Loss of lutropin/human choriogonadotropin receptor messenger ribonucleic acid during ligand-induced down-regulation occurs post transcriptionally.

Abstract

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.

Isolation and Characterization of a Novel trans-Factor for Luteinizing Hormone Receptor mRNA from Ovary

“…We have shown that the decline in cell surface LHR expression seen after hCG administration is paralleled by a specific loss of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) in the ovary (9). Furthermore, the loss of LHR mRNA does not result from decreased transcription but occurs post-transcriptionally with a 3-fold decrease in half-life (8).…”

“…The cell surface expression of LHR varies during different stages of the ovarian cycle. Its expression in granulosa cells and luteal cells is greatly decreased by an endogenous preovulatory LH surge or by the administration of a pharmacological dose of human chorionic gonadotropin (hCG), a placental counterpart of LH (5)(6)(7)(8). We have shown that the decline in cell surface LHR expression seen after hCG administration is paralleled by a specific loss of all four LHR mRNA transcripts (6.7, 4.4, 2.6, and 1.8 kb) in the ovary (9).…”

Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary

“…The loss of LH receptor mRNA occured through post-transcriptional mechanisms as the transcription rate, determined by nuclear run on assays, remained identical in hCG treated group compared to the control. In fact, the nuclei isolated from the hCG treated ovaries was found to incorporate 3 H uridine into total RNA at much higher levels than that seen in control ovaries (Lu, et al 1993). Thus, we concluded that the loss of steady state LH receptor mRNA seen in the hCG treated group was due to increased mRNA degradation rather than a decrease in the rate of its synthesis.…”

“…Previous studies have shown that in the rat, preovulatory LH surge produces a rapid decline in LH receptor expression as well as an uncoupling of LH receptor from the cognate G proteins (Hoffman, et al 1991, Lapolt, et al 1990, Lu, et al 1993. The loss of LH receptor from the cell surface may be explained on the basis of the rapid endocytosis of the LH-receptor complex, causing its depletion.…”

“…Ovaries were collected at different time intervals up to 72 h (Hoffman, et al 1991, Lu, et al 1993, Peegel, et al 1994) and were processed for Northern blot analysis for the expression of LH receptor mRNA, for in situ hybridization analysis in tissue sections to visualize the changes in mRNA expression, and for solution hybridization assay to quantitate changes in mRNA levels. The results showed that within 6 h of hCG injection, there was a steady decline in the expression levels of LH receptor mRNA that reached almost non-detectable levels by 24 h (Hoffman, et al 1991, Lu, et al 1993, Peegel, et al 1994). This was followed by a gradual recovery to control levels by 72 h, as shown in the solution hybridization analysis data presented in Fig 1. The loss of mRNA was rather unexpected, since the decrease of cell surface receptor expression was thought to occur through rapid internalization of the ligand-bound receptor.…”