Heterologous expression and characterization of rat liver glucokinase regulatory protein.
Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414-419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.
Mookhtiar
K A
KA
Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
Kalinowski
S S
SS
Brown
K S
KS
Tsay
Y H
YH
Smith-Monroy
C
C
Robinson
G W
GW
D048868:Adaptor Proteins, Signal Transducing; D000494:Allosteric Regulation; D000818:Animals; D002352:Carrier Proteins; D011232:Chemical Precipitation; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D015870:Gene Expression; D005941:Glucokinase / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; C485100:GCKR protein, human; C499484:Gckr protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C080906:glucokinase regulatory protein; D005941:Glucokinase