FURTHER CHARACTERIZATION OF THE GLUCOCORTICOID RESPONSE UNIT IN THE PHOSPHOENOLPYRUVATE CARBOXYKINASE GENE - THE ROLE OF THE GLUCOCORTICOIDRECEPTOR-BINDING SITES

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the rate-limiting step of gluconeogenesis. The activity of this enzyme is controlled by several hormones, including glucocorticoids, glucagon, retinoic acid, and insulin, that principally affect the rate of transcription of the PEPCK gene. Glucocorticoids induce PEPCK gene transcription through a complex glucocorticoid response unit that consists of, from 5' to 3', accessory factor elements AF1 and AF2; two noncanonical glucocorticoid receptor-binding sites, GR1 and GR2; a third accessory factor element , AF3; and a cAMP-response element, CRE. A complete glucocorticoid res ponse is dependent on the presence of both GR-binding sites, all three accessory elements, and the CRE. In this study we assess the relative roles of GR1 and GR2 in the context of the glucocorticoid response un it and use a combination of binding and function assays to compare GR1 and GR2 to glucocorticoid response elements (GREs) that conform close ly to the consensus sequence. The relative binding affinity of GR foll ows the order: consensus GRE >> GR1 > GR2. Mutations that disrupt the binding of GR to GR1 result in a major reduction of the glucocorticoid response, whereas similar mutations of GR2 have a much smaller effect . Unlike the simple consensus GRE, neither GR1 nor GR2 mediate a gluco corticoid response through a heterologous promoter. The accessory elem ents appear to have different functional roles. AF2 is still needed fo r a maximal glucocorticoid response when GR1 is converted to a high-af finity GR-binding element, but AF1 and AF3 are not required.