Lowenna J. Holt

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life.

ABSTRACT 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 (Grb10Δ2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Δ2-4 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 Grb10Δ2-4 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.

Imprinted genes that regulate early mammalian growth are coexpressed in somatic stem cells

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.

Dual Ablation of Grb10 and Grb14 in Mice Reveals Their Combined Role in Regulation of Insulin Signaling and Glucose Homeostasis

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.

Grb10 regulates the development of fiber number in skeletal muscle.

Grb10 is an intracellular adaptor protein that acts as a negative regulator of insulin and insulinlike 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<0.01). There is no change in myofiber type proportions between WT and Grb10‐deficient muscles, nor are the metabolic properties of the muscles altered on Grb10 deletion. Notably, the weight and cross‐sectional area of hindlimbs from neonatal mice are increased in Grb10‐deficient animals (198 and 137% of WT, respectively, both P<0.001). Functional gene signatures for myogenic signaling and proliferation are up‐regulated in Grb10‐deficient neonatal muscle. Our findings indicate that Grb10 plays a previously unrecognized role in regulating the development of fiber number during murine embryonic growth. In addition, Grb10‐ablated muscle from adult mice shows coordinate gene changes that oppose those of muscle wasting pathologies, highlighting Grb10 as a potential therapeutic target for these conditions.— Holt, L. J., Turner, N., Mokbel, N., Trefely, S., Kanzleiter, T., Kaplan, W., Ormandy, C. J., Daly, R. J., Cooney, G. J. Grb10 regulates the development of fiber number in skeletal muscle. FASEB J. 26, 3658–3669 (2012). www.fasebj.org

Molecular Determinants of Grb14-Mediated Inhibition of Insulin Signaling

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 alphaC-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.

Adapter protein connections: The MRL and Grb7 protein families

The growth factor receptor bound (Grb)7 family of src homology (SH)2 domain-containing adapter proteins contains three members: Grb7, 10 and 14 (Daly 1998, Han et al. 2001). An observation that has intrigued researchers in the field for a decade is the close similarity between the central regions of Grb7 and a Caenorhabditis elegans cell migration protein, Mig10 (Stein et al. 1994, Manser et al. 1997). This conserved region encompasses a Ras-association (RA) and a pleckstrin homology (PH) domain. The recent characterization of two mammalian orthologues of Mig10, Rap1-GTPinteracting adapter molecule (RIAM)/proline-rich EVH1 ligand (PREL)1 and Lpd (Lamellipodin) now sheds light on the relationship between the Grb7 family and Mig10 (Lafuente et al. 2004, Krause et al. 2004, Jenzora et al. 2005). Sequence and domain structure comparisons reveal that two related protein families have arisen during evolution: the Grb7 family, and the newly-termed Mig10/RIAM/Lpd (MRL) family (Lafuente et al. 2004) (Figure 1A). Since Grb7 family orthologues are absent from C. elegans, it is likely that the corresponding gene families evolved from a common ancestral gene encoding a Mig10-like protein. In this review, we describe how the addition of different protein modules or motifs to a common core domain structure has given rise to two families of adapter proteins with distinct molecular connectivities and signalling functions (Figure 1B).

Growth factor receptor-bound protein 14 undergoes light-dependent intracellular translocation in rod photoreceptors: functional role in retinal insulin receptor activation.

Growth factor receptor-bound protein 14 (Grb14) is involved in growth factor receptor tyrosine kinase signaling. Here we report that light causes a major redistribution of Grb14 among the individual subcellular compartments of the retinal rod photoreceptor. Grb14 is localized predominantly to the inner segment, nuclear layer, and synapse in dark-adapted rods, whereas in the light-adapted rods, Grb14 redistributed throughout the entire cell, including the outer segment. The translocation of Grb14 requires photoactivation of rhodopsin, but not signaling through the phototransduction cascade, and is not based on direct Grb14-rhodopsin interactions. We previously hypothesized that Grb14 protects light-dependent insulin receptor (IR) activation in rod photoreceptors against dephosphorylation by protein tyrosine phosphatase 1B. Consistent with this hypothesis, we failed to observe light-dependent IR activation in Grb14(-/-) mouse retinas. Our studies suggest that Grb14 translocates to photoreceptor outer segments after photobleaching of rhodopsin and protects IR phosphorylation in rod photoreceptor cells. These results demonstrate that Grb14 can undergo subcellular redistribution upon illumination and suggest that rhodopsin photoexcitation may trigger signaling events alternative to the classical transducin activation.

Grb10 deletion enhances muscle cell proliferation, differentiation and GLUT4 plasma membrane translocation

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. J. Cell. Physiol. 229: 1753–1764, 2014.

Ablation of Grb10 Specifically in Muscle Impacts Muscle Size and Glucose Metabolism in Mice

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.

O51 Analyse moléculaire du mode d’action de Grb14 sur la signalisation de l’insuline

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.