The overall aim of this thesis is to investigate strategies to aid in the measurement of G-protein coupled receptor (GPCR) activity for high-throughput screening and sensing applications. GPCRs are cell surface receptors which have seven membrane spanning domains. They are the largest family of membrane proteins in the human genome and are involved in a number of physiological and pathophysiological pathways. They are the most widely targeted protein family for therapeutics being the target for over 30% of the currently available prescription drugs (Jacoby et al. 2006). For this reason commercial interest and investment into compound screening using these receptors as targets is of high importance in lead drug discovery. Additionally, the extensive ligand range of the GPCR superfamily, which includes light, odorants/ volatiles, neurotransmitters and hormones, make them an attractive biological recognition element in biosensor applications. This thesis demonstrates the functional expression of the H1-histamine, M2-muscarinic and α₂ₐ-adrenergic receptors of the G-protein coupled receptor family, along with their associated G-proteins (Gα, Gβ and Gγ). Expression was achieved using the Sf9/baculovirus expression system. The G-proteins were successfully incorporated into an assay system using time-resolved fluorescence resonance energy transfer (TRFRET). TR-FRET was used in order to create a homogeneous assay format capable of monitoring GPCR activation through the movement of the G-protein subunits. Fluorescence changes in the TR-FRET assay indicated a change in distance between the Gα subunit and Gβγ dimer. The separation of the Gα subunit and the Gβγ dimer after activation resulted in a significant decrease in TR-FRET measurement. The homogeneous set-up of the TR-FRET assay could potentially be adaptable to an array based format. This thesis describes the capture of vesicles containing functional GPCRs onto a solid substrate via the specific interaction between complementary oligonucleotides. GPCR presence and function within the immobilized vesicles, was demonstrated using fluorescent ligands. Further to this, alternative lipid hosts (to the vesicles), known as cubosomes, were introduced. When tagged with an oligonucleotide, these cubosome particles were also shown to immobilize site specifically onto a complementary oligonucleotide surface. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369537 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009
| Identifer | oai:union.ndltd.org:ADTP/290606 |
| Date | January 2009 |
| Creators | Bailey, Kelly |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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