| Summary: | 碩士 === 國立中央大學 === 系統生物與生物資訊研究所 === 98 === G-protein-coupled receptors (GPCRs) that belong to seven transmembrane receptors, mediate a variety of extracellular signals to induce intracellular responses. Only 10% of GPCRs are targeted bt 50% of current marked drugs, emphasizing the potential of the remaining 90% of the GPCR superfamily for drug targets. The difficulties in large producing GPCRs in vitro and in generating crystal structures hinder structural studies of GPCRs. More complicatedly, GPCRs can form homo- or heteromers for function. The responses between homomers and heteromers or between oligomers and monomers are different while using the same agonist to stimulate. The oligomeric potential of GPCRs allows for more complex ligand-receptor relationships and signaling pathways. Four proton-sensing GPCRs (OGR1, GPR4, TDAG8, G2A) are identified to have fully activation at pH6.4~pH6.8 and important to pH homeostasis and acid-induced pain. Among these genes, primary sequence of G2A is less close to the others, and four of five critical histidine residues that are involved in pH sensing of OGR1 are replaced by other amino acids in G2A. Unexpectedly, G2A is the only proton-sensing GPCR that does not generate any significant responses after acid stimulation in cells overexpressing G2A gene, but did so in OGR1. Whether G2A does respond proton or forms heteromers with other family receptors (such as OGR1) to be functional, remains unclear. The objective of this thesis are (1) to purify proton-sensing GPCRs using bacteria expression system for structural analysis and (2) to explore whether G2A responds acid stimulation using ligand-mediated internalization technique and whether G2A form a functional heteromer with OGR1 to respond acid stimulation using FRET acceptor photobleaching technique. I have found that (1) bacteria expression system was not suitable for in vitro expression of proton-sensing GPCRs; (2) G2A did not internalize into the cytosol in response to acid stimulation, but OGR1 and TDAG8 did so; (3) G2A and OGR1 can form heteromers in resonse to acid stimulation.
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