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Nicotinic acetylcholine receptor subunits ?4 and ?5 associated with smoking behaviour and lung cancer are regulated by upstream open reading frames.

ABSTRACT: Nicotinic acetylcholine receptor subunits (nAChR) are associated with different aspects of smoking behaviour as well as with smoking related disorders. Several of these subunits have been found to be upregulated in smokers or differentially expressed in lung tumor cells. The mechanisms behind these observations are not known but assumed to be mainly post-transcriptional. Many post-transcriptional mechanisms are initiated by functionally relevant sequence motifs within untranslated gene regions, such as upstream open reading frames (uORFs). We performed a systematic search in all smoking-associated neuronal nAChR subunits and identified functionally relevant uORFs in CHRNA4 and CHRNA5. Luciferase experiments showed that these uORFs are able to significantly decrease protein expression. Our quantitative real-time PCR (qPCR) results strongly suggest that the observed effects originate at the translation rather than at the transcription level. Interestingly, the CHRNA4 uORF was only functionally relevant when expressed in the shorter isoform of this gene. Therefore, the data presented in this study strongly points towards an important role of uORFs within the 5'UTR of CHRNA4-isoform 1 and CHRNA5 as regulators of protein translation. Moreover, the shared uORF of CHRNA4-isoform 1/isoform 2 represents the first example of a sequence context-dependent uORF.

SUBMITTER: Eggert M

PROVIDER: S-EPMC3699600 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Nicotinic acetylcholine receptor subunits α4 and α5 associated with smoking behaviour and lung cancer are regulated by upstream open reading frames.

Eggert Marlene M Aichinger Eric E Pfaffl Michael W MW Steinlein Ortrud K OK Pfob Martina M

PloS one 20130702 7

Nicotinic acetylcholine receptor subunits (nAChR) are associated with different aspects of smoking behaviour as well as with smoking related disorders. Several of these subunits have been found to be upregulated in smokers or differentially expressed in lung tumor cells. The mechanisms behind these observations are not known but assumed to be mainly post-transcriptional. Many post-transcriptional mechanisms are initiated by functionally relevant sequence motifs within untranslated gene regions, ...[more]

PMID: 23843950

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Project description:Selected nicotinic agonists were used to activate and desensitize high-sensitivity (HS) (α4)2(β2)3) or low-sensitivity (LS) (α4)3(β2)2) isoforms of human α4β2-nicotinic acetylcholine receptors (nAChRs). Function was assessed using (86)Rb(+) efflux in a stably transfected SH-EP1-hα4β2 human epithelial cell line, and two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing concatenated pentameric HS or LS α4β2-nAChR constructs (HSP and LSP). Unlike previously studied agonists, desensitization by the highly selective agonists A-85380 [3-(2(S)-azetidinylmethoxy)pyridine] and sazetidine-A (Saz-A) preferentially reduced α4β2-nAChR HS-phase versus LS-phase responses. The concatenated-nAChR experiments confirmed that approximately 20% of LS-isoform acetylcholine-induced function occurs in an HS-like phase, which is abolished by Saz-A preincubation. Six mutant LSPs were generated, each targeting a conserved agonist binding residue within the LS-isoform-only α4(+)/(-)α4 interface agonist binding site. Every mutation reduced the percentage of LS-phase function, demonstrating that this site underpins LS-phase function. Oocyte-surface expression of the HSP and each of the LSP constructs was statistically indistinguishable, as measured using β2-subunit-specific [(125)I]mAb295 labeling. However, maximum function is approximately five times greater on a "per-receptor" basis for unmodified LSP versus HSP α4β2-nAChRs. Thus, recruitment of the α4(+)/(-)α4 site at higher agonist concentrations appears to augment otherwise-similar function mediated by the pair of α4(+)/(-)β2 sites shared by both isoforms. These studies elucidate the receptor-level differences underlying the differential pharmacology of the two α4β2-nAChR isoforms, and demonstrate that HS versus LS α4β2-nAChR activity can be selectively manipulated using pharmacological approaches. Since α4β2 nAChRs are the predominant neuronal subtype, these discoveries likely have significant functional implications, and may provide important insights for drug discovery and development.

Dataset Information

Nicotinic acetylcholine receptor subunits ?4 and ?5 associated with smoking behaviour and lung cancer are regulated by upstream open reading frames.

Publications

Nicotinic acetylcholine receptor subunits α4 and α5 associated with smoking behaviour and lung cancer are regulated by upstream open reading frames.

Similar Datasets

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