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The obesity epidemic is associated with an increase in the prevalence of a number of chronic diseases including type 2 diabetes, cardiovascular disease, hypertension and some cancers and has been described by the World Health Organisation as one of the greatest public health challenges of the 21st century. Obesity is characterised by excessive expansion of adipose tissue mass underpinned by adipocyte hyperplasia. Central to this is the process of adipogenesis, which encompasses the proliferation and terminal differentiation of fibroblastic preadipocytes, contained within adipose tissue, to mature adipocytes. Despite the pivotal role of this process in obesity our understanding of the regulatory mechanisms governing adipocyte development, either in physiological or pathophysiological settings, is limited. Studies aimed at understanding this complex process are integral to development of more effective strategies for the prevention and/or treatment of obesity and obesity related diseases. Our laboratory recently identified a putative role for inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, in the dynamic regulation of lipid accumulation. Upon treatment of a variety of cell types with insulin or oleic acid IMPDH translocates to lipid droplets and inhibition of this translocation is correlated with reduced lipid accumulation. As lipid droplet formation and lipid accretion are defining features of adipogenesis, it was hypothesised that IMPDH may facilitate efficient lipid accumulation during adipose conversion of preadipocytes. In vitro systems have been used extensively to dissect the molecular and cellular events involved in adipogenesis. Therefore the aim of this project was to extend these investigations to examine the requirement for IMPDH activity during adipogenesis, using the well characterised murine 3T3-L1 cell line and primary human preadipocytes (phPAs). IMPDH expression and activity were transiently increased during differentiation of the 3T3-L1 cells although IMPDH did not associate with lipid droplets under these conditions. Pharmacological inhibition of IMPDH, using mycophenolic acid (MPA; 1 µM), reduced intracellular GTP by 60%, and blocked mitotic clonal expansion (MCE) and adipogenesis. Supplementation with guanosine (60 µM), a substrate in the nucleotide salvage pathway, restored both GTP levels and adipogenesis. These observations indicated that IMPDH activity is required for efficient differentiation of 3T3-L1 preadipocytes. Preliminary studies, involving differentiation of phPAs in standard serum-free medium (SFM) suggested that phPAs were resistant to MPA. To afford better comparison between the phPAs and the 3T3-L1 cells, which are differentiated in serum-containing medium (SCM), a modified 3T3-L1 like protocol facilitating efficient differentiation of the phPAs in SCM was established. Under these conditions phPAs displayed considerable variation in sensitivity to MPA which gave a trend towards decreased differentiation (reduced by 26%; p=0.07). Supplementation with guanosine significantly reduced adipogenesis (by 37%; p<0.05) in the phPAs independent of MPA. Furthermore, cells that were MPA resistant were also refractory to guanosine suggesting greater plasticity of guanine metabolism in phPAs from those subjects. A major difference between the cell types was that phPAs differentiated with high efficiency in the absence of MCE. Collectively, these data indicate that MCE is required for efficient differentiation of 3T3-L1 cells but not phPAs, even when differentiated under similar conditions, and suggest that the involvement of MCE underpins the differences in sensitivity to MPA between cell types. The differential effects of guanosine suggest there are additional differences with respect to the effects of manipulation of guanine nucleotides between cell types. In summary, the work presented in this thesis demonstrated inhibition of IMPDH blocked adipogenesis of murine 3T3-L1 cells and reduced differentiation of phPAs in some subjects. These observations provided novel insights into differences between differentiation of 3T3-L1 cells and phPAs, including their relative sensitivities to alterations in guanine nucleotides, and have implications for adipose tissue biology especially those factors involved in guanine metabolism. Ultimately this knowledge may form the basis for development of novel therapeutic strategies aimed at reduction of obesity and associated complications such as insulin resistance and type 2 diabetes.

AMPA receptors mediate excitatory synaptic transmission throughout the central nervous system via activation by their natural agonist glutamate. Several other molecules have been recognized as receptor agonist or antagonist, and recently allosteric modulators have been developed that potentiate the currents generated by these receptors. The goal of this thesis has been to address specific and as yet unresolved questions regarding the binding interactions between the AMPA receptors and these classes of molecules. For instance AMPA receptors are seemingly converted to have lower affinity for agonist as they move towards synapses and we evaluate two hypotheses put forward to explain the molecular mechanisms responsible for this. Additionally, guanine nucleotides competitively inhibit AMPA receptors and a second goal has been to further characterize guanine nucleotide binding, and to create mutations that selectively diminish this so that the function of the inhibition can be evaluated. A third goal has been to characterize the molecular factors that influence the effects of the allosteric modulators in order to explain why their efficacy differs greatly between brain regions. Experiments pertaining to these three goals were carried out sequentially and are described below as Projects 1 (guanine nucleotide inhibition), Project 2 (agonist affinity), and Project 3 (allosteric modulators). Project 1. Guanine nucleotides competitively inhibit AMPA-Rs (AMPA receptors) and because this inhibition is ubiquitous among virtually all types of glutamate receptors from fish to mammals, it likely serves a physiological function. Evaluation of this would be greatly facilitated if nucleotide binding could be eliminated through mutations without altering other aspects of receptor function, or if compounds were discovered that selectively prevent nucleotide binding. It was previously reported that a lysine in the chick kainate binding protein (cKBP) is specifically involved in guanine nucleotide binding. Therefore we mutated the homologous lysine (K445) in AMPA-R subunit GluR1 plus 12 additional residues around the glutamate binding pocket with the expectation that this would reduce nucleotide binding even further. Nucleotide affinity was determined by measuring the displacement of [3H]fluorowillardiine. As expected, the guanine nucleotide affinity was decreased about five-fold in R1-K445A mutants and the agonist affinity was seemingly unchanged. However, when tested by electrophysiology, characteristics of the mutant such as desensitization and the EC50 for glutamate were found to be altered. None of the other mutations were more successful at decreasing nucleotide affinity selectively. Nonetheless, these studies have given new insight into the docking mode of guanine nucleotides. The loss of binding in R1-K445A was much larger for GTP and GDP than for GMP, and guanosine binding, which is much lower, was unaffected by the mutation. These data suggest that the first phosphate of GMP determines the higher affinity of the phosphorylated nucleotides, and that K445 stabilizes the binding of the second and third phosphates of GDP and GTP. This along with various other observations suggest that the guanine base docks deep within the agonist binding pocket and that bulky additions, such as the phosphates, are accommodated by projecting out of the cleft in the vicinity of lysine 445. However, the exact docking mode of guanine nucleotides would have to be determined by crystallography. Project 2. Agonist binding to AMPA-R in brain consists of a high and low affinity components with KDs of 9-28 nM and 190-700 nM. Previous studies have suggested that newly synthesized receptors have high affinity and are converted to lower affinity by a secondary process. Two particular processes have been implicated, namely the conversion of receptor glycosylation from immature to complex, and modulation by receptor associated proteins. Both hypotheses were evaluated in this project using homomeric receptors GluR1-4 expressed in HEK 293 cells. The role of glycosylation was tested mostly with GluR4 receptors because they are expressed in distinct populations that exhibit either immature or complex glycans and their binding consists of high and low affinity components similar to those previously seen in brain receptors. Cells were treated with castanospermine or deoxymannojirimycin to decrease the proportion of receptors with complex glycosylation, or with cycloheximide plus chloroquine to increase the number of receptors with complex glycosylation. Although 70% of receptors from cells treated with cyloheximide/chloroquine exhibited complex glycans compared to <5% with other treatments, the affinity decreased at most 2-fold. Also, the low affinity component was nearly 80% of the total binding in receptors that exhibited virtually no complex glycans. Taken together these data indicate that complex glycosylation is not the key factor that confers low affinity. To test the second hypothesis GluR1i or GluR2i were co-expressed with stargazin which associates to receptors in neurons and affects their kinetics and trafficking. Considering the affinities of the two components seen in brain, we expected stargazin to cause a 20-fold or greater decrease in binding affinity. This was not the case, however our results did suggest that stargazin caused the appearance of a low affinity component but this was small and remained largely masked by the more abundant high affinity component. Recently, experiments with brain membranes have revealed preliminary evidence that an associated protein of ~85kDa may cause receptors to have low affinity. This hypothesis is currently under investigation. Project 3. Ampakines are cognitive enhancers that potentiate AMPA receptor currents at excitatory synapses. The efficacy of these drugs varies substantially among neurons in different brain regions, being for example about three times larger in the hippocampus than in the thalamus. Binding assays have shown that these compounds also increase the affinity of receptors for agonists. Importantly, the efficacy of these drugs to increase synaptic responses and agonist binding exhibit a positive correlation. Indeed, we have found that the increase in agonist binding (Emax) induced by the prototypical ampakine CX546 is highly variable across eight brain regions and that there is a 3-fold difference between the hippocampus and the thalamus which is similar to the difference reported for physiological efficacy. Therefore, binding assays or receptor autoradiography can potentially be used to predict the physiological efficacy of these drugs in a particular brain region. An important goal of this project has been to identify factors that may be responsible for the regionally different efficacies. Ampakines show some preference for receptor subunits but various considerations suggest that other factors must be involved. In this project we evaluated the role of a novel class of proteins called TARPs (transmembrane AMPA receptor regulatory proteins) that have recently been discovered to be tightly associated with AMPA receptors and to regulate their kinetics. Four of these proteins, named lambda;2(stargazin),λ3,λ4,and λ8 are abundant in the brain, but they exhibit highly selective regional distribution. We determined the maximum increase in agonist binding (Emax ) caused by saturating CX546 in three different AMPA receptor subunits, GluR1i, GluR2i, and GluR4i without and with co-expression of the four TARPs. Without TARPs, both Glu2i and GluR4i showed an Emax value of 100% over baseline binding. Co-expression of TARPs increased the Emax in GluR2i and this was largest for λ3 and λ8 (~130%). However, TARPs decreased the Emax of CX546 in GluR4i and this was most notable with λ2 and λ4 (~72%). Agonist binding in GluR1i was increased by only 15% and it was not significantly changed by TARPs. The expression patterns of TARPs and AMPA-R subunits in the brain have been partially characterized in the literature. Thus, it was previously reported that GluR4i transcripts are abundant in the thalamus but minor in the hippocampus. Using western blots we confirmed that this is also true for protein content; in the thalamus expression of GluR1, GluR2, GluR3, and GluR4 was 4%, 33%, 40%, and 147% respectively, of that in the hippocampus. When considering the known expression patterns of TARP variants, the hippocampus can be described as being enriched in GluR2, λ3 and λ8 while GluR4, λ2 and λ4 are prevalent in the thalamus. In comparison between these specific subunit/TARP combinations, the Emax values for those representative of the hippocampus (GluR2i/λ3 or λ8) were ~2-times larger than the Emax values of thalamic combinations (R4i/λ2 or λ4). Thus we can conclude that the differences in the expression of both TARP variants and AMPA-R subunits are critical factors for determining the variable efficacy of ampakines across brain regions.

The SRP and SRP receptor have long been recognized as essential components of the protein translocation machinery in higher eukaryotes. The biochemical studies discussed in this thesis demonstrate that the signal recognition particle (SRP) mediated transport of proteins across the mammalian endoplasmic reticulum requires the participation of guanine ribonucleotides, in a capacity distinct from their role in polypeptide elongation. The requirement for guanine ribonucleotides during translocation was detected by experimentally separating the synthesis and transport phases of the translocation reaction. Here, the initial targeting of ribosomes to the membrane required SRP and an SRP receptor, but not GTP. However, the insertion of the nascent chain into the membrane required the presence of both SRP and SRP receptor, as well as, GTP. Further biochemical characterization of the initially targeted translocation intermediate demonstrated that SRP remains bound to targeted nascent signal sequences, unless GTP is present. The SRP-receptor catalyzed displacement of SRP from ribosomes was GTP-dependent both with intact membranes and with the purified SRP receptor preparations. GTP specific binding localized to the α subunit of the receptor by photoaffinity labeling and by probing nitrocellulose blots of the receptor with GTP. In addition, an analysis of the α subunit primary sequence revealed elements which are similar, yet not identical, to guanine ribonucleotide binding site consensus sequence elements. These results, taken together, indicate that the SRP receptor represents a novel class of GTP binding protein and is responsible for the guanine ribonucleotide mediated displacement of SRP from nascent signal sequences. A more detailed biochemical investigation of the GTP hydrolysis cycle of the SRP receptor demonstrated that the affinity between SRP and the SRP receptor is substantially greater in the presence of bound GTP and that the subsequent hydrolysis of bound GTP by SRα is necessary to recycle SRP to the cytoplasm. Purified SRP receptor was shown to hydrolyze GTP slowly. However, the GTP hydrolysis rate was substantially increased when both the SRP receptor and SRP were present in equimolar quantity. SRP does not hydrolyze GTP under these assay conditions. Moreover, free SRP was found not to compete effectively with SRP-ribosome complexes for the receptor, implying that the conformation of SRP is altered upon binding to a signal sequence. This result suggests that the affinity between SRP and the SRP receptor may be exquisitely regulated in order to prevent futile GTP hydrolysis cycles from occurring in the absence of secretory protein synthesis. Furthermore, the demonstration that the SRP receptor is a GTP binding protein provides fundamental insight into the mechanism of protein translocation. The displacement of SRP appears to be tightly coupled to the membrane insertion of nascent signal sequences. The membrane inserted intermediate in nascent chain translocation can be characterized by i) a resistance to extraction from the membrane with either EDTA or 0.5M KOAc; ii) an insensitivity to protease digestion, even after dissolution of the membrane with nonionic detergent. These results indicate that SRP displacement allows the nascent chain to interact with an additional membrane bound, protein component of the cellular translocation apparatus. Once in contact with this additional component, the nascent chain is shown to be capable to transverse the membrane bilayer in the absence of ribonucleotide hydrolysis or the continued elongation of the polypeptide. Thus, the results are incompatible with postulated mechanisms of protein translocation requiring that energy be derived from the continued elongation of the nascent polypeptide or from the direct interaction of a hydrophobic signal sequence with the lipid bilayer.