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.
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Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0278.
Peegel
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Mosier
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Menon
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D000818:Animals; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D015342:DNA Probes; D015536:Down-Regulation; D005260:Female; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010053:Ovary / Q000378:metabolism / Q000648:ultrastructure; D011555:Pseudopregnancy / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D011974:Receptors, LH / Q000235:genetics*; D014158:Transcription, Genetic
D006063:Chorionic Gonadotropin; D015342:DNA Probes; D012333:RNA, Messenger; D011974:Receptors, LH