Compartmentalization and in vivo insulin‐induced translocation of the insulin‐signaling inhibitor Grb14 in rat liver

Abstract

Grb14 is a member of the Grb7 ⁄ Grb10 ⁄ Grb14 family of adaptor proteins, which lack intrinsic enzymatic activity and share a common multidomain structure.These adaptors bind to several receptor tyrosine kinases and signaling proteins, and are involved in the regulation of various processes, including cell growth and The molecular adaptor Grb14 binds in vitro to the activated insulin receptor (IR) and inhibits IR signaling. In this study, we have used rat liver subcellular fractionation to analyze in vivo insulin effects on Grb14 compartmentalization and IR phosphorylation and activity. In control rats, Grb14 was recovered mainly in microsomal and cytosolic fractions, but was also detectable at low levels in plasma membrane and Golgi ⁄ endosome fractions. Insulin injection led to a rapid and dose-dependent increase in Grb14 content, first in the plasma membrane fraction, and then in the Golgi ⁄ endosome fraction, which paralleled the increase in IR b-subunit tyrosine phosphorylation. Upon sustained in vivo IR tyrosine phosphorylation induced by high-affinity insulin analogs, in vitro IR dephosphorylation by endogenous phosphatases, and in vivo phosphorylation of the IR induced by injection of bisperoxo(1,10 phenanthroline)oxovanadate, a phosphotyrosine phosphatase inhibitor, we observed a striking correlation between IR phosphorylation state and Grb14 content in both the plasma membrane and Golgi ⁄ endosome fractions. In addition, coimmunoprecipitation experiments provided evidence that Grb14 was associated with phosphorylated IR b-subunit in these fractions. Altogether, these data support a model whereby insulin stimulates the recruitment of endogenous Grb14 to the activated IR at the plasma membrane, and induces internalization of the Grb14-IR complex in endosomes. Removal of Grb14 from fractions of insulin-treated rats by KCl treatment led to an increase of in vivo insulin-stimulated IR tyrosine kinase activity, indicating that endogenous Grb14 exerts a negative feedback control on IR catalytic activity. This study thus demonstrates that Grb14 is a physiological regulator of liver insulin signaling.Abbreviations BPS, between plekstrin homology and SH2; bpV(phen), bisperoxo(1,10-phenanthroline) oxovanadate; EGF, epidermal growth factor; ER, endoplasmic reticulum; GST, glutathione S-transferase; IR, insulin receptor; IRS-1, insulin receptor substrate-1; IRb, insulin receptor b-subunit; PDK-1, 3-phosphoinositide-dependent kinase-1; PI3-kinase, phosphoinositide-3-kinase; PIR, phosphorylated insulin receptorinteracting region; WGA, wheat germ agglutinin.

Endosomal proteolysis of internalised [ArgA0]-human insulin at neutral pH generates the mature insulin peptide in rat liver in vivo

“…Moreover, an increase in IR transit through endosomes was observed with [Arg A0 ]-HI, which may originate from: (1) the conversion of [Arg A0 ]-HI into HI, which may delay the dissociation of insulin peptides from the endosomal IR and its subsequent recycling; (2) a reduced dissociation of [Arg A0 ]-HI-receptor complex at the fluctuating endosomal pH compared with HI-receptor complex; and (3) a serum half-life of [Arg A0 ]-HI longer than that of HI. Interestingly, as previously reported for the protease-resistant H2-analogue [15,24], [Arg A0 ]-HI induced an increase in the kinetics of MAP kinase activation and a more sustained association of GRB14 with the hepatic endosomes.…”

“…Administration of HI [24] or arginyl-insulins did not cause detectable changes in the levels of IRS-1 or SHC isoforms at the endosomal locus. In contrast, the three ligands led to a rapid increase in the content of the molecular adaptor GRB14 within EN fractions [15]. Endosomal association of GRB14 was more sustained after the administration of [Arg A0 ]-HI, which paralleled its effect on phosphorylated IR-β content.…”

“…Polyclonal IgG against human phospho-p44/42 mitogen-activated protein (MAP) kinase and rat p44/42 MAP kinase proteins were from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibody against growth factor receptor-bound protein 14 (GRB14) has been previously described [15]. Rabbit antimouse cathepsin D R291 [16,17], sheep anti-human cathepsin D M8147 [16,17] and rabbit anti-rat cathepsin B 7183 [18] were obtained from J. S. Mort (Shriners Hospital for Crippled Children, Montreal, Canada).…”

Modulation de la sensibilité à l’insuline par les adapteurs moléculaires de la famille de Grb7

“…Une injection d'insuline induit en 30 secondes la fixation de Grb14 sur le récepteur dans le foie de rats, et l'interaction se poursuit tant que le récepteur est activé et phosphorylé. En effet, une analyse par fractionnement cellulaire de ces foies a démontré que Grb14 reste associée au récepteur de l'insuline depuis son activation à la membrane plasmique jusqu'aux endosomes où le récepteur est finalement séparé de l'insuline et retourne à sa conformation inactive [40]. Il est bien établi que le récepteur de l'insuline est rapidement internalisé dans les endosomes après l'activation par la liaison de son ligand, et que les récepteurs présents dans les endosomes sont actifs en termes de transduction du signal [41,42].…”

Novel Grb14-Mediated Cross Talk between Insulin and p62/Nrf2 Pathways Regulates Liver Lipogenesis and Selective Insulin Resistance

“…The Grb14 expression level is thus inversely correlated with insulin sensitivity in human and animal models of insulin resistance. We previously reported that Grb14 is recruited to the activated insulin receptor and inhibits its catalytic activity and downstream insulin signaling (5,11,12). Furthermore, Grb14 expression is stimulated by insulin, suggesting that it might be involved in a negative feedback loop of insulin signaling and action (9).…”