To investigate the function of the Grb10 adapter protein, we have generated mice in which the Grb10 gene was disrupted by a gene-trap insertion. Our experiments confirm that Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele. Consistent with this, disruption of the maternal allele results in overgrowth of both the embryo and placenta such that mutant mice are at birth Ϸ30% larger than normal. This observation establishes that Grb10 is a potent growth inhibitor. In humans, GRB10 is located at chromosome 7p11.2-p12 and has been associated with Silver-Russell syndrome, in which Ϸ10% of those affected inherit both copies of chromosome 7 from their mother. Our results indicate that changes in GRB10 dosage could, in at least some cases, account for the severe growth retardation that is characteristic of Silver-Russell syndrome. Because Grb10 is a signaling protein capable of interacting with tyrosine kinase receptors, we tested genetically whether Grb10 might act downstream of insulin-like growth factor 2, a paternally expressed growth-promoting gene. The result indicates that Grb10 action is essentially independent of insulinlike growth factor 2, providing evidence that imprinting acts on at least two major fetal growth axes in a manner consistent with parent-offspring conflict theory.adapter protein ͉ cell signaling ͉ genomic imprinting ͉ growth factor receptor-bound protein ͉ insulin-like growth factor

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 development1, but also post-natal functions including energy homeostasis2 and behaviour3, 4. 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)5, 6, imprinted genes may evolve to modulate social behaviour, although to date no such instance is known. Predominantly expressed from the maternal allele during embryogenesis, Grb10 encodes an intracellular adapter protein that can interact with a number of receptor tyrosine kinases and downstream signalling molecules7. 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 to date a unique imprinted gene, able to influence distinct physiological processes, fetal growth and adult behaviour, due to actions of the two parental alleles in different tissues.

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10⌬2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10⌬2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10⌬2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.Insulin controls glucose homeostasis by regulating protein, lipid, and carbohydrate metabolism. Cellular responses to insulin in target tissues, such as skeletal muscle, adipose tissue, and liver, are mediated via the insulin receptor (Insr) (reviewed in reference 51). Activation of the Insr results in tyrosine phosphorylation of intracellular docking proteins such as Shc and IRS-1 through IRS-4, which then bind specific Src homology 2 (SH2) domain-containing enzymes and adapters, leading to the activation of downstream signaling cascades. A critical event mediating insulin regulation of metabolic endpoints is the activation of phosphatidylinositol 3-kinase (PI3K). This stimulates the synthesis of phosphatidylinositol 3,4,5-triphosphate, which induces plasma membrane recruitment and subsequent phosphorylation of protein kinase B (also known as Akt), a key player in the regulation of glucose uptake and glycogen synthesis. Activation of the Insr and downstream signaling results in increased glucose uptake, utilization, and storage in adipose tissue and skeletal muscle, while decreased gluconeogenesis and glycogenolysis and increased glycogen synthesis occur in the liver (reviewed in reference 51). Resistance to these effects of insulin is a defining feature of type 2 diabetes, a polygenic disease afflicting over 110 million people worldwide. Impaired insulin action is also a feature of obesity and predisposes people to arteriosclerosis and cardiovascular diseases, facts which highlight its importance in human health. The fundamental role of the Insr in insulin action was demonstrated following targeted disruption of the receptor (1, 29). To dissect the contribution of individual tissues to gl...

A small sub-set of mammalian genes are subject to regulation by genomic imprinting such that only one parental allele is active in at least some sites of expression. Imprinted genes have diverse functions, notably including the regulation of growth. Much attention has been devoted to the insulin-like growth factor signalling pathway that has a major influence on fetal size and contains two components encoded by the oppositely imprinted genes, Igf2 (a growth promoting factor expressed from the paternal allele) and Igf2r (a growth inhibitory factor expressed from the maternal allele). These genes fit the parent-offspring conflict hypothesis for the evolution of genomic imprinting. Accumulated evidence indicates that at least one other fetal growth pathway exists that has also fallen under the influence of imprinting. It is clear that not all components of growth regulatory pathways are encoded by imprinted genes and instead it may be that within a pathway the influence of a single gene by each of the parental genomes may be sufficient for parent-offspring conflict to be enacted. A number of imprinted genes have been found to influence energy homeostasis and some, including Igf2 and Grb10, may coordinate growth with glucose-regulated metabolism. Since perturbation of fetal growth can be correlated with metabolic disorders in adulthood these imprinted genes are considered as candidates for involvement in this phenomenon of fetal programming.

The control of foetal growth is poorly understood and yet it is critically important that at birth the body has attained appropriate size and proportions. Growth and survival of the mammalian foetus is dependent upon a functional placenta throughout most of gestation. A few genes are known that influence both foetal and placental growth and might therefore coordinate growth of the conceptus, including the imprinted Igf2 and Grb10 genes. Grb10 encodes a signalling adapter protein, is expressed predominantly from the maternally-inherited allele and acts to restrict foetal and placental growth. Here, we show that following disruption of the maternal allele in mice, the labyrinthine volume was increased in a manner consistent with a cell-autonomous function of Grb10 and the enlarged placenta was more efficient in supporting foetal growth. Thus, Grb10 is the first example of a gene that acts to limit placental size and efficiency. In addition, we found that females inheriting a mutant Grb10 allele from their mother had larger litters and smaller offspring than those inheriting a mutant allele from their father. This grandparental effect suggests Grb10 can influence reproductive strategy through the allocation of maternal resources such that offspring number is offset against size.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting Bismuth payload.

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