Chromium is a micronutrient that potentiates the action of insulin and is an essential component of "glucose tolerance factor" discovered over 40 years ago. Chromium could have beneficial effects for the >15 million type-2 diabetics in the USA, and many Americans already take Cr (III) as a daily dietary supplement, alone or in multivitamin formulations. Nonetheless, there is scant information and continuing controversy regarding the biology of Cr (III), including uptake, bioavailability, target of action and even whether the metal ion itself or some organo-metallic complex is the bioactive species. The goal of this project is to define the biochemical basis for chromium enhancement of insulin action. In preliminary studies various organic and inorganic forms of Cr (III) were found to be equally efficacious in potentiating initial signaling events triggered by insulin. Chromium added at nanomolar concentrations to intact living cells in culture increased insulin-stimulated Tyr phosphorylation at sub-optimal doses of insulin. The effect was attributed to impaired insulin receptor (IR) dephosphorylation, assayed by addition of chromium to purified membranes. This project will extend these studies to test the hypothesis that Cr (III) inhibits the dephosphorylation of the activated IR by protein Tyr phosphatases (PTP). This could occur by interaction of the Cr (III) either with PTP, blocking the enzyme activity by interacting with an active site cysteine, or with the substrate, the Tyr phosphorylated IR, protecting it from dephosphorylation. Experiments will use dephosphorylation assays to identify the target protein (PTP or IR). Analysis of reaction products will show whether chromium action is selective for different Tyr phosphorylation sites in the IR, which would enhance specific downstream signaling pathways. Structural determinants for interaction with chromium will be determined by mutagenesis and Cr(III) binding assays with recombinant target protein. The results will provide new knowledge of the molecular actions of Cr (III), provide a basis for understanding the biological effects of dietary chromium and open new opportunities for interventions to combat type-2 diabetes.