Subject(s)
Genetic Predisposition to Disease/genetics , Melanoma/genetics , Skin Neoplasms/genetics , Adult , Aged , Aged, 80 and over , Amino Acid Substitution/genetics , Cadherins/genetics , Cell Cycle Proteins/genetics , Chromosomes, Human, Pair 1/genetics , Chromosomes, Human, Pair 9/genetics , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p16/chemistry , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinases/genetics , Female , Genes, BRCA2 , Genes, Neoplasm/genetics , Genetic Linkage/genetics , Humans , Italy , Male , Melanoma/diagnosis , Melanoma/enzymology , Middle Aged , Models, Molecular , Pedigree , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
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.