Abstract:
The adapter protein Grb14 (Growth Factor Receptor Bound 14) has been shown to directly interact with the cytoplasmic domain of the activated insulin receptor. To date, the function of Grb14 and the purpose of its association with the IR are unclear. To aid in the elucidation of Grb14's function, human Grb14 (hGrb14) interacting proteins were isolated from an adult human liver cDNA library using a modified yeast two-hybrid system. Three interacting proteins were identified: a novel protein named Gip1 (G_rb14 I_nteracting P_rotein-1), the gene product of 'KIAA0093' (a 'NEDD4' homologue), and Tax1bp1/Txbp151 (Tax1-binding protein-1/Tax1-binding protein 151). The hGrb14:Tax1bp1 interaction was studied. The Tax1bp1 primary sequence was found to encode an internal coiled-coil domain, a carboxyl-terminal LIM domain and a novel domain shared only with the Ndp52 protein. In the two-hybrid system, the amino acid residues 236 to 593 of the Tax1bp1 coiled-coil domain mediated its association with hGrb14. Furthermore, this portion of Tax1bp1 could also mediate self-association in the two-hybrid system. In a genetically modified CHO (Chinese hamster ovary) cell line constitutively overexpressing the human insulin receptor and human Grb14 and transiently overexpressing Tax1bp1, an interaction between Tax1bp1 and hGrb14 was observed. Structural characterization of the TAXBP1 gene was also performed. The 'TAX1BP1' gene, mapped to 7p14-7p15, contained 16 exons that spanned 70 KB. These results provide evidence that Tax1bp1 is a self-associating human Grb14 interacting protein.
