G protein coupled receptors (GPCRs) are the largest family of transmembrane receptors in the genome and are activated by a multitude of ligands including neuropeptides, hormones and sensory signals. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important mediators in homeostatic control. Many modulators of PVN/SON activity, including neurotransmitters and hormones act via GPCRs - in fact over 100 non-chemosensory GPCRs have been detected in either the PVN or SON. The introduction to this thesis begins with a comprehensive summary of GPCR expression within the PVN/SON, with a critique of the detection techniques used within the literature. Also discussed are some aspects of the regulation and known roles of GPCRs in the PVN/SON, as well the possible functional significance of orphan GPCRs. Particular interest is paid to the recently 'deorphanised' G protein-coupled oestrogen (E2) receptor, GPER, which is the first receptor to be acknowledged as a steroid binding GPCR (although there are conflicting studies regarding its affinity for E2) and is expressed in the PVN and SON. Steroids are known to have fast non-genomic effects that are thought to be mediated in-part by membrane-associated forms of the traditional steroid receptors (members of a family of transcription factors). However, the possible discovery of a fast E2 GPCR has raised speculation regarding the existence of other steroid binding GPCRs. Thus the experimental Chapters were undertaken to explore the concept of fast steroid receptors, with particular emphasis on their possible roles in neuroendocrine systems. Firstly, the distribution of the putative E2 receptor was investigated to give further insight into its possible in vivo roles. In the rodent, high levels of GPER gene and protein expression were detected in the oxytocin and vasopressin neurones in the PVN and SON, the anterior and intermediate lobe of the pituitary, adrenal medulla and renal medulla and pelvis, suggesting roles for GPER in multiple functions including hormone release. To clarify the controversy surrounding GPER as an E2 receptor, we investigated GPER function in vitro using a series of cell signalling assays. However, E2 did not stimulate GPER-mediated signalling, suggesting that either GPER remains an orphan GPCR, or the cell lines used in this study lacked a vital component for E2 activation of GPER. As the rapid effects of glucocorticoid have been reported in numerous brain regions (including the PVN and SON), endocrine, and other tissues, the second part of this thesis focussed on the search for a possible fast glucocorticoid receptor. We compared the tissue distribution gene expression profiles of approximately 125 orphan GPCRs common to human and rodent with tissues that are known to exhibit fast effects of steroids (e.g., hippocampus, PVN, SON, thymus, kidney, etc.). Of the 125 orphans,3 GPCRs (GPR108, GPR146, and TMEM87B) had distribution profiles that closely matched the regions/tissues of interest. These orphans were tested for glucocorticoid activation using a universal deorphanisation assay. However, the identity of the fast glucocorticoid receptor remains unknown, as none of the candidate orphan GPCRs responded to glucocorticoids.
|Date of Award||2011|
|Supervisor||Stephen Lolait (Supervisor) & Anne-Marie O'Carroll (Supervisor)|