EAR2 and EAR3/COUP-TFI regulate transcription of the rat LH receptor

Our previous studies demonstrated regulation of the human LH receptor (hLHR ) promoter by nuclear orphan receptors EAR2, EAR3/COUP-TFI (repression), an d TR4 (activation) through a direct-repeat motif (hDR). The current studies investigated the differential binding of orphan receptors to rat (rLHR) an d hLHR promoters, and their modulation of rLHR gene transcription in rat gr anulosa cells. The rLHR DR with one nucleotide difference from hDR at its c ore sequence mediated inhibition of the rLHR transcription, to which EAR2 a nd EAR3/COUP-TFI but not TR4 bound. The A/C mismatch was responsible for th e lack of TR4 binding and function, but had no effect on EAR2 and EAR3/COUP -TFI. EAR2 and EAR3/COUP-TF bound to the rLHR DR with lower affinity than t o the hDR, and exhibited lesser inhibitory capacity. This difference result ed from the lack of a guanine in the rDR, which is present 3' next to the h DR core. These studies have identified sequence-specific requirements for t he binding of EAR2, EAR3/COUP-TFI, and TR4 to the DRs that explain their di fferential regulation of rat and human LHR genes. In addition, hCG treatmen t significantly reduced the inhibition of rLHR gene in granulosa cells and also decreased EAR2 and EAR3/COUP-TFI protein levels. These results indicat e that hormonally regulated expression of EAR2 and EAR3/COUP-TFI contribute s to gonadotropin-induced derepression of LHR promoter-activity in granulos a cells.