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

FRANCINI F; BALTRUSCH S; TIEDGE M; LENZEN S

Hanover

Congreso; III Workshop of the Islet Research European Network (IREN): Bioengineering of the Ideal Surrogate Cell for Diabetes Insulin-Replacement Therapy; 2001

Background and Aims: The low-affinity glucose phosphorylating enzyme 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 a regulatory protein. The glucokinase regulatory protein (GRP) 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 was the aim of this study to characterize the molecular mechanisms of interaction between GK and GRP. Materials and Methods: Site-directed mutagenesis of selected amino acids in the GK and the GRP protein was performed by the Altered Sites II in vitro Mutagenesis System. Protein interactions of wildtype and mutant proteins were characterized by the MATCHMAKER GAL4 Two-Hybrid System 2 and verified by growth selection and quantitative chemiluminescence ß-galactosidase assays. Results: The interaction between GK and the GRP resulted in a tenfold increase of ß-galactosidase activity above the background level. Replacement of  the mostly aliphatic amino acids inside the binding motif of the GRP (181-188) by acid or short chain ones revealed that the amino acids Ser-182, Val-186, Ala187 significantly reduced the interaction with GK to the background level. The asparagine-leucine consensus motif for GRP interaction could be localized in three areas of the GK protein in the C-terminal lobe as well as in the substrate-binding site. Only the double mutation of the amino acids Leu-58/Asp-204 and Leu-355/Asp-350 inside the GK protein showed a significant decrease in ß-galactosidase activity in comparison to the wildtype protein. Conclusion: Mutation of the aliphatic residues Ser-182, Val-186 and Ala-187 of the GRP significantly reduced the interaction with GK. Thus this amino acid motif is crucial for the GK binding of the GRP. Importantly the complementary GK binding sequence is part of a proposed nuclear export signal in the GK protein which would be masked through GK-GRP interaction. These molecular aspects 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.