Mutational analysis of the glucose regulatory element in the promoter of the glucagon receptor gene.
Recently, we identified a glucose regulatory element in the promoter of the rat glucagon receptor gene. The effect of glucose is centered on a highly palindromic sequence of 19 nucleotides that we called the G box (Portois et al., 1999, J. Biol. Chem. 274: 8181-8190). This sequence contains two E boxes. Recently, we investigated the role of each individual E box, as well as the contribution of the sequences located upstream and downstream from this G box. (1) Mutation of nucleotides "CA" to "GT" in the first E box (position -543 to -542) suppressed the activation of the CAT reporter gene by glucose. In contrast, mutation of the nucleotides "CA" to "GT" in the second E box (position -534 to -533) had no effect on this glucose activation. (2) Deletion of a sequence upstream from the G box (nucleotides -579 to -555) suppressed the activation by glucose, whereas deletion of a sequence located downstream from the G box (nucleotides -501 to -443) had no effect on this parameter. (3) Subcloning of a small promoter fragment of only 49 nucleotides (-560 to -512) into the pCat5 plasmid conferred to transfected cells sensitivity to glucose in terms of CAT activity. Consequently, all transactivation factors required for this glucose effect must act via this short gene fragment.
Portois
L
L
Laboratory of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Brussels, Belgium.
Tastenoy
M
M
Svoboda
M
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D005809:Genes, Regulator / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D051381:Rats; D018027:Receptors, Glucagon / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D018027:Receptors, Glucagon; D004247:DNA; D005947:Glucose