Call number: LD2668 .T4 1984 S88 / Master of Science

No description available.

Prototoxin proteins have been identified as members of the Ly6/uPAR super family whose three-finger motif resembles that of α-bungarotoxin. Though they are known to modify the function of nAChRs, their specificity is still unclear. Our lab identified three prototoxin proteins in the chicken ciliary ganglion: Ch3ly, Ch5ly, and Ch6ly. Ch6ly was later identified as prostate stem cell antigen (PSCA), and specifically decreased the amount of calcium influx through the homomeric α7 nAChR subtype. I then identifiedCh3ly and Ch5ly as LY6E and LYPD6B, respectively. I focused my attention onLYPD6B because of its expression in the brain. This dissertation tests whether LYPD6Bis a prototoxin protein that specifically co-localizes with and modifies the function of the heteromeric α3β4* nAChRs (the other nAChR subtype expressed in the chicken ciliary ganglia). In the first part of my dissertation I performed intracellular two-electrode voltage clamp on Xenopus oocytes co-expressing human LYPD6B and different stoichiometries of the α3β4* nAChR, these included two (α3)2(β4)3 withβ4−α3−β4−β4−α3 and β4−α3−β4−α3−β4 stoichiometries, two (α3)3(β4)2 with stoichiometries β4−α3−α3−β4−α3 and β4−α3−β4−α3−α3, two (α3β4)2(α5D)β4−α3−α5D−β4−α3 and β4−α3−β4−α3−α5D, and (α3β4)2(α5N) with stoichiometries β4−α3−α5N−β4−α3 and β4−α3−β4−α3−α5N. Concatemeric constructs are designed to link nAChR subunits, thus when translated it is done so as a single polypeptide. LYPD6Bincreased the acetylcholine (ACh) potency and desensitization rate, but decreased the maximum current response (Imax) for the (α3)3(β4)2 nAChR subtype. Yet, LYPD6Bonly decreased the Imax for the (α3β4)2α5 D-variant and not the N-variant (associated with increase nicotine consumption). For the second part of my dissertation, I determined if the expression of LYPD6B correlated with nAChRs in an activity dependent manner. Though LYPD6B mRNA expression correlates with nAChR subunit mRNA expression levels, it seemed to be independent of nAChR activity. To determine if fluorescent colocalization occurs between LYPD6B and a specific nAChR subtype, I genetically engineered LYPD6B to express a human influenza hemagglutinin (HA) epitope tag and cloned into a chicken retrovirus. LYPD6B was shown to co-localize only with the α3β4*heteromeric and not the homomeric α7 nAChRs, in a nAChR activity dependent manner. This study adds to the complexity of a prototoxin’s function by suggesting that the specificity is dependent on nAChR type and stoichiometry. It is the first in identifying a prototoxin protein, LYPD6B, which specifically modulates the function of the(α3)3(β4)2 and (α3β4)2(α5 D-variant) heteromeric nAChR subtypes. For the (α3β4)2(α5D-variant) nAChR subtype LYPD6B decreased the Imax. Such observation may be telling of a novel mechanism involved with nicotine dependence. For the(α3)3(β4)2 nAChR subtype LYPD6B increases its ACh sensitivity, desensitization rate, while decreasing Imax. Additionally, the co-localization of LYPD6B and α3β4* nAChRsin the lack of nAChR activity highlights the relevance of the functional effects α3β4*nAChRs exhibit due to LYPD6B. Such relevance may be the utilization of limiting Ach amounts.

Neuromuscular synapse formation is driven by two nerve-derived molecules, agrin and neuregulin. Agrin is believed to signal through a Muscle Specific Kinase (MuSK)/Lrp4 receptor complex to cluster existing acetylcholine receptors (AChRs) in the postsynaptic membrane via a rapsyn-mediated mechanism, while neuregulin signals via ErbB kinase receptors to induce synaptic gene transcription. Recent studies suggest that neuregulin-ErbB signalling may also cross-signal to the agrin-MuSK pathway to modulate agrin’s ability to cluster AChRs. This thesis aimed to further elucidate this idea. Results of this thesis present two novel findings. First, there is a direct interaction between two tyrosine kinase signalling pathways at the neuromuscular synapse and second, neuregulin plays an important role in modulating, modifying and refining AChRs at developing synapses. Here I show that neuregulin can modulate two distinct processes. In the presence of agrin, neuregulin was able to potentiate both agrin-induced AChR clustering and agrin-induced AChR cluster dispersal, and this modulation by neuregulin occurred independently of any transcriptional mechanism. In vitro, I observed a marked effect by neuregulin on the number and size of AChR clusters that were induced by agrin. Treatment of myotubes for 4hrs with agrin and neuregulin led to a significant potentiation in agrin-induced AChR clustering compared to agrin treatment alone. Neuregulin on its own had no measurable effect on AChR clustering. When incubation times were much longer (12hrs), neuregulin promoted a further significant decrease in AChR cluster number compared to agrin treatment alone. Thus at 12hrs, rather than inhibit AChR clustering, as has been previously suggested, neuregulin promoted the dispersal of AChRs from pre-existing agrin-induced clusters. Follow-up in vivo studies into the potentiating ability of neuregulin in agrin-induced AChR clustering showed that the injection of exogenous neuregulin into developing mouse sternomastoid musculature led to an increase in the size of AChRs. Collectively, these data suggest interactions between the signalling pathways initiated by agrin and neuregulin. Subsequent investigation into the second messengers downstream of agrin-MuSK and neuregulin-ErbB signalling revealed that cyclin-dependent kinase 5 (Cdk5) and Shp2 played a role in neuregulin’s modulation of AChR cluster formation and dispersal. It appears that neuregulin enhances the phosphorylation status of MuSK by inhibiting the Shp2-dependent negative feedback loop on MuSK phosphorylation, thereby leading to an increase in AChR cluster numbers. By contrast, the way in which neuregulin disperses agrin-induced AChR clusters seems to occur partially, via a Cdk5 signalling-dependent mechanism. While it is accepted that neuregulin acts in a transcriptional manner during neuromuscular synapse formation, real-time PCR and immunoblot results suggest that transcriptional regulation was not involved in neuregulin’s modulation of agrin-induced AChR clustering.

No description available.

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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 132-149). Also available in electronic version. Access restricted to campus users.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 132-154). Also available in electronic version. Access restricted to campus users.

Thesis--Ph. D., University of Hong Kong.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.