“Studio della regolazione della funzione mitocondriale da parte del frammento C-terminale del recettore muscarinico M2 “

In eukaryotes the translation process occurs in most cases through a cap-dependent mechanism which starts the mRNA scanning process up to the first AUG start codon. The AUG codon is not always read in the correct open reading frame (ORF) context, in these cases, alternative translation initiation mechanisms are activated, including the cap-independent translation process mediated by nucleotide sequences that serves as the internal ribosome entry site (IRES). The IRES sequences form long and ordered RNA structures which recruit ribosomes to initiate translation at an internal start codon of the mRNA sequence, bypassing the canonical scanning mechanism. This IRES-dependent translation had been shown that it is important in guiding protein synthesis in particular cellular contexts where cap-dependent translation is impaired, such as stressful conditions (apoptosis, response to hypoxia and ischemia), disease or during embryogenesis and development. IRES elements have also been identified in G protein-coupled receptors (GPCRs), a family of transmembrane receptors involved in numerous physiological and pathological processes, present in different tissues and systems, that are essential for the proper functioning of the living organism. GPCRs have a typical serpentine structure, with a single polypeptide chain crossing the membrane seven times; the seven transmembrane domains are connected to each other by intracellular and extracellular loops. The third cytoplasmic loop and the carboxy-terminal domain are responsible for the interaction with the G protein, determining its activation, which causes a series of phosphorylation in order to promote a cellular response. To the GPCRs family belongs the subfamily of the mAChR, muscarinic acetylcholine receptors, activated by the endogenous neurotransmitter acetylcholine. Mutations in the DNA sequence corresponding to the third cytoplasmic loop of the M2 receptor, allowed us to identify the third in-frame methionine, Met368, as the alternative starting point of the translation of C-terminal portion, triggered by the IRES sequence. Therefore in this thesis, starting from this assumption, we have seen through fluorescence microscopy that the M2tail doesn’t follow the normal route to the plasma membrane, as the wild-type receptor does, but it is sorted into the mitochondria. The presence in the mitochondria induces a considerable decrease in oxygen consumption by impairing oxidative phosphorylation that leads to a reduced cell growth and affects the oxygen radical formation. In this thesis I present data concerning the expression of the C-terminal region of M2 muscarinic receptor, as well as of other GPCRs, that can regulate mitochondrial function, that represents a novel mechanism that cells could activate for controlling their metabolism under variable environmental conditions.