Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins

Abstract

To identify proteins interacting in the insulin-signaling pathway that might define new pathways or regulate existing ones, we have employed the yeast two-hybrid system. In a two-hybrid screen of a human liver cDNA library, we identified the human growth factor receptor bound 14 (hGrb14) adaptor protein as a partner of the activated insulin receptor. Additional analysis of the insulin receptor--hGrb14 interaction in the yeast two-hybrid system revealed that the SH2 domain of hGrb14 was not the sole region involved in binding the activated insulin receptor. The insulin-stimulated interaction between hGrb14 and the insulin receptor was also observed in different mammalian cultured cell lines. This association was detected at 1 min of insulin stimulation and was maximal at 10 nM and greater concentrations of insulin. Chinese hamster ovary cells stably expressing the insulin receptor (CHO-IR) and hGrb14 were used to examine the effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation of proteins; in general, increasing levels of hGrb14 expression resulted in a reduction in tyrosine phosphorylation. This decrease was demonstrated for the specific proteins src homology-containing and collagen-related protein (Shc), insulin receptor substrate-1 (IRS-1), and Downstream of tyrosine Kinase (Dok). The broad effects of hGrb14 overexpression on insulin-stimulated tyrosine phosphorylation suggest that it acts early in the insulin-signaling pathway.

Regulation of insulin and type 1 insulin‐like growth factor signaling and action by the Grb10/14 and SH2B1/B2 adaptor proteins

“…This involves a non-competitive mechanism with an order of potency Grb14 > Grb10 > Grb7, and IR activity more potently inhibited than IGFR activity [97]. Consistent with these in vitro effects, over-expression of Grb10 [81,92,[103][104][105] and Grb14 [38,94,97] in cells inhibits insulin-and/or IGF-1-stimulated tyrosine phosphorylation of endogenous substrates (IRS1, IRS2, Shc and p62Dok). Conversely, depletion of endogenous Grb10 by small interfering RNA (siRNA) interference potentiates insulin/IGF-induced substrate phosphorylation [81,103,106,107].…”

“…First detected by yeast two-hybrid and glutathione S-transferase (GST) pull-down assays, interactions of Grb7 [87], Grb10 [27,36,[88][89][90][91][92][93] and Grb14 [12,38,94] with IR and of Grb10 with IGFR [36,91,93,95,96] have been demonstrated by co-immunoprecipitation and bioluminescence resonance energy transfer of over-expressed proteins in CHO, COS and HEK293 cells. Endogenous Grb14 also associates with ligandactivated IR in 3T3L1 adipocytes and rat liver [38,57] and with light-activated IR in rat retina [21].…”

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

“…On the basis of the crystal structure of the tyrosine kinase domain of the IR in complex with the PIR ⁄ BPS domain of Grb14, Depetris et al [8] have shown that Grb14 binds as a pseudosubstrate inhibitor to the peptide-binding groove of the kinase and thus acts as a selective inhibitor of insulin signaling. Consistent with this observation, overexpression of Grb14 in CHO-IR cells impairs Akt and ERK insulin signaling pathways and inhibits distal effects of insulin on glycogen and DNA synthesis [6][7][8][9], and microinjection of Grb14 into Xenopus laevis oocytes inhibits insulin-induced oocyte maturation [10]. Conversely, disruption of the Grb14 gene in mice ameliorates glucose tolerance in vivo and insulin signaling in both liver and skeletal muscle [11].…”

A Genome-Wide SNP Scan Reveals Novel Loci for Egg Production and Quality Traits in White Leghorn and Brown-Egg Dwarf Layers

“…In human and mammals, GRB14 mRNA was found to be expressed at high level in the ovary, liver, kidney, skeletal muscle and so on [25], [26]. It interacts with insulin receptors (IR) and insulin-like growth-factor receptors (IGFR), and may play an inhibiting role in tyrosine kinase receptor (Tkr) signaling pathways [27], [28]. IGF and IGFR were reported to regulate ovarian functions and follicular developments in chickens [29], [30].…”