Identification of Fructose 6-Phosphate- and Fructose 1-Phosphate-binding Residues in the Regulatory Protein of Glucokinase

Veiga‐da‐Cunha, Maria; Schaftingen, Emile Van

doi:10.1074/jbc.m105984200

Cited by 59 publications

(74 citation statements)

References 51 publications

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“…We established a co-immunoprecipitation assay using recombinant proteins in which we can quantitatively measure the amount of GK bound to GKRP. A mixture of recombinant Histagged human hepatic GK and FLAG-tagged human GKRP in the ratio of 4:1 (16:4 g) yielded a nearly 1:1 complex in the presence of 5.5 mM glucose and 0.4 mM fructose 6-phosphate, which is known to enhance the association of GK and GKRP (17). Under this condition, compound A inhibited the interaction between GK and GKRP with an IC 50 value of 16.4 M (Fig.…”

Section: Effect Of Glucokinase Activator On Gk-gkrp Interaction In VImentioning

confidence: 99%

An Allosteric Activator of Glucokinase Impairs the Interaction of Glucokinase and Glucokinase Regulatory Protein and Regulates Glucose Metabolism

Futamura

Hosaka

Kadotani

et al. 2006

Journal of Biological Chemistry

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Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V max ) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRPassociated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.There are three key aspects of type 2 diabetes pathogenesis, which are the focus of current and future therapies: insulin resistance, defective insulin secretion, and increased hepatic glucose production. Glucokinase (GK) 2 is the predominant glucose phosphorylation enzyme in pancreatic ␤-cells and hepatocytes. GK plays an important role as a glucose sensor for controlling plasma glucose homeostasis by enhancing insulin secretion from pancreatic ␤-cells and glucose metabolism in the liver (1, 2), which provides rational expectations that enhancement of GK activity would be a novel therapeutic strategy for type 2 diabetes. Consistent with this rationale, recently discovered small molecule allosteric activators of GK have been demonstrated to have antidiabetic efficacy in rodents (3, 4).To investigate the mechanism of action of GK activators, the interaction between GK and glucokinase regulatory protein (GKRP) is a key aspect. It is well known that hepatic GK activity is modulated by the endogenous inhibitor, glucokinase regulatory protein (GKRP) (5-8). GK is localized in the nucleus as an inactive complex with GKRP at low glucose concentrations and is dissociated from the GK⅐GKRP complex and translocated to the cytoplasm at high glucose concentrations, which triggers glucose disposal (9). Modulators of the GK-GKRP interaction have been shown to enhance hepatic glucose disposal (7). We recently solved the co-crystal structure of hepatic GK complex with GK activator, in which GK undergoes a large conformational change between the active and inactive forms at diffe...

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Section: Effect Of Glucokinase Activator On Gk-gkrp Interaction In VImentioning

confidence: 99%

An Allosteric Activator of Glucokinase Impairs the Interaction of Glucokinase and Glucokinase Regulatory Protein and Regulates Glucose Metabolism

Futamura

Hosaka

Kadotani

et al. 2006

Journal of Biological Chemistry

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“…The proteins were expressed at 18°C in M9 salts medium and partially purified by polyethylene glycol precipitation and DEAE-Sepharose chromatography [33]. The purity of GKRP in the DEAESepharose pools was about 70% and its concentration was calculated by multiplying the total concentration of protein by the purity factor.…”

Section: Choice Of Obese Patients Included In the Primary Screeningmentioning

confidence: 99%

“…The purity of GKRP in the DEAESepharose pools was about 70% and its concentration was calculated by multiplying the total concentration of protein by the purity factor. GKRP was assayed based on its ability to inhibit recombinant human liver glucokinase in the presence of 5 mmol/l glucose and the indicated concentrations of fructose 6-phosphate or fructose 1-phosphate, using a pyruvate kinase/ lactate dehydrogenase coupled assay [33,35].…”

Section: Choice Of Obese Patients Included In the Primary Screeningmentioning

confidence: 99%

“…Except for the Arg227Stop mutant, the mutants of rat liver GKRP were prepared with a PCR-based technique using back-to-back primers [32], Pwo polymerase and the expression vector of rat liver GKRP as a template [33]. The expression plasmid for the Arg227Stop mutant was constructed by PCR amplification of a rat liver cDNA using one primer that contained the initiator ATG flanked at its 5′-end by a NdeI restriction site and a second one that replaced the Arg227 codon by a TGA terminator flanked at its 3′-end by a BamHI restriction site.…”

Section: Choice Of Obese Patients Included In the Primary Screeningmentioning

confidence: 99%

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Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians

et al. 2003

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Aims/Hypothesis. Glucokinase regulatory protein (GKRP) controls the activity of glucokinase in liver but possibly also in some areas of the central nervous system, suggesting that it could play a role in body mass control. Its gene is located in a region (2p21-23) linked to serum leptin levels. Our goal was to investigate whether mutations in the GKRP gene were associated with obesity. Methods. Mutations were sought in the GKRP gene of 57 patients from the families of the French genomewide scan for obesity that contributed most to the positive LOD score with 2p21-23. The identified mutations were further sought in 720 unrelated obese individuals and 384 individuals of normal weight and their effect on the properties of recombinant GKRP were investigated. Results. The most frequent mutation (Pro446Leu) had a similar allele frequency in the obese (0.63) and normal weight (0.64) subjects and did not affect the properties of GKRP. Similarly, no effect on the properties of GKRP was observed with Arg590Tyr, found in 10 out of 720 obese subjects and in 2 out of 384 control subjects (p=0.18). Mutation Arg227Stop was found in one obese family and in 1 out of 384 control subjects and led to an insoluble protein. Mutation Arg518Gln, replacing a conserved residue, led to a marked decrease in the affinity of GKRP for both fructose 6-phosphate and fructose 1-phosphate and to a destabilization of GKRP. However, this mutation did not co-segregate with obesity in the single family in which it was found. Conclusions/interpretation. Mutations that affect the properties of GKRP are found in the French population, but they do not seem to account for the linkage between the 2p23 locus and quantitative markers of obesity. [Diabetologia (2003) 46:704-711]

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“…The binding affinity of GKRP for GK is increased by fructose 6-phosphate and decreased by fructose 1-phosphate, which binds to the same site on GKRP (17). GKRP also determines the subcellular location of GK and sequesters the enzyme in the nucleus in the fasted state (18).…”

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confidence: 99%

Stimulation of Hepatocyte Glucose Metabolism by Novel Small Molecule Glucokinase Activators

et al. 2004

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Glucokinase (GK) has a major role in the control of blood glucose homeostasis and is a strong potential target for the pharmacological treatment of type 2 diabetes. We report here the mechanism of action of two novel and potent direct activators of GK: 6-[(3-isobutoxy-5-isopropoxybenzoyl)amino]nicotinic acid (GKA1) and 5-({3-isopropoxy-5-[2-(3-thienyl)ethoxy] benzoyl}amino)-1,3,4-thiadiazole-2-carboxylic acid (GKA2), which increase the affinity of GK for glucose by 4-and 11-fold, respectively. GKA1 increased the affinity of GK for the competitive inhibitor mannoheptulose but did not affect the affinity for the inhibitors palmitoylCoA and the endogenous 68-kDa regulator (GK regulatory protein [GKRP]), which bind to allosteric sites or to N-acetylglucosamine, which binds to the catalytic site. In hepatocytes, GKA1 and GKA2 stimulated glucose phosphorylation, glycolysis, and glycogen synthesis to a similar extent as sorbitol, a precursor of fructose 1-phosphate, which indirectly activates GK through promoting its dissociation from GKRP. Consistent with their effects on isolated GK, these compounds also increased the affinity of hepatocyte metabolism for glucose. GKA1 and GKA2 caused translocation of GK from the nucleus to the cytoplasm. This effect was additive with the effect of sorbitol and is best explained by a "glucose-like" effect of the GK activators in translocating GK to the cytoplasm. In conclusion, GK activators are potential antihyperglycemic agents for the treatment of type 2 diabetes through the stimulation of hepatic glucose metabolism by a mechanism independent of GKRP. Diabetes 53:535-541, 2004

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Identification of Fructose 6-Phosphate- and Fructose 1-Phosphate-binding Residues in the Regulatory Protein of Glucokinase

Cited by 59 publications

References 51 publications

An Allosteric Activator of Glucokinase Impairs the Interaction of Glucokinase and Glucokinase Regulatory Protein and Regulates Glucose Metabolism

An Allosteric Activator of Glucokinase Impairs the Interaction of Glucokinase and Glucokinase Regulatory Protein and Regulates Glucose Metabolism

Mutations in the glucokinase regulatory protein gene in 2p23 in obese French caucasians

Stimulation of Hepatocyte Glucose Metabolism by Novel Small Molecule Glucokinase Activators

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