to associate with several transmembrane tyrosine kinase receptors, binds to the intracellular portion of LRP6 and negatively regulates Wnt signaling. GRB10 overexpression suppressed Wnt3a-, and LRP6-induced but not beta-catenin-induced TCF-dependent-reporter activities in HEK293T cells, suggesting that GRB10 functions upstream of beta-catenin. Actually, GRB10 overexpression attenuated the Wnt3a-induced accumulation of beta-catenin. In addition, RNAi-mediated down-regulation of endogenous GRB10 stimulated Wnt3a-induced reporter activities, indicating that GRB10 is indeed a novel negative regulator of the Wnt signaling pathway. The finding that GRB10 interferes with the binding of Axin to LRP6 indicated a possible molecular mechanism by which the overexpression of GRB10 suppresses Wnt signaling.

Mouse Grb10 is a tissue-specific imprinted gene with promoter-specific expression. In most tissues, Grb10 is expressed exclusively from the major-type promoter of the maternal allele, whereas in the brain, it is expressed predominantly from the brain type promoter of the paternal allele. Such reciprocally imprinted expression in the brain and other tissues is thought to be regulated by DNA methylation and the Polycomb group (PcG) protein Eed. To investigate how DNA methylation and chromatin remodeling by PcG proteins coordinate tissue-specific imprinting of Grb10, we analyzed epigenetic modifications associated with Grb10 expression in cultured brain cells. Reverse transcriptase PCR analysis revealed that the imprinted paternal expression of Grb10 in the brain implied neuron-specific and developmental stage-specific expression from the paternal brain type promoter, whereas in glial cells and fibroblasts, Grb10 was reciprocally expressed from the maternal major-type promoter. The cell-specific imprinted expression was not directly related to allele-specific DNA methylation in the promoters because the major-type promoter remained biallelically hypomethylated regardless of its activity, whereas gametic DNA methylation in the brain type promoter was maintained during differentiation. Histone modification analysis showed that allelic methylation of histone H3 lysine 4 and H3 lysine 9 were associated with gametic DNA methylation in the brain type promoter, whereas that of H3 lysine 27 regulated by the Eed PcG complex was detected in the paternal major-type promoter, corresponding to its allele-specific silencing. Here, we propose a molecular model that gametic DNA methylation and chromatin remodeling by PcG proteins during cell differentiation cause tissue-specific imprinting in embryonic tissues.

Growth factor receptor-bound protein 10 (Grb10) is an adapter protein that interacts with a number of tyrosine-phosphorylated growth factor receptors, including the insulin receptor (IR). To investigate the role of Grb10 in insulin signaling, we generated cell lines in which the expression levels of Grb10 are either overexpressed by stable transfection or suppressed by RNA interference. We found that suppressing endogenous Grb10 expression led to increased IR protein levels, whereas overexpression of Grb10 led to reduced IR protein levels. Altering Grb10 expression levels had no effect on the mRNA levels of IR, suggesting that the modulation occurs at the protein level. Reduced IR levels were also observed in cells with prolonged insulin treatment, and this reduction was inhibited in Grb10-deficient cells. The insulin-induced IR reduction was greatly reversed by MG-132, a proteasomal inhibitor, but not by chloroquine, a lysosomal inhibitor. IR underwent insulin-stimulated ubiquitination in cells, and this ubiquitination was inhibited in the Grb10-suppressed cell line. Together, our results suggest that, in addition to inhibiting IR kinase activity by directly binding to the IR, Grb10 also negatively regulates insulin signaling by mediating insulin-stimulated degradation of the receptor.