Abstract
G protein coupled receptors (GPCRs) are transmembrane proteins involved in cell signalling, and are effective drug targets. Following GPCR activation and phosphorylation on serine/threonine residues by one or more of six GPCR kinases (GRKs), two different arrestin proteins bind to the C-terminus. The main consequence of arrestin binding is GPCR desensitisation and endocytosis. However, these proteins are increasingly appreciated for their ability to facilitate signalling cascades that may be independent of the typical ‘G protein-mediated’ effects.
Biased ligands are a novel class of drugs that exploit differences in signalling consequences mediated by independent effectors to reduce on-target adverse effects by improving the specificity of the GPCR response. Activation of the cannabinoid type-1 receptor (CB1) is associated with on-target adverse effects that may be overcome with biased ligands. However, little is known about whether biased signalling of this receptor (whether G protein or arrestin) is possible. Unique phosphorylation ‘barcodes’ that cause selective interaction with different arrestins have been previously documented, and could provide a strategy for biased ligands. This thesis investigated whether the six different GRKs can cause differences in the interaction between CB1 and either of the two non-visual arrestin. Site-directed mutagenesis was employed to identify specific phosphorylation sites important in arrestin interaction. This study also determined the contribution of each arrestin subtype to cell signalling consequences, such as G protein activation, cAMP inhibition, ERK phosphorylation, and receptor endocytosis.
This study revealed that regulation of ligand-mediated CB1 interaction with either of the two non-visual arrestins is more homogeneous than previously assumed. Arrestin-2/3 translocation of a range of ligands was similarly modulated by most GRKs. The GRK2/3 subtypes exerted the largest control over translocation in a mostly G protein activation-dependent manner. This thesis therefore identifies the GRK2/3-dependent component of arrestin translocation as a core limitation of arrestin-biased ligand development. This leads to the conclusion that G protein biased ligands must have inherently low efficacy. Furthermore, this thesis does not identify an arrestin-mediated pERK response downstream of CB1 in HEK293 cells, which suggests other (as yet unidentified) proteins may be critical to this response beyond GRKs/arrestins.