AGONIST-MEDIATED DESTABILIZATION OF M1 MUSCARINIC ACETYLCHOLINE-RECEPTOR MESSENGER-RNA - ELEMENTS INVOLVED IN MESSENGER-RNA STABILITY ARE LOCALIZED IN THE 3'-UNTRANSLATED REGION

The effects of chronic agonist exposure on receptor number (down-regul ation) have been shown, in part, to be due to effects on mRNA levels. Agonist-mediated effects on muscarinic acetylcholine receptor (mAChR) mRNA were investigated in Chinese hamster ovary (CHO) cells stably tra nsfected with mi mAChR gene constructs containing the open reading fra me and a series of deletions of the flanking 3'-untranslated region (3 '-UTR). Carbachol (CBC) down-regulated m1 mAChRs encoded by the constr uct m1C1, an mi mAChR transcript containing the entire flanking 3'UTR (nucleotides 1526-2622), in a time dependent fashion with maximal decr eases occurring by 12 h. Steady-state levels of m1C1 mRNA declined in a parallel fashion beginning 6 h after CBC pretreatment. Similar findi ngs were obtained with m1C2, a construct which is missing all but 261 bases of flanking 3'-UTR (nucleotides (nt) 1526-1786). Since the rate of mRNA degradation represents an important potential regulatory mecha nism to control the level of gene expression, we investigated the effe cts of CBC treatment on m1C1 and m1C2 mRNA stability. The half-life of either transcript in untreated cells was approximately 14 h, whereas m1C1 and m1C2 transcript half-lives decreased to similar to 3 h in cel ls treated with CBC. Agonist-induced destabilization of m1C2 mRNA coul d be mimicked by phorbol esters in a concentration-dependent manner an d blocked by the protein kinase inhibitor, H-7. In contrast, mi mAChR mRNA constructs missing nt 1526-1786 of the 3'-UTR (m1C3 and m1C4) did not undergo agonist or phorbol ester-induced destabilization. In the neuroblastoma cell line IMR-32, endogenous mi mAChR mRNA was down-regu lated and destabilized following CBC treatment. These results demonstr ate that agonist-induced mRNA destabilization is a potential mechanism for regulating mi mAChR levels. Furthermore, deletion studies identif y a 261 base region of the 3'-UTR having the potential to form stable stem-loop structures which likely harbors element(s) responsible for m essage destabilization.