Abstract

The influence of an upstream open reading frame (ORF) in the 5′-untranslated region (UTR) of the mRNA on corticotropin-releasing hormone receptor type 1 (CRHR1) translation was studied in constructs containing the 5′-UTR of CRHR1, with or without an ATG-to-ATA mutation in the upstream ORF, and the main ORF of luciferase or CRHR1. Upstream mutation in luciferase constructs increased luciferase activity when transfected into COS-7 or AtT20 cells compared with the native 5′-UTR. Transfection of CRHR1 constructs containing the upstream mutation into AtT20 or LVIP2.0zc reporter cells, resulted in higher¹²⁵I-Tyr-oCRH binding and corticotropin-releasing hormone-stimulated cAMP production, without changes in CRHR1 mRNA levels (measured by RNase protection assay). In vitro translation of luciferase or CRHR constructs with or without mutation of the upstream ATG, and Western blot analysis with anti-luciferase and anti-CRHR1 antibodies confirmed that mutation of the upstream ATG increases translation of the main ORF. The mechanism by which the upstream ORF inhibits translation may involve translation of the upstream peptide, because in vitro translation, or transfection into LVIP2.0zc cells of a fusion construct of the upstream ORF and green fluorescent protein (GFP) yielded a band consistent with the molecular size of GFP protein. The study shows that the upstream AUG in 5′-UTR of CRHR1 mRNA inhibits receptor expression by inhibiting mRNA translation and suggests the short open reading frame in the 5′-UTR plays a role in regulating translation of the CRH receptor.