Novel interactions of the 5-HT₂ₐ and related receptors with intracellular signalling proteins

Abstract

The aim of this project is to further elucidate the pathways involved in the intracellular signalling mechanisms of the 5-HT₂ₐ and related receptors. The Gprotein coupled receptors (GPCRs) are named after their ability to interact with and signal through the trimeric G-proteins. The 5-hydroxytryptamine 2A receptor (5- HT₂ₐR) is a member ofthe group I family of rhodopsin-related GPCRs. The receptor is known to activate phospholipase C (PLC) via the heterotrimeric G proteins Gaq/n, but has been shown to also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner, that appears to be independent of Gq/n. The M3 muscarinic receptor, another member of the group I GPCRs, has also been shown to signal through both PLC (via Got) and the alternative pathway of PLD activation via ARF. In this thesis, it has been shown that both these receptors interact directly with members of the ADP-ribosylation Factor (ARF) family of small G-proteins. Not only is there evidence to show that these receptors activate PLD signalling through the ARF family of proteins, as shown by in vivo signalling assays, but it can also be shown that the receptors interact directly with ARF. The 5-FIT₂ₐ receptor associates with ARF1, and the third intracellular loop domain of the M3 muscarinic receptor associates with both ARF1 and ARF6, as shown by in vitro GST interaction assays.

Experiments undertaken to elucidate the exact criteria for this interaction suggest that a complex of proteins involving GPy for the M3 muscarinic receptor, and arrestin for the 5-HT₂ₐ receptor. The GDP/GTP status of the ARF involved plays a role in the ability of this interaction to take place. The conserved N/DPxxY motif in transmembrane domain 7 (tm7) ofthe Group I GPCRs also seems to affect the ability of the receptor to signal through ARF. Thus changing this motif altered the binding of ARF isoforms to the 5-FIT₂ₐ receptor.

The binding of novel interaction partners to the 5-HT₂ₐ receptor was also investigated, with the discovery ofthat the glial protein S100B bound to the carboxy terminal domain ofthe 5-HT₂ₐ receptor in a calcium dependent manner.

These findings have implications for the investigation of the signalling pathways of these and other related Group I type GPCRs