University of Manitoba Researchers

Permanent URI for this community

http://hdl.handle.net/1993/2761

Browse

Browsing University of Manitoba Researchers by Subject "2-HYBRID SYSTEM"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Restricted
    Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins
    (2001-02-28) Hemming, R; Agatep, R; Badiani, K; Wyant, K; Arthur, G; Gietz, RD; Triggs-Raine, B
    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.