MECHANISM OF REGULATION OF THYROTROPIN-RELEASING-HORMONE RECEPTOR MESSENGER-RIBONUCLEIC-ACID IN STABLY TRANSFECTED RAT PITUITARY-CELLS

We showed previously that the level of TRH receptor (TRH-R) mRNA in ra t pituitary GH3 cells is down-regulated by TRH. Here, we study the mec hanism of regulation of TRH-R mRNA in a line of GH3 cells that are sta bly transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH -mTRHR-1 cells were found to have 2.4 times the number of TRH-Rs and t o stimulate a 2.5-fold greater increase in inositol phosphates in resp onse to TRH than the parent cell line and to show TRH-induced down-reg ulation of TRH-R number. GH-mTRHR-1 cells contained 26 +/- 1.6 molecul es of mouse TRH-R mRNA/cell and 230 +/- 31 molecules of mRNA for the n eomycin resistance gene (NEO) with which it was cotransfected. In GH-m TRHR-1 cells, TRH caused a dose-dependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contra st, TRH did not affect NEO mRNA or glyceraldehyde phosphate dehydrogen ase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did n ot affect total poly(A) RNA synthesis. Most importantly, TRH caused a 4-fold increase in the rate of degradation of mouse TRH-R mRNA, but di d not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 an d more than 20 h in cells treated with 1-mu-M TRH for 1.5 h, respectiv ely. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GH-mTRHR-1 cells is to enhance the rate of its degradation. W e suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degr adation.