Obesity and its associated metabolic complications are major burdens upon health systems globally highlighting the need for basic research into therapeutic targets to treat such disorders. The circulating hormone adiponectin has protective effects against several obesity-associated metabolic complications including insulin-resistance, hypertension and cardiovascular disease. Recent studies have elucidated the beneficial metabolic effects of adiponectin yet the molecular mechanisms behind its actions remain poorly characterised. The recent discovery of two receptors of adiponectin in 2003, AdipoR1 and AdipoR2, promises to further our understanding of how adiponectin mediates such effects. AdipoR1 and AdipoR2 are predicted to be seven transmembrane domain proteins (7TMs), however, they are considered unique to other 7TMs such as G-protein coupled receptors (GPCRs) since they have been shown to exhibit a reversed topology and also activate a distinct set of signalling molecules. Owing to their recent discovery and a lack of essential tools to permit basic research into adiponectin receptor (AdipoR) biology, our knowledge of these receptors remains extremely limited. The aim of this thesis was to generate tools and further characterise AdipoR structure and function in order to advance our understanding of how adiponectin’s effects are mediated. Early chapters in this thesis focus upon the generation and characterisation of fundamental molecular tools such as AdipoR mammalian expression plasmids and cell lines. Using these tools, we confirm that each AdipoR localises to the plasma membrane (PM) and does indeed possess a reversed topology to GPCRs; having an intracellular amino terminus and an extracellular carboxyl terminus. AdipoR1 and AdipoR2 are able to form both homo and hetero-multimers, as shown for numerous GPCR proteins, but neither receptor appears to be modified by N-linked glycosylation, a common modification of GPCRs. AdipoR-specific polyclonal antibodies were also generated and characterised in early chapters which allowed detection of endogenous AdipoR1 and AdipoR2 protein in multiple cell lines as well as primary human adipocytes. These studies revealed that AdipoRs are not only expressed in a variety of cell lines but also exhibit differential expression profiles suggesting AdipoR1 and AdipoR2 may mediate distinct functions of adiponectin. A major finding of this thesis was the discovery of a novel AdipoR1-interacting protein, ERp46. This is the first AdipoR1-specific interacting protein identified and only the second AdipoR-interacting protein described to date. ERp46 (also known as EndoPDI and pcTRP) has been previously described although knowledge of its function and cell biology is very limited. ERp46 co-immunoprecipitated with AdipoR1 in several different cell lines and showed no evidence of interaction with AdipoR2. Via immunofluorescence microscopy, ectopically expressed ERp46 localised to both the PM and intracellular structures, likely to be the endoplasmic reticulum (ER) whilst subcellular fractionation revealed that endogenous ERp46 localised to the ER-containing and PM-containing fractions in both HeLa and HEK cell lines. In silico analyses predict that ERp46 has a transmembrane domain and exhibits an extracellular / luminal carboxyl terminus, the latter of which was confirmed by immunofluorescence microscopy studies using epitope-tagged ERp46 constructs. Given that ERp46 primarily localises to the ER, we assessed whether ERp46 was involved in regulation of AdipoR trafficking. ShRNA knockdown of ERp46 resulted in increased AdipoR1 and AdipoR2 expression at the PM providing evidence to support a functional role in AdipoR biology. The presence of ERp46 at the PM, together with the data suggesting ERp46 regulates AdipoR subcellular localisation, led to studies investigating whether ERp46 contributes to adiponectin signalling. We demonstrate that adiponectin treatment results in the phosphorylation of key adiponectin targets (such as AMPK and p38MAPK) in HeLa cells and present data to support the requirement of ERp46 for specific adiponectin-induced signalling events. Furthermore, our studies show that ERp46 is not only involved in adiponectin-induced signalling but also in specific signalling events induced by insulin and epidermal growth factor (EGF) treatment. In summary, the work presented in this thesis confirms the findings of the initial publication in which the receptors were described and also identifies several novel features of the adiponectin receptors previously not reported. The identification of an AdipoR1-specific interacting protein that modulates both the localisation and signalling events of adiponectin is of particular interest. The data described in this thesis supports emerging evidence which suggests that AdipoR1 and AdipoR2 have distinct roles in adiponectin signalling and as such, this work has provided further insights into the mechanisms behind adiponectin action and provides a basis for future studies.

Currently the mammalian endoplasmic reticulum (ER) is known to contain at least 20 different protein disulphide isomerase (PDI) family members. The oxidoreductases in the PDI family are thought to catalyse the formation and rearrangement of disulphide bonds in newly synthesised proteins. The focus of this work was to characterise two of the PDI family members: PDI and ERp46. In vitro translation reactions of major histocompatibility complex (MHC), β1-integrin (β1-I), haemagglutinin (HA), procollagen α1(III) and preprolaction (pPL) were carried out in untreated or PDI-depleted cells. The depletion of PDI decreased the rate of folding of MHC and β1-I and also prevented the oligomerisation of HA, suggesting a role for PDI in folding these putative substrates. However, when PDI was depleted neither the folding of pPL or HA was affected, implying that they may not be substrates for PDI. To determine the role of ERp46 in the cell, a substrate-trapping approach was used. Here substrates interacting with ERp46 were trapped as mixed disulphides isolated by immunoprecipitation, separated by 2D SDS-PAGE and identified by mass spectrometry. It was demonstrated that ERp46 forms mixed disulphides with at least 23 proteins, including heavily secreted proteins such as laminins, integrins and collagens. In particular, interactions with Ero1, Prx IV, EDEM3 and ERAP2 were found and confirmed by immunoprecipitation of radiolabelled in vitro translated protein. Notably nine of these clients of ERp46 have previously been identified as substrates of ERp57 (Jessop, Watkins et al. 2009). This would support the hypothesis that several different oxidoreductases, working in concert, are required to fold certain substrate proteins. Also, it was confirmed that Prx IV and Ero1 each form a mixed disulphide with PDI. These results highlight the importance of PDI family members in recruiting co-factors to substrates. Additionally, the over-expression of ERp46 led to increased cell survival following DTT treatment, yet after depletion of ERp46, cells were less able to grow, perhaps suggesting a role for ERp46 in maintaining ER redox homeostasis and cell survival. This suggestion was supported by the finding that ERp46 is able to catalyse the reduction of Prx IV in the presence of glutathione. These results suggest that Prx IV provides a novel mechanism for the transfer of disulphide bonds to nascent proteins in the ER via PDI family members such as ERp46 and PDI.