Citation

  • Authors: Baltrusch, S., Francini, F., Lenzen, S., Tiedge, M.
  • Year: 2005
  • Journal: Diabetes 54 2829-37
  • Applications: in vitro / DNA / jetPEI, jetPEI-Hepatocyte
  • Cell types:
    1. Name: COS-1
      Description: African green monkey kidney cells
      Known as: COS, COS1
    2. Name: Rat primary hepatocytes
      Description: Primary rat hepatocytes

Abstract

The glucokinase regulatory protein (GRP) plays a pivotal role in the regulation of metabolic flux in liver by the glucose-phosphorylating enzyme glucokinase. Random peptide phage display library screening for binding partners of GRP allowed the identification of an asparagine-leucine consensus motif. Asparagine-leucine motifs of glucokinase located in the hinge region, as well as in the large domain, were changed by site-directed mutagenesis. The L58R/N204Y and the L309R/N313Y glucokinase mutants showed a significantly reduced interaction with GRP. The L355R/N350Y mutant had a fivefold-higher binding affinity for GRP than wild-type glucokinase. Imaging of glucokinase and GRP fluorescence fusion proteins revealed that the L58R/N204Y glucokinase mutant lacked glucose-dependent translocation by GRP, whereas the L355R/N350Y glucokinase mutant was trapped in the nucleus due to high affinity for GRP. The results indicate that the L58/N204 motif in the hinge region confers binding to GRP, while the L355/N350 motif may modulate the binding affinity for GRP. This latter motif is part of the alpha10 helix of glucokinase and accessible to GRP in the free and complex conformation.

Pubmed