Juvenile hormone regulation of Drosophila Epac: A guanine nucleotide exchange factor for Rap1 small GTPase

A microchip array encompassing probes for 14,010 genes of Drosophila melanogaster was used to analyze the effect of (10R) juvenile hormone III (JH) on genome-wide gene abundance in Drosophila S2 cells. Treatment with JH yielded a collection of 32 known or putative genes that demonstrated a significant change in abundance with hormone treatment when compared to a control treatment of methyl linoleate (MLA) which has a similar structure to JH but displays no hormonal activity. The relative expression ratio (RER) of these loci was further analyzed by real-time quantitative reverse transcription PCR (qRT-PCR) using three stable reference genes that exhibited constant expression in the presence or absence of JH III. Among the genes confirmed to increase genes was Epac (Exchange Protein directly Activated by Cyclic AMP), a guanine nucleotide exchange factor for the small GTPase Rap1. Epac transcripts displayed a rapid, dose-dependent increase with JH treatment. Epac transcriptional activity induced by JH was also verified by qRT-PCR using an intron-specific probe. S2 cells were also challenged with various JH homologs, analogs, and metabolites to determine the specificity of the enhanced RER of Epac. The molting hormone, 20 hydroxyecdysone, did not increase Epac RERs above control levels. The response of late third instar (96 h) Drosophila to the potent agonist methoprene was tested by exposing the insects through diet. Significantly higher levels of Epac RERs were noted at 12 and 18 h after exposure. JH had no effect on Epac RERs in the human cell line, HEK-293.