Abstract:
The neuropeptide PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide) is suggested to activate the PAC1 receptor (PACAP Type I Receptor). This receptor is part of the Class B family of GPCRs (G Protein-Coupled Receptors), which also includes the glucagon and corticotropin-releasing factor (CRF) receptors. Activation of PAC1 is hypothesized to occur during the onset of migraine attacks. Therefore, blocking PACAP binding to the PAC1 receptor may prevent these attacks. This may prove to be an alternative treatment strategy for migraine patients. However, PAC1 has several splice variants and very little is known about how PACAP interacts with these isoforms. Evidence from structural and computational modelling studies of the extracellular domain (ECD) of the PAC1s splice variant has suggested two conflicting models of PACAP binding. Within these models, the involvement of specific residues in the PAC1 ECD for receptor activation is poorly characterised. Further, it remains unclear if interactions between the different PAC1 receptor splice variants and PACAP are conserved. This thesis aimed to determine the importance of amino acids in the PAC1 ECD for receptor activation. Based on the homology between other ligand bound Class B GPCRs, several potentially important regions of the PAC1 ECD were identified. Alanine mutagenesis of selected residues in these regions was performed to determine their roles in receptor activation. These mutant receptors were transiently transfected into COS-7 cells and the cAMP signalling response to PACAP38, PACAP27, and VIP were measured. Cell surface expression of mutant receptors was measured by ELISA. Alanine mutagenesis of 13 PAC1n and 2 complementary PAC1s residues showed no major effect on PACAP38, PACAP27, or VIP potency. No significant differences in potency exceeded a 3-fold shift. Mutation did not decrease cell surface expression, however HA-PAC1n M57A and HA-PAC1n Y130A expression was increased. This data suggests that the residues examined in the PAC1 ECD are not important for receptor activation or cell surface expression. Further, it is apparent that current models of PAC1 binding require additional refinement. It is proposed that the contacts between the PAC1 ECD and PACAP may be distinct from that observed in the glucagon and CRF receptors. Overall, these results provide further insights into the activation of PAC1 and will aid the future development of antagonists which block PAC1.