Diabetologia (2012) 55:114–122 DOI 10.1007/s00125-011-2348-5 ARTICLE Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk M. G. Rees & S. Wincovitch & J. Schultz & R. Waterstradt & N. L. Beer & S. Baltrusch & F. S. Collins & A. L. Gloyn Received: 26 August 2011 / Accepted: 28 September 2011 / Published online: 25 October 2011 # Springer-Verlag 2011 Abstract Aims/hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. Methods Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently trans- fected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency. M. G. Rees : N. L. Beer : A. L. Gloyn (*) Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LJ, UK e-mail: anna.gloyn@drl.ox.ac.uk M. G. Rees : S. Wincovitch : F. S. Collins National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA J. Schultz : R. Waterstradt : S. Baltrusch Institute for Medical Biochemistry & Molecular Biology, University of Rostock, Rostock, Germany Results P446L-GKRP had a decreased degree of nuclear localisation, ability to sequester GCK and direct interaction with GCK as measured by FRET compared with WT- GKRP. Decreased interaction was observed between WT- GKRP and GCK at high compared with low glucose, but not between P446L-GKRP and GCK. Rat WT-GKRP and P446L-GKRP behaved quite differently: both variants responded to high glucose by diminished sequestration of GCK but showed no effect of the P446L variant on nuclear localisation or GCK sequestration. Conclusions/interpretation Our study suggests the common human P446L-GKRP variant protein results in elevated hepatic glucose uptake and disposal by increasing active cytosolic GCK. This would increase hepatic lipid biosyn- thesis but decrease fasting plasma glucose concentrations and provides a potential mechanism for the protective effect of this allele on type 2 diabetes risk. Keywords Association study . Genetics . Glucokinase . Molecular mechanism Abbreviations GKRP Glucokinase regulatory protein hGKRP Human GKRP rGKRP Rat GKRP GCK Glucokinase ECFP Enhanced cyan fluorescent protein EGFP Enhanced green fluorescent protein EYFP Enhanced yellow fluorescent protein FRET Fluorescence resonance energy transfer FRETN Sensitised emission-based FRET efficiency F6P Fructose 6-phosphate F1P Fructose 1-phosphate WT Wild-type SNP Single nucleotide polymorphism