The 3′-untranslated region of the α2C-adrenergic receptor mRNA impedes translation of the receptor message

We report that tyro subtypes of alpha(2)-adrenergic receptors (alpha(2A/D)- and alpha(2C)-AR) are ectopically expressed with dramatically different efficiencies and that this difference is due to a 288-nucleotide (nt) segment in the 3'-untranslated region (3'-UTR) of the alpha(2C)-AR mRNA that impairs translational processing, NIH-3T3 fibroblasts mere transfected with receptor constructs (coding region plus 552 nt, alpha(2C)-AR; coding region plus 1140 nt, alpha(2A/D)-AR) and a vector conferring G418 resistance. Transcription was driven by the murine sarcoma virus promoter element, and the receptor gene segment was upstream of an SV40 polyadenylation cassette. Drug-resistant transfectants were evaluated for expression of receptor mRNA and protein, 90% of the NIH-3T3 alpha(2C)-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell Lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha(2A/D)-AR transfectants expressed receptor protein (200-5000 fmol/mg). Similar results were obtained following transfection of DDT1MF-2 cells with the two receptor constructs. The role of the 3'-UTR of the alpha(2C)-AR in mRNA processing was determined by generating new constructs in which the 3'-UTR. was progressively truncated from 552 to 470, 182, 143, or 74 nt 3' to the stop codon. Truncation of the 3'-UTR resulted in the expression of receptor protein in the G418-resistant transfectants (nt 74, 100%; nt 143, 80%; nt 182, 50%). The level of mRNA in the transfectants expressing the receptor protein was not greater than that in nonexpressing clones, and the differences in protein expression did not reflect altered mRNA stability in the truncated construct. The alpha(2C)-AR mRNA with the longer 3'-UTR underwent translational initiation as it was found in the polysome fraction, indicating that the lack of receptor protein was due to impaired translational elongation or termination. These data suggest that translational efficiency is a hey mechanism for regulating alpha(2C)-AR expression and associated signaling events.Biophysical Structural Chemistr