This thesis is concerned with the study of the collagen receptor glycoprotein VI (GPVI). GPVI has been studied in a number of human haematopoietic cell lines and found to be expressed only in those showing mekaryocytic features. Moreover, differentiation of the human cell lines HEL and CMK to a megakaryocytic lineage using the phorbol ester PMA revealed upregulation of GPVI together with the associated FcR γ-chain. Further, primary cultures of mouse marrow cells demonstrated up-regulation of GPVI towards the end of differentiation, therefore confirming data obtained with cell lines and pointing to GPVI as a possible marker of megakaryocytic differentiation. Structure/function studies of GPVI were carried out by means of transient and stable transfection of the receptor into COS-7 or K562 cells. These studies demonstrated the necessity of the transmembrane argine and the cytoplasmic tail of GPVI for association to the FcR γ-chain. Lack of association or absence of FcR γ-chain rendered GPVI unable to signal, despite binding to convulxin, a GPVI-specific ligand which causes activation of the receptor. K562 cells expressing GPVI and the FcR γ-chain were able to reconstitute the proximal but not distal events in GPVI signaling. A detailed analysis demonstrated impairment in phosphorylation and translocation of SLP-76 to the membrane, despite the presence and activation of others proteins known to be necessary for this phosphorylation/translocation to occur. Stable expression of GPVI in K562 cells, which display low levels of expression of the collagen receptors α2βl and CD36, does not confer signaling properties in response to collagen. However, the cells respond to a collagen related peptide (CRP) which is specific for GPVI and to the snake venoms convulxin, alborhagin and alboaggregin-A, demonstrating that GPVI is one important component through which these venoms are acting.

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The neuromuscular junction is a complex structure resulting from the interaction of an innervating neuron and skeletal muscle fiber. The neuron releases a molecule called agrin which acts via a muscle specific tyrosine kinase, MuSK, to initiate the localized differentiation and specialization of the muscle membrane at the synapse. A defining characteristic of the postsynaptic aspect neuromuscular junction is the concentration of acetylcholine receptors (AChRs) at the crests of junctional folds. Also clustered to these sites is the dystrophin associated protein (DAP) complex, a collection of proteins previously associated with muscular dystrophy and synapse formation. A central component of this complex, dystroglycan, had previously been shown to bind to agrin with high affinity. Inhibition studies that blocked agrin binding to dystroglycan indicated that dystroglycan might be required for synapse formation. In addition, several properties of dystroglycan, including its ability to co-cluster with the AChR and to bind both the neural and muscle forms of agrin, made it an obvious candidate as the putative co-receptor for MuSK. Based on these results we have attempted to define the role of dystroglycan in synaptogenesis. / We studied nerve muscle co-cultures derived from Xenopus embryos and found that dystroglycan is present at almost all neural agrin and AChR clusters. This holds true even in developing synapses as well as in extrasynaptic clusters of AChRs. AChR and dystroglycan aggregates can be induced in vitro, by treating myotubes with agrin and/or exogenous laminin. The AChR clusters formed by laminin application were larger and more dense then those formed by agrin treatments and could be inhibited by the addition of anti-laminin antiserum or laminin fragments that do not self-polymerize. In addition, laminin, unlike agrin, was found to induce AChR and dystroglycan clustering independent of MuSK activation. The introduction of an antisense dystroglycan construct into the C2C12 muscle cell line and the resulting reduction in expressed dystroglycan on myotubes also had little effect on phosphorylation but instead reduced the number of agrin induced AChR clusters on myotubes. These finding suggested that dystroglycan is not the co-receptor for MuSK but rather functions at a point in AChR clustering downstream of agrin-MuSK signaling. When similar experiments were conducted on dystroglycan null myotubes we found that these myotubes respond to agrin in a manner similar to wild-type myotubes but that the AChR clusters formed on null myotubes were two to three times larger, half as dense and significantly less stable. Synapses in chimeric mice with dystroglycan deficient muscle were similarly affected. In culture and in vivo the absence of dystroglycan also resulted in the disruption of laminin, perlecan and acetylcholinesterase localization to AChR clusters but did not affect rapsyn or agrin localization. Finally, I present unpublished observations that suggest that the close association of dystroglycan with rapsyn and AChRs may not result from a direct interaction between dystroglycan and rapsyn. From these results we propose that dystroglycan is a key element of a "trap" requir

The lumen of the endoplasmic reticulum (ER) provides a specialized environment to assure the folding and oligomerization of secretory proteins to their native conformations. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a biosensor in the ER that detects the folding state of glycoproteins. UGGT-catalyzed monoglucosylation of incompletely folded glycoproteins leads to their continued retention in the ER through their association with the lectins calnexin and calreticulin for further folding or for degradation. Purified recombinant UGGT from rat liver and glycoprotein substrates from a mutant strain of Saccharomyces cerevisiae were used in an in vitro system to examine the peptide components recognized by UGGT in unfolded glycoproteins and glycopeptides. Mass spectrometry was used to measure and quantitate the levels of glucose incorporation into these substrates that was directly related to their level of recognition by UGGT. To assess the capacity of UGGT for sensing non-native structures in glycoprotein substrates, Exo-1,3-beta-glucanase (beta-Glc) from S. cerevisiae was crystallized and its structure determined. A mutagenesis strategy was used to mutate solvent-exposed residues to yield the beta-Glc F280S point mutant that retained enzymatic activity while still being recognized by UGGT. These data suggest that UGGT recognizes solvent-exposed hydrophobic patches in the primary and tertiary structure of glycoproteins even in near-native conformations.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 181-198).

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Thesis (Ph.D.) -- University of Adelaide, Dept of Anatomical Sciences, 2003? / Bibliography: leaves 262-298.

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract and vita. Includes bibliographical references (leaves 130-146).