An internal ribosome entry site (IRES) is a nucleotide sequence that allows cap independent translation initiation by recruiting the ribosome in proximity of an internal initiation codon. Here, we describe an IRES driven mechanism of translation initiation for the human muscarinic M2 receptor. The insertion of a stop codon in the N-terminal of the third cytoplasmic loop of the M2 receptor, resulted in a mutant receptor (M2-stop) that was still able to bind muscarinic ligands and to function. The insertion of the stop codon resulted in the expression of a N-terminal receptor fragment, constituted by the transmembrane regions I-V, and a C-terminal receptor fragment, constituted by the transmembrane regions V and VI. The two fragments could bind to each other and reconstitute the functional muscarinic receptor. Translation of the C-terminal fragment was not due to leaky scanning and reinitiation of translation, as the insertion of a stable hairpin loop did not prevent its translation. Mutagenesis of the three methionines in frame after the stop codon, revealed that translation initiation starts at the third methionine (codon 368). Western blot analysis showed that the C-terminal fragment of the M2-stop receptor has an apparent molecular mass of ~15.000 Dalton. Remarkably, a fragment of the same molecular weight was found also in the cells transfected with the wild type M2 receptor, suggesting that the IRES driven mechanism of translation initiation is physiologically functioning.

An internal ribosome entry site (IRES), present within the third cytoplasmic loop, drives the expression of the Carboxyl-terminal domain of the human muscarinic M2 receptor

MAGGIO, Roberto;FLATI, VINCENZO;
2015

Abstract

An internal ribosome entry site (IRES) is a nucleotide sequence that allows cap independent translation initiation by recruiting the ribosome in proximity of an internal initiation codon. Here, we describe an IRES driven mechanism of translation initiation for the human muscarinic M2 receptor. The insertion of a stop codon in the N-terminal of the third cytoplasmic loop of the M2 receptor, resulted in a mutant receptor (M2-stop) that was still able to bind muscarinic ligands and to function. The insertion of the stop codon resulted in the expression of a N-terminal receptor fragment, constituted by the transmembrane regions I-V, and a C-terminal receptor fragment, constituted by the transmembrane regions V and VI. The two fragments could bind to each other and reconstitute the functional muscarinic receptor. Translation of the C-terminal fragment was not due to leaky scanning and reinitiation of translation, as the insertion of a stable hairpin loop did not prevent its translation. Mutagenesis of the three methionines in frame after the stop codon, revealed that translation initiation starts at the third methionine (codon 368). Western blot analysis showed that the C-terminal fragment of the M2-stop receptor has an apparent molecular mass of ~15.000 Dalton. Remarkably, a fragment of the same molecular weight was found also in the cells transfected with the wild type M2 receptor, suggesting that the IRES driven mechanism of translation initiation is physiologically functioning.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/33656
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