Regulators of G protein signaling (RGSs) are negative regulators of G protein coupled receptors (GPCRs). Our lab has demonstrated that yeast Saccharomyces cerevisiae is a useful system to study RGS and G protein signaling. Mammalian RGSs can be expressed in yeast and favored to interact with mammalian GPCRs as well. / Based on the observation that human RGS1 causes yeast cell growth arrest, I therefore used RGS1 expressing yeast cells to screen a mouse T cell cDNA library in order to find potential interacting proteins. From the screen, I identified a mouse sphingomyelin synthase 1 (SMS1) cDNA. By using a series of different apoptotic stimuli, such as hydrogen peroxide, osmotic stress, exogenous ceramide and its precursors, high temperature etc., SMS1 expression was found to suppress cell growth arrest and prevent viability decline, indicating that SMS1 represents an anti-apoptotic protein that functions by decreasing the intracellular level of pro-apoptotic ceramide. / Gene analysis further indicated that the SMS1 gene consists of 16 exons spread over a 256kb portion of mouse chromosome 19. It is alternatively spliced to produce 4 different transcripts (SMS1alpha1, SMS1alpha2, SMS1beta and SMS1gamma) and encode 3 different proteins (SMS1alpha, SMS1beta and SMS1gamma). Notably, I found that SMS1beta protein does not interfere with SMS1alpha anti-apoptotic function, although both of these two proteins contain the protein-protein interaction domain, sterile alpha motif (SAM), at their N-terminus. / I also carried out a study to examine GPCR-RGS interactions using the yeast expression system. Our lab had noticed that there was an extra RGS5 related protein that was detected by western blot analysis in the protein extracts prepared from yeast and HEK293 cells expressing RGS5. The size of the band was approximately 2 times the molecular weight of RGS5, indicating the possibility that RGS5 forms a dimer. To further examine this hypothesis, I, therefore, performed a series of experiments, included yeast 2 hybrid assays, to demonstrate that RGS5 does interact with itself. This is the first report that RGS can form a dimer. The implications for this finding are discussed in detail.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.111875 |
| Date | January 2007 |
| Creators | Yang, Zhao, 1970- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Anatomy and Cell Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002651418, proquestno: AAINR38665, Theses scanned by UMI/ProQuest. |
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