Regulation of the number of insulin receptors on the cell surface plays a critical role in determining insulin sensitivity. In order to study the mechanism of transcriptional regulation of the insulin receptor gene, we have cloned the 5'-end of the human and mouse insulin receptor genes. Recently, we have characterized trans-acting factors that bind to a 26 base pair enhancer located 503-528 base pairs upstream from the translation start site. This enhancer accounts for a two- to threefold increase in the level of gene expression. Using electrophoretic mobility shift analysis, we have demonstrated at least two proteins that bind to overlapping sequences in the enhancer. Although the enhancer sequence does not contain a perfect match to the consensus Sp1 binding motif, there appears to be a relatively low affinity binding site for Sp1. In addition, a second protein binds to the enhancer sequence. Although the identity of this second DNA binding protein has not been determined, it appears to be a novel factor. While the insulin receptor mediates the first step in insulin action, other proteins function downstream in the insulin action pathway. We have initiated studies into the regulation of the gene encoding pp120/HA4, a membrane glycoprotein in rat liver that undergoes insulin-stimulated tyrosine phosphorylation. Consistent with the tissue-specific expression in liver, the pp120/HA4 gene has a TATA box containing promoter. In addition, the 5'-flanking region of the gene contains putative binding sites for several factors including a glucocorticoid response element and an insulin response element. We have initiated studies to investigate the regulation of expression vectors in which the promoter plus 5'-flanking region of the pp120/HA4 gene drive the expression of the luciferase gene. Using this approach, we have begun to map cis-acting elements responsible for the ability of insulin and dexamethasone to increase gene expression.
