The role of Grb10 in insulin receptor signalling

• Main Author: Holt, L. J.
• Published: University of Cambridge 2003
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604199

To facilitate the study of Grb10 function, polyclonal antibodies were produced. They were directed against the SH2 domain of Grb10, which is common to all isoforms. The purified antibody was highly specific for Grb10 and able to detect both mouse and human forms of the protein. To examine the effects of Grb10 proteins on the insulin signalling pathway, they were expressed exogenously in CHO-T cells, in both a transient and stable manner. Comparison were made of the mouse and human isoforms of Grb10 at the genomic, RNA and protein levels. Differential expression of isoforms was observed in murine cells. Use of the information generated by the Human Genome Project allowed the genomic organisation of the human Grb10 isoforms to be determined. Bioinformatics tools were also employed to predict binding motifs and phosphorylation sites within Grb10. These elucidated similarities between the isoforms, and differences, in their potential regulation. In examining its effects on the insulin signalling system in CHO-T cells, hGrb10x was shown to associate with the activated insulin receptor in a rapid and sustained manner. Various isoforms of Grb10 bound equally well to the receptor. The global tyrosine phosphorylation state of the insulin receptor was not altered in the presence of hGrb10x.  However, insulin-stimulated activation of IRS-1 and ERK were inhibited. The tyrosine phosphorylation state of a broad array of proteins was also affected by the expression of hGrb10x in CHO-T cells. hGrb10x induced an apparent increase in total IRS-1 protein level. The studies presented in this thesis are consistent with hGrb10x acting as a negative regulator of the insulin signalling pathway in CHO-T cells.