title: Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.
abstract: Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.
authors.affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.
authors.forename: Bogumil
authors.initials: B
authors.lastname: Zelent
authors.affiliation: †Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, Oxford, U.K.
authors.forename: Anne
authors.initials: A
authors.lastname: Raimondo
authors.affiliation: †Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, Oxford, U.K.
authors.forename: Amy
authors.initials: A
authors.lastname: Barrett
authors.affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.
authors.forename: Carol W
authors.initials: CW
authors.lastname: Buettger
authors.affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.
authors.forename: Pan
authors.initials: P
authors.lastname: Chen
authors.affiliation: †Oxford Centre for Diabetes Endocrinology & Metabolism, University of Oxford, Oxford, U.K.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Gloyn
authors.affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.
authors.forename: Franz M
authors.initials: FM
authors.lastname: Matschinsky
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D005636:Fructosephosphates / Q000378:metabolism; D005941:Glucokinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D058849:Protein Refolding; D055550:Protein Stability; D021381:Protein Transport; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D005636:Fructosephosphates; C485100:GCKR protein, human; D008024:Ligands; D050505:Mutant Proteins; D011994:Recombinant Proteins; C032284:fructose-1-phosphate; D014364:Tryptophan; D005941:Glucokinase; D005947:Glucose