Abstract:
Migraine is a prevalent and debilitating neurological disorder that afflicts ~15% of the population. There is increased release of the neuropeptide, calcitonin gene-related peptide (CGRP) during a migraine attack which binds two G protein-coupled receptors (GPCRs): the CGRP and AMY₁ receptors. CGRP receptor regulation has been previously researched. However, there is very little information on the AMY₁ receptor regulation. The aim of this thesis is to compare the regulation mechanisms of these two CGRP-responsive receptors, primarily through investigating receptor internalisation. Internalisation of the receptors was investigated using fluorescently labelled CGRP. The CGRP receptor internalised robustly and was present in the cytosol. In comparison, the AMY₁ receptor remained at the cell surface. The location of the receptors were further investigated using immunofluorescence. The CGRP receptor colocalised with early endosome antigen 1 (EEA1), indicating its presence in early endosomes. There was little colocalisation seen for the AMY₁ receptor and EEA1. In addition, the interaction between a key internalisation protein, βarrestin, was investigated. Stimulation with CGRP saw translocation of the fluorescently labelled β-arrestin2 to the cell surface and then to the cytosol for the CGRP receptor. βarrestin2 remained diffuse in the cytosol for the AMY₁ receptor. Receptor interactions with βarrestin were further investigated using chemical cross-linking. β-arrestin was cross-linked to the CGRP receptor with an increase in β-arrestin binding upon stimulation. Internalisation was further explored using sucrose density centrifugation, where the CGRP receptor was isolated from early endosomes and β-arrestin was also found in the early endosome fraction. The findings in this thesis indicate two distinct regulation mechanisms for the CGRP and AMY₁ receptors. The CGRP receptor internalised into early endosomes through an interaction with β-arrestin whereas the AMY₁ receptor did not robustly internalise and did not appear to have an interaction with β-arrestin. This research is able to give insight into the cellular processes that may be occurring in migraine. Understanding the difference in regulation of these receptors is important as it will help provide more effective migraine drug development through knowledge of which receptors may be active in different cellular compartments.