Molecular characterization of the interaction between glucokinase and glucokinase regulatory protein.

FRANCINI F

Bonn

Congreso; Einführungstagug Alexander vos Humboldt Stiftung; 2001

Glucokinase (GK) couples changes in the millimolar glucose concentration range to glucose metabolism in pancreatic ß-cells and liver. In addition to transcriptional mechanisms hepatic GK activity is modulated on the posttranslational level through an interaction with the glucokinase regulatory protein (GRP). This protein binds and inhibits GK competitively with respect to glucose and the inhibition is enhanced by fructose-6-phosphate and suppressed by fructose-1-phosphate. Most recent studies also demonstrate, that the GRP is responsible for the intracellular movement of GK. Binding motifs in both proteins were recently discovered by Phage Display library screenings to be important for the interaction. It is the aim of this study to characterize the molecular mechanisms of interaction between GK and GRP. To cope with this aim site-directed mutagenesis of selected amino acids in the GK and the GRP protein, performed by the Altered Sites II in vitro Mutagenesis System, will be used. Protein interactions will be characterized by the MATCHMAKER GAL4 Two-Hybrid System 2 and verified by growth selection and quantitative chemiluminescence ß-galactosidase assays. Molecular characterization of GK-GRP binding will contribute to the understanding of the functional role of GK in intermediary metabolism with particular emphasis upon the mechanisms of nuclear translocation in liver and the so far unknown corresponding mechanisms in pancreatic ß-cells.