Despite extensive studies over many years, the molecular and biochemical mechanisms of insulin action remain to be elucidated. The first step in this mechanism is the binding of insulin to a specific receptor on the plasma membrane of target cells. Analysis of the structure and function of the insulin receptor (IR) is hampered by the fact that all cells tested have detectable levels of endogenous IRs. We propose to take a novel approach by using homologousrecombination, following microinjection of mutated genomic DNA fragments, to inactivate the endogenous IR genes. Specifically, both single and double allele mutants will be produced in HepG2 hepatoma, and 3T3-F442A adipocyte cell lines. The single allele mutants will be used for studies involving the regulation of individual alleles, the double allele mutants, which will be receptor null cells, will be used for transfection studies with mutant IRs generated by in vivo site-directed mutagenesis. Furthermore, we will make functional mutations by in vivo mutagenesis, in one allele only to study, the interaction between IRs in a heterozygote cell as a model for NIDDM.
Resnik, J L; Reichart, D B; Huey, K et al. (1998) Elevated insulin-like growth factor I receptor autophosphorylation and kinase activity in human breast cancer. Cancer Res 58:1159-64 |
Webster, N J; Kong, Y; Sharma, P et al. (1997) Differential effects of Wilms tumor WT1 splice variants on the insulin receptor promoter. Biochem Mol Med 62:139-50 |
Webster, N J; Resnik, J L; Reichart, D B et al. (1996) Repression of the insulin receptor promoter by the tumor suppressor gene product p53: a possible mechanism for receptor overexpression in breast cancer. Cancer Res 56:2781-8 |
Kosaki, A; Pillay, T S; Xu, L et al. (1995) The B isoform of the insulin receptor signals more efficiently than the A isoform in HepG2 cells. J Biol Chem 270:20816-23 |
Kosaki, A; Webster, N J (1993) Effect of dexamethasone on the alternative splicing of the insulin receptor mRNA and insulin action in HepG2 hepatoma cells. J Biol Chem 268:21990-6 |