Loss of Lutropin/human Choriogonadotropin Receptor Messenger Ribonucleic Acid During Ligand-induced Down-regulation Occurs Post Transcriptionally

Previous studies have demonstrated that ligand-induced down-regulation of the LH/hCG receptor in rat corpus luteum results in a loss of ligand-binding activity and a parallel decline in steady state levels of receptor mRNA. The purpose of the present study was to determine whether this loss of receptor mRNA during receptor down-regulation is due to inhibition of transcription or increased degradation of the receptor mRNA. To differentiate between these two possibilities, nuclear run-off assays were performed to study transcription rates of nuclei isolated from control and hCG down-regulated rat ovaries. A 750-mer LH/hCG receptor cDNA probe, spanning the carboxy-terminal region of the peptide and the 3'-untranslated region of the receptor cDNA, was constructed by polymerase chain reaction. RNA transcripts were synthesized from existing nuclear RNA from control and down-regulated nuclei and hybridized to the receptor cDNA probe immobilized on nylon membranes and subjected to autoradiography. Hybridization to actin cDNA was also run alongside as a control. The results of run-off transcription analysis indicated that during down-regulation, the transcription rates of LH/hCG receptor mRNA are not decreased compared to those in saline-treated controls. Although no decrease in the transcription rates of the receptor mRNA was seen in the down-regulated state, the steady state levels of the receptor mRNA showed a decline when assayed by either solution hybridization or Northern blot analysis. Furthermore, hCG induced down-regulation appears to increase the transcriptional activity of the nuclei. Estimates of steady state LH/hCG receptor mRNA turnover rates indicate that the half-life of the message in down-regulated ovaries is markedly reduced compared to that in the control. It is concluded that the loss of steady state LH/hCG receptor mRNA is not due to a decrease in transcription, but probably represents an increased degradation of the receptor mRNA.