| Popis: |
The aim of this project is to elucidate the protein:protein interactions that underly some of the intracellular signalling mechanisms of the 5-hydroxytryptamine 2A receptor (5-HT2AR) and related receptors. The 5-HT2AR is a member of the group I family of rhodopsin-related GPCRs. The receptor is known to activate phospholipase C (PLC) via the hetertrimeric G proteins Gαq/11, but has been shown to also signal through the phospholipase D (PLD) pathway in a novel ADP-ribosylated factor (ARF)-dependent manner, that appears to be independent of Gαq/11. The M3 muscarinic receptor, another member of the group I GPCRs, has also been shown to signal through both PLC and the alternative pathway for PLD activation via ARF. This thesis, provides novel evidence for physical interaction of both these receptors with members of the ARF family of small G-proteins in a differential manner. Not only is there evidence from in vivo signalling assays that these receptors can activate the PLD signalling pathway through the ARF family of proteins, also, in vitro GST interaction assays demonstrate that distinct domains of the receptors are involved in interactions with ARF isoforms. The 5-HT2A receptor associates selectively with ARF1 via the carboxy terminal tail of the receptor, whereas the third intracellular loop domains of the M3 muscarinic receptor, and to a lesser extent the carboxy terminal (ct) tail, associates with both ARF1 and ARF6. Experiments undertaken to elucidate the molecular complexes involved in these interactions suggest the involvement of Gβγ for the M3 muscarinic receptor, and arrestin for the 5-HT2A receptor. The binding of other novel interaction partners to the 5-HT2A receptor was also investigated, by use of a functional proteomic approach utilising MALDI-TOF mass spectrometry to identify binding partners. These studies led to the discovery that the glial protein S100β binds to the carboxy-terminal domain of the 5-HT2A receptor in a calcium dependent manner. |
