Compartmentalization and in vivo insulin-induced translocation of the insulin-signaling inhibitor Grb14 in rat liver.
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 beta-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 beta-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.
Desbuquois
Bernard
B
Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Paris, France. bernard.desbuquois@inserm.fr
Béréziat
Véronique
V
Authier
François
F
Girard
Jean
J
Burnol
Anne-Françoise
AF
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002451:Cell Compartmentation; D004305:Dose-Response Relationship, Drug; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D010766:Phosphorylation; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C490976:Grb14 protein, rat; D007328:Insulin; D011506:Proteins; D014443:Tyrosine; D011972:Receptor, Insulin