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Grb10 is an adaptor-type signaling protein most highly expressed in tissues involved in insulin action and glucose metabolism, such as muscle, pancreas, and adipose. Germline deletion of Grb10 in mice creates a phenotype with larger muscles and improved glucose homeostasis. However, it has not been determined whether Grb10 ablation specifically in muscle is sufficient to induce hypermuscularity or affect whole body glucose metabolism. In this study we generated muscle-specific Grb10-deficient mice (Grb10-mKO) by crossing Grb10flox/flox mice with mice expressing Cre recombinase under control of the human α-skeletal actin promoter. One-year-old Grb10-mKO mice had enlarged muscles, with greater cross-sectional area of fibers compared with wild-type (WT) mice. This degree of hypermuscularity did not affect whole body glucose homeostasis under basal conditions. However, hyperinsulinemic/euglycemic clamp studies revealed that Grb10-mKO mice had greater glucose uptake into muscles compared with WT mice. Insulin signaling was increased at the level of phospho-Akt in muscle of Grb10-mKO mice compared with WT mice, consistent with a role of Grb10 as a modulator of proximal insulin receptor signaling. We conclude that ablation of Grb10 in muscle is sufficient to affect muscle size and metabolism, supporting an important role for this protein in growth and metabolic pathways.

Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes.

Phosphoinositide 3-kinase kinase (PI3K) is at the heart of one of the major signal transduction pathways (1–4). The signals mediated by this enzyme influence a wide variety of cellular functions, including cell growth, differentiation and survival, glucose metabolism and cytoskeletal organization. The PI3K is expressed in photoreceptor cells and is regulated through the light-induced tyrosine phosphorylation of the insulin receptor (IR) in vivo (5;6). We have reported that light-induced tyrosine phosphorylation of IR requires the photoactivation of rhodopsin, but not transducin signaling (7). We also found that photoreceptor-specific deletion of IR resulted in stress-induced photoreceptor degeneration, suggesting the importance of IR in the survival of photoreceptor neurons (8). The molecular mechanism behind the light-induced activation of retinal IR is not known. Retinal IR has a high basal level of autophosphorylation compared to liver IR (9) and retinal IR autophosphorylation is light-dependent (5). These observations led us to hypothesize that retinal IR phosphorylation could be modulated by soluble factor(s) in the retina. To identify the regulators of IR, yeast two-hybrid screening of a bovine retinal cDNA library with the cytoplasmic domain of retinal IR (10) identified growth factor receptor-bound protein 14 (Grb14) (11;12), which binds to various tyrosine kinase receptors including IR (13–16). The crystal structure of the tyrosine kinase domain in complex with the IR-interacting domain of Grb14 has been resolved and revealed that Grb14 acts as a pseudo-substrate inhibitor that binds in the peptide binding groove of the kinase, and thus functions as a selective inhibitor of insulin signaling (17). In vitro experiments have shown that Grb14 impairs the tyrosine kinase activity of the IR towards exogenous substrates and protects the tyrosine-phosphorylation from dephosphorylation by protein tyrosine phosphatase-1B (PTP1B) (18). In liver, Grb14 deletion resulted in decreased IR phosphorylation due to increased dephosphorylation of the IR by PTP1B (19). The precise functional role of Grb14 in the retina is not known. Here we report that Grb14 undergoes light-dependent intracellular redistribution upon illumination of rod photoreceptor cells. In the dark, Grb14 was found to occupy all subcellular compartments of the rod, except for the outer segment. Following thirty minutes of light exposure, Grb14 was found evenly distributed throughout the entire rod cell, including the outer segment. This process is triggered by the photoexcitation of rhodopsin but is not mediated by transducin signaling. Ablation of Grb14 in the retina resulted in the loss of light-dependent activation of retinal IR and our studies suggest Grb14 translocates to photoreceptor outer segments following photobleaching of rhodopsin and protects the IR phosphorylation in rod photoreceptors cells. These findings demonstrate that Grb14 can undergo subcellular redistribution upon illumination and suggest that rhodopsin may have additional previously uncharacterized signaling functions in photoreceptors.

Extracellular ATP suppressed the growth of HL-60 leukemia cells and induced their differentiation as revealed by N-formyl-methionyl-leucyl-phenylalanine-induced beta-glucuronidase release. ATP degraded to ADP, AMP, and adenosine, and the effect of ATP on cell growth was mimicked by these metabolites added to the cultures. The stable analog alpha,beta-methylene ATP, however, had only a weak inhibitory effect on cell growth. Adenine nucleotide-induced growth suppression was reversed by uridine, suggesting the involvement of intracellular pyrimidine starvation secondary to adenosine accumulation. Consistent with this, ATP induced intracellular starvation of pyrimidine nucleotides, and this effect was also prevented by pretreatment of cells with uridine. The order of effectiveness of ATP-induced differentiation of HL-60 cells, unlike that for growth suppression, was ATP > ADP > AMP, and adenosine had no effect. Furthermore, uridine had no effect and the stable analog, alpha,beta-methylene ATP also induced HL-60 cell differentiation, suggesting that differentiation was due to ATP per se. We tested the hypothesis that ATP-induced differentiation arises from activation of adenylyl cyclase by the novel P2Y(11) receptor using the cell-permeable inhibitor of protein kinase A, Rp-CPT-cAMPS (8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphorothioate, Rp isomer). Rp-CPT-cAMPS (1-100 microM) prevented ATP-induced differentiation of HL-60 cells as assessed by fMLP-induced beta-glucuronidase release. However, Rp-CPT-cAMPS did not prevent ATP-induced growth suppression. Taken together, the data indicate that extracellular ATP suppresses HL-60 growth and induces their differentiation by distinct mechanisms. Growth suppression arises from adenosine generation and consequent pyrimidine starvation. Differentiation arises, at least in part, from a distinct mechanism involving the activation of cell surface P2 receptors coupled to cAMP generation and activation of protein kinase A.

The adsorption of BSA and RNA onto hydrophilic and thermosensitive poly(N-isopropyl-acrylamide) (NIPAM) latex particles was described as a function of pH, ionic strength and temperature. The hydrogel poly(NIPAM) latex was synthesized by precipitation polymerization in the presence of a cationic amino-containing monomer. The latex obtained was characterized in terms of particle size, and electrophoretic mobility as a function of pertinent variables: pH, temperature and ionic strength. The adsorption of BSA onto the latex was investigated to identify the conditions at which the adsorbed amount of BSA was negligible. The adsorption of RNA was studied to establish the conditions which give rise to maximal adsorption of RNA. In order to favor the desorption of RNA, desorption was investigated by changing the pH, ionic strength, and temperature. The adsorption of BSA was found to be lower at 20 than at 40 degrees C. However, the adsorption of RNA is drastically affected by the pH and the ionic strength of the medium. Maximal adsorbed amounts were obtained at acidic pH, 20 degrees C, and low ionic strength. The adsorption is shown to decrease when the pH, temperature and ionic strength increase, implying that the adsorption was mainly governed by electrostatic interactions. Maximal release of RNA molecules was obtained at high ionic strength and basic pH.

Grb10 is an intracellular adaptor protein which binds directly to several growth factor receptors, including those for insulin and insulin-like growth factor receptor-1 (IGF-1), and negatively regulates their actions. Grb10-ablated (Grb10(-/-) ) mice exhibit improved whole body glucose homeostasis and an increase in muscle mass associated specifically with an increase in myofiber number. This suggests that Grb10 may act as a negative regulator of myogenesis. In this study, we investigated in vitro, the molecular mechanisms underlying the increase in muscle mass and the improved glucose metabolism. Primary muscle cells isolated from Grb10(-/-) mice exhibited increased rates of proliferation and differentiation compared to primary cells isolated from wild-type mice. The improved proliferation capacity was associated with an enhanced phosphorylation of Akt and ERK in the basal state and changes in the expression of key cell cycle progression markers involved in regulating transition of cells from the G1 to S phase (e.g., retinoblastoma (Rb) and p21). The absence of Grb10 also promoted a faster transition to a myogenin positive, differentiated state. Glucose uptake was higher in Grb10(-/-) primary myotubes in the basal state and was associated with enhanced insulin signaling and an increase in GLUT4 translocation to the plasma membrane. These data demonstrate an important role for Grb10 as a link between muscle growth and metabolism with therapeutic implications for diseases, such as muscle wasting and type 2 diabetes.

Grb10 is an intracellular adaptor protein that acts as a negative regulator of insulin and insulin-like growth factor 1 (IGF1) receptors. Since global deletion of Grb10 in mice causes hypermuscularity, we have characterized the skeletal muscle physiology underlying this phenotype. Compared to wild-type (WT) controls, adult mice deficient in Grb10 have elevated body mass and muscle mass throughout adulthood, up to 12 mo of age. The muscle enlargement is not due to increased myofiber size, but rather an increase in myofiber number (142% of WT, P

Lifelong, many somatic tissues are replenished by specialized adult stem cells. These stem cells are generally rare, infrequently dividing, occupy a unique niche, and can rapidly respond to injury to maintain a steady tissue size. Despite these commonalities, few shared regulatory mechanisms have been identified. Here, we scrutinized data comparing genes expressed in murine long-term hematopoietic stem cells with their differentiated counterparts and observed that a disproportionate number were members of the developmentally-important, monoallelically expressed imprinted genes. Studying a subset, which are members of a purported imprinted gene network (IGN), we found their expression in HSCs rapidly altered upon hematopoietic perturbations. These imprinted genes were also predominantly expressed in stem/progenitor cells of the adult epidermis and skeletal muscle in mice, relative to their differentiated counterparts. The parallel down-regulation of these genes postnatally in response to proliferation and differentiation suggests that the IGN could play a mechanistic role in both cell growth and tissue homeostasis.

Grb14 belongs to the Grb7 family of molecular adapters and was identified as an inhibitor of insulin signaling. Grb14 binds to activated insulin receptors (IR) and inhibits their catalytic activity. To gain more insight into the Grb14 molecular mechanism of action, we generated various mutants and studied the Grb14-IR interaction using coimmunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments. Biological activity was further analyzed using the Xenopus oocyte model and a functional complementation assay measuring cellular proliferation rate in Grb14 knockout mouse embryonic fibroblasts. These studies identified two important interaction sites, Grb14 L404-IR L1038 and Grb14 R385-IR K1168, involving the IR αC-helix and activation loop, respectively. Interestingly, the former involves residues that are likely to be crucial for the specificity of IR binding with regard to other members of the Grb7 family. In addition, mutation of the Grb14-S370 residue suggested that its phosphorylation status controlled the biological activity of the protein. We further demonstrated that insulin-induced Grb14-PDK1 interaction is required in addition to Grb14-IR binding to mediate maximal inhibition of insulin signaling. This study provides important insights into the molecular determinants of Grb14 action by demonstrating that Grb14 regulates insulin action at two levels, through IR binding and by interfering with downstream pathways. Indeed, a precise knowledge of the molecular mechanism of insulin signaling inhibition by Grb14 is a prerequisite for the development of insulin-sensitizing molecules to treat pathophysiological states such as obesity or type 2 diabetes.

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.

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 (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 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 Grb10Delta2-4 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.

The Grb proteins (growth factor receptor-bound proteins) Grb7, Grb10 and Grb14 constitute a family of structurally related multidomain adapters with diverse cellular functions. Grb10 and Grb14, in particular, have been implicated in the regulation of insulin receptor signalling, whereas Grb7 appears predominantly to be involved in focal adhesion kinase-mediated cell migration. However, at least in vitro, these adapters can bind to a variety of growth factor receptors. The highest identity within the Grb7/10/14 family occurs in the C-terminal SH2 (Src homology 2) domain, which mediates binding to activated receptors. A second well-conserved binding domain, BPS [between the PH (pleckstrin homology) and SH2 domains], can act to enhance binding to the IR (insulin receptor). Consistent with a putative adapter function, some non-receptor-binding partners, including protein kinases, have also been identified. Grb10 and Grb14 are widely, but not uniformly, expressed in mammalian tissues, and there are various isoforms of Grb10. Binding of Grb10 or Grb14 to autophosphorylated IR in vitro inhibits tyrosine kinase activity towards other substrates, but studies on cultured cell lines have been conflicting as to whether Grb10 plays a positive or negative role in insulin signalling. Recent gene knockouts in mice have established that Grb10 and Grb14 act as inhibitors of intracellular signalling pathways regulating growth and metabolism, although the phenotypes of the two knockouts are distinct. Ablation of Grb14 enhances insulin action in liver and skeletal muscle and improves whole-body tolerance, with little effect on embryonic growth. Ablation of Grb10 results in disproportionate overgrowth of the embryo and placenta involving unidentified pathways, and also impacts on hepatic glycogen synthesis, and probably on glucose homoeostasis. This review discusses the extent to which previous studies in vitro can account for the observed phenotype of knockout animals, and considers evidence that aberrant function of Grb10 or Grb14 may contribute to disorders of growth and metabolism in humans.

Introduction La proteine Grb14, qui appartient a la famille Grb7 d’adaptateurs moleculaires, a ete identifiee comme etant un inhibiteur specifique de la signalisation de l’insuline. En reponse a l’insuline, elle se fixe sur le recepteur de l’insuline (IR) active et inhibe son activite catalytique. Une analyse precise du mecanisme moleculaire de l’action de Grb14 sur la signalisation de l’insuline est necessaire pour permettre le developpement de molecules qui en ciblant Grb14 permettraient d’augmenter la transmission du signal de l’insuline et d’ameliorer les etats de resistance a l’insuline associes a l’obesite et au diabete de type 2. Materiels et methodes Afin de mieux comprendre le mecanisme moleculaire de l’action de Grb14 nous avons cree differents mutants ponctuels de Grb14 et du IR, et analyse leur interaction par des d’experiences de co-immunoprecipitation et de Bret. L’activite biologique de ces mutants a ete etudiee dans le modele des ovocytes de Xenope, ainsi que dans des experiences de complementation fonctionnelle mesurant la proliferation cellulaire de fibroblastes d’embryons de souris KO pour Grb14 transfectes avec les differents mutants de la proteine. Resultats Nous avons ainsi identifie 2 sites d’interaction, l’un dans l’helice αC du recepteur et impliquant les residus L1038 du IR et L404 de Grb14, et l’autre dans la boucle d’activation du recepteur et impliquant les residus K1168 du IR et R385 de Grb14. De facon interessante, les residus responsables du premier site d’interaction ne sont pas conserves dans les proteines homologues, recepteur de l’IGF-1 et proteines Grb7/10, suggerant ainsi que ce pourrait etre une des cles de la specificite de l’effet inhibiteur de Grb14 sur le IR. Nous avons egalement analyse les consequences de la mutation du residu S370 de Grb14 et montre que sa phosphorylation controle l’interaction avec le IR ainsi que l’effet biologique de l’insuline. Grb14 interagit aussi avec la proteine PDK1, kinase majeure de la signalisation de l’insuline activant Akt. Nous avons montre que l’insuline stimule l’interaction Grb14-PDK1, et que cette interaction est importante pour l’effet inhibiteur de Grb14 sur la signalisation de l’insuline. Conclusion Cette etude a permis de demontrer que Grb14 inhibe l’action de l’insuline en agissant a 2 niveaux, par son interaction avec IR mais aussi en interferant avec des voies de signalisation en aval comme le recrutement de PDK1.

Introduction Grb14 est un inhibiteur de la signalisation insulinique qui se fixe sur le recepteur de l’insuline et inhibe son activite tyrosine kinase. L’etude de Grb14 chez des patients diabetiques de type 2, ainsi que dans des modeles animaux d’insulinoresistance, a permis de mettre en evidence une correlation inverse entre le niveau d’expression de Grb14 dans le tissu adipeux et la sensibilite a l’insuline. Pour elucider le role de Grb14 dans ce tissu, nous avons etudie les consequences de modifications de l’expression de Grb14 sur le metabolisme et la differenciation adipocytaires. Materiels et methodes Nous avons genere une lignee d’adipocytes 3T3-L1 surexprimant Grb14 de facon inductible, et nous avons etudie parallelement des fibroblastes embryonnaires issus de souris Grb14-/- (MEF KO). La differenciation adipocytaire, la signalisation et les effets metaboliques de l’insuline ainsi que la production et la secretion d’adipokines ont ete etudiees dans ces deux lignees cellulaires. Resultats La differenciation adipocytaire ne semble pas alteree par la surexpression de Grb14. Par contre, les MEF KOpour Grb14 montrent a la fois une augmentation de la proliferation cellulaire et une amelioration de la differenciation adipocytaire. La surexpression de Grb14 induit une inhibition de la stimulation par l’insuline des effecteurs Akt et ERK1/2, bien que cela ne se traduise pas par une modification significative de l’effet de l’insuline sur le transport de glucose. Au niveau endocrine, on observe une diminution de 50 % de la secretion de leptine dans les adipocytes qui surexpriment Grb14, alors que la production et la secretion d’adiponectine ne semblent pas alterees. Conclusion Ces experiences suggerent qu’au-dela de son role inhibiteur de la signalisation insulinique Grb14 semble egalement agir sur d’autres fonctions cellulaires majeures : la proliferation cellulaire et la differenciation adipocytaire.