Heterologous Expression of G-Protein Coupled Receptors Leads to Activation of Cellular Stress Response Pathways in Saccharomyces Cerevisiae
| Author(s) | Mancini, J. Dominic | |
| Date Accessioned | 2009-09-30T21:33:58Z | |
| Date Available | 2009-09-30T21:33:58Z | |
| Publication Date | 2009-05 | |
| Abstract | G-protein coupled receptors (GPCRs), a diverse class of therapeutically relevant proteins, are implicated in regulating nearly every aspect of our physiology. High-resolution structural data exists only for a small handful of GPCRs, inhibiting the efficient structure-based design of new pharmaceuticals. Heterologous expression of GPCRs is an effective method of obtaining the large quantities of purified protein necessary for structural analysis via x-ray crystallography. S. cerevisiae is an economical expression system, capable of producing active human A2a receptor in mg quantities per liter of culture, and has tremendous potential as an expression system for the structural analysis of other GPCRs. The human adenosine A1 (hA1R), human adenosine A2a (hA2aR), human adenosine A2b (hA2bR), human adenosine A3 (hA3R), human β 2 adrenergic β2A (hβ2AR), human cannabinoid CB1 (hCB1), human cannabinoid CB2 (hCB2), human chemokine CCR5 (hCCR5R), human chemokine CXCR4 (hCXCR4R), human dopamine D2 (short isoform) (hD2R), human follicle stimulating hormone FSH (hFSHR), human neurokinin NK1 (hNK1R), and human neurokinin NK2 (hNK2R) receptors were individually cloned into pITy expression plasmids, which were stably transformed into BJ5464 cells. The unfolded protein response (UPR) and the heat shock response (HSR) associated with expressing each of these GPCRs was determined by transforming each strain with a UPR and HSR reporter plasmid and conducting time-course experiments. The UPR and HSR results were analyzed in conjunction with localization data obtained from confocal images and GPCR activity results obtained from radio-ligand binding experiments to determine a correlation between HSR, UPR, and protein mislocalization. Protein mis-localization was accompanied by UPR activation and in most cases HSR activation in the GPCR expressing strains. Expression of human A2a did not activate either response. Monitoring cellular stress responses may be an efficient method of finding other GPCRs that properly express in S. cerevisiae. | en |
| Advisor | Anne Skaja Robinson | |
| Program | Chemical Engineering | |
| Sponsor | University of Delaware | en |
| URL | http://udspace.udel.edu/handle/19716/4390 | |
| Publisher | University of Delaware | en |
| dc.subject.lcsh | Saccharomyces cerevisiae | |
| dc.subject.lcsh | G proteins | |
| dc.subject.lcsh | Plasmids | |
| Title | Heterologous Expression of G-Protein Coupled Receptors Leads to Activation of Cellular Stress Response Pathways in Saccharomyces Cerevisiae | en |
| Type | Undergraduate Thesis |