Molecular studies on neuropeptide Y receptors involved in the regulation of feeding behaviour

The regulation of energy and nutrient homeostasis is a complex procedure involving interactions between numerous neuropeptides, neurotransmitters and hormones implicated in the control of this fundamental behaviour. The PP-fold peptide family member Neuropeptide Y (NPY) is a well documented stimulator of food intake and is believed to play a role in the physiological regulation of ingestive behaviour. At the start of the work presented in this thesis the NPY receptor subtype responsible for mediating the characteristic augmentation in feeding was thought to be a novel, previously unidentified receptor subtype, as the pharmacology of the cloned NPY receptor subtypes failed to mimic the pharmacology known to stimulate NPY-elicited eating. The therapeutic implications for the development of an anti-obesity drug based on an antagonist to the "feeding" receptor for NPY made the cloning of this receptor subtype a rapidly evolving and highly competitive field. Initial efforts were therefore directed at cloning the feeding receptor for NPY using various novel receptor cloning strategies. The structural diversity exhibited by the members of the NPY receptor gene family made cloning using a homology screening strategy difficult, as demonstrated by the difficulties encountered in designing degenerate primers pairs for PCR or GenetrapperTM technology. As a result, functional expression cloning strategies were employed as an alternative strategy to clone novel NPY receptor gene family members, in particular the feeding receptor for NPY. Two different COS cell expression cloning strategies were used to identify NPY receptor gene family members following ligand binding and screening for cells expressing the desired cDNA by different detection methods. During the course of this work the structure of the NPY Y5 receptor was reported using an expression cloning strategy identical to one described in this thesis, and this initial report proposed that this receptor was the feeding receptor for NPY. However, subsequent reports describing a role for the NPY Y1 receptor in the regulation of NPY- induced feeding have generated considerable controversy regarding the identity of the NPY receptor subtype involved in feeding behaviour regulation. The cloning of the NPY Y5 receptor enabled further studies to be directed toward examining the regulation of the Y5 gene, with a view to elucidating further the role of both the Y1 and Y5 receptors in the control of feeding behaviour. Hybridisation screening of a rat genomic library permitted the identification of a partial rat Y5 genomic clone, and enabled further efforts to characterise the promoter region to be carried out. Additional studies were carried out to investigate the regulation of Y1 and Y5 receptors by examining changes in the levels of receptor mRNA expression