Human growth factor receptor bound 14 binds the activated insulin receptor and alters the insulin-stimulated tyrosine phosphorylation levels of multiple proteins.
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.
Department of Biochemistry & Medical Genetics, University of Manitoba, Winnipeg, Canada.
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R
Hemming
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R
Agatep
K
K
Badiani
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K
Wyant
G
G
Arthur
R D
RD
Gietz
B
B
Triggs-Raine
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; C099655:GRB14 protein, human; D007328:Insulin; D011506:Proteins; D011972:Receptor, Insulin