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Abstract
We report that two subtypes of alpha2-adrenergic receptors (alpha2A/D- and alpha2C-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 alpha2C-AR mRNA that impairs translational processing. NIH-3T3 fibroblasts were transfected with receptor constructs (coding region plus 552 nt, alpha2C-AR; coding region plus 1140 nt, alpha2A/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 alpha2C-AR transfectants expressed receptor mRNA, but only 14% of the clonal cell lines expressed receptor protein. In contrast, 90% of the NIH-3T3 alpha2A/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 alpha2C-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 alpha2C-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 key mechanism for regulating alpha2C-AR expression and associated signaling events.
Highlights
The response of the cell to hormones/neurotransmitters is an integrated process that involves varying numbers of molecules
The dissociation between ␣2C-AR protein and mRNA was observed following stable transfection of DDT1MF-2 cells derived from hamster smooth muscle, indicating that the failure to process the receptor mRNA is not restricted to a fibroblast cell line (Fig. 1, B and C)
The processing of mRNA to the mature protein is subject to regulation at several steps including translational initiation, elongation, and termination (21–23)
Summary
The response of the cell to hormones/neurotransmitters is an integrated process that involves varying numbers of molecules. In contrast to the ␣2A/D-AR, there is an apparent dissociation between ␣2C-AR protein expression and receptor mRNA observed in discrete areas of the central nervous system and the NG108-15 neuroblastoma ϫ glioma cell hybrid (7–10), suggesting that translation of the ␣2C-AR mRNA is a regulated event.
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